*Aggregatibacter Actinomycetemcomitans***: Clinical Significance of a Pathobiont Subjected to Ample Changes in Classification and Nomenclature**

**Niels Nørskov-Lauritsen 1, Rolf Claesson 2, Anne Birkeholm Jensen 3, Carola Höglund Åberg <sup>4</sup> and Dorte Haubek 3,\***


Received: 2 October 2019; Accepted: 13 November 2019; Published: 18 November 2019

**Abstract:** *Aggregatibacter actinomycetemcomitans* is a Gram-negative bacterium that is part of the oral microbiota. The aggregative nature of this pathogen or pathobiont is crucial to its involvement in human disease. It has been cultured from non-oral infections for more than a century, while its portrayal as an aetiological agent in periodontitis has emerged more recently. *A. actinomycetemcomitans* is one species among a plethora of microorganisms that constitute the oral microbiota. Although *A. actinomycetemcomitans* encodes several putative toxins, the complex interplay with other partners of the oral microbiota and the suppression of host response may be central for inflammation and infection in the oral cavity. The aim of this review is to provide a comprehensive update on the clinical significance, classification, and characterisation of *A. actinomycetemcomitans*, which has exclusive or predominant host specificity for humans.

**Keywords:** adherence; endocarditis; fimbriae; JP2; leukotoxin; periodontitis

#### **1. Introduction**

*Aggregatibacter actinomycetemcomitans* is the type species of genus *Aggregatibacter*, which is part of bacterial family *Pasteurellaceae*. [*Bacterium actinomycetem comitans*] was cultured from actinomycotic lesions of humans in the early 20th century. The absence of related microorganisms rendered it difficult to classify this Gram-negative, fastidious rod, and isolates cultured from invasive infections were referred to national reference institutions. The expanding field of oral microbiology with a focus on periodontitis, particularly the localized, severe form that affects adolescents, caused a renewed interest in the bacterial species. In 2006, the current species name was adopted, and *A. actionomycetemcomtians* became type species of a new bacterial genus, *Aggregatibacter*. Influential events in the narrative of *A. actinomycetemcomitans* are listed in Table 1.


**Table 1.** Seminal events in the history of *Aggregatibacter actinomycetemcomitans*.

*A. actinomycetemcomitans* is one species among a plethora of microorganisms that constitute the oral microbiota. It has been estimated that at least 500 different bacterial species colonise the oral cavity [13–15], and half of these may have been cultivated and validly named because of vigorous efforts directed to the cultivation of oral bacteria. Analysis of a large number of 16S rRNA gene clones from studies of the oral microbiota increased the number of taxa to 619 [16], and the number is steadily increasing (www.homd.org). Bacterial species cannot be validly named in the absence of a cultured type strain [17]. Although "taxa", "phylotypes" or "operative taxonomic units" revealed by deep sequencing of polymerase chain reaction (PCR)-amplified 16S rRNA genes have relevance for recognition of microbial fluctuations in health and disease, only cultivable microbiota can be made subject to extensive characterisation, including adherence, animal experiments, antimicrobial susceptibility, co-culture, generation of mutants, and growth characteristics.

Carriage of *A. actinomycetemcomitans* appears to be highly host-specific. Although the spread and dissemination of bacterial clones occur, these are not frequent events; hosts tend to carry their strain from teething to edentulous old age [18]. Yet, the species encompasses properties that sometimes reveal its significance in human disease. Particularly, a single serotype b clonal lineage designated the JP2 clone is associated with a severe form of localised periodontitis and tooth loss in adolescents [12]. But rather than being the causative agent of aggressive periodontitis, *A. actinomycetemcomitans* may be necessary for the action of a consortium of bacterial partners by suppressing host defences [19]. It may be classified as a low abundance oral pathobiont, defined as a member of the microbiota that exerts specific effects on the host's mucosal immune system associated with the development of disease [20]. Although *A. actinomycetemcomitans* may accompany (*comitans*) *Actinomyces*, the narrative of a pathobiont is not valid for other invasive infections such as infectious endocarditis, where *A. actinomycetemcomitans*—when identified—is detected as the sole pathogen by culture and/or PCR on removed heart valves. Severe periodontitis and infective endocarditis are two prominent diseases of very different prevalence, symptoms, and outcome. Although they may share a causative microorganism, a number of conditions is still unknown, and host factors, oral hygiene, and incidental circumstances may be instrumental.

The aim of the present review is to provide a comprehensive update on the characterisation, classification and clinical significance of *A. actinomycetemcomitans* with a particular focus on selected clinical entities. Adhesion, persistence, and inactivation of immune cells are probably essential for the understanding of the intimate association with the host, and these factors are detailed for the purpose

of the elucidation of pathogenicity. A number of relevant publications and reviews of other important biochemical mechanisms of this bacterial species are listed in the relevant sections.

#### **2. Taxonomy, Classification, Serotype (St) and Population Structure**

More than 100 years ago, [*Bacterium actinomycetem comitans*] was co-isolated with *Actinomyces* from actinomycotic lesions of humans [1] (*Actinomyces*, ray fungus, referring to the radial arrangement of filaments in *Actinomyces bovis* sulfur granules; actinomycosis, a chronic disease characterised by hard granulomatous masses). Ample changes have occurred in the classification and nomenclature of this species. Despite the limited similarity with *Actinobacillus lignieresii*, it was reclassified as [*Actinobacillus actinomycetemcomitans*] in a seminal textbook from 1929 [2]. According to Cowan [21], the bacterium was placed in this genus because 'neither Topley nor Wilson could think where to put it'. In 1962 the phenotypic resemblance of [*Actinobacillus actinomycetemcomitans*] with [*Haemophilus aphrophilus*] was described [3], and a subsequent relocation of [*Actinobacillus actinomycetemcomitans*] to genus *Haemophilus* occurred [22]. Nomenclatural classification as [*Haemophilus actinomycetemcomitans*] within the genus *Haemophilus* permitted antimicrobial susceptibility testing according to standards outlined by the US Clinical and Laboratory Standards Institute. Disk diffusion could be performed and interpreted on *Haemophilus* test medium (HTM) in 5% CO2, and HTM broth microdilution testing was carried out in ambient air [23]. However, the nomenclatural relocation did not result in a satisfying classification, because neither [*Actinobacillus actinomycetemcomitans*] nor [*Haemophilus aphrophilus*] are adequately related to *Haemophilus influenzae*, the type species of the genus *Haemophilus*. Finally, in 2006 the new genus *Aggregatibacter* was created to accomodate *Aggregatibacter actinomycetemcomitans*, *Aggregatibacter aphrophilus* and *Aggregatibacter segnis* [11]. A fourth *Aggregatibacter* species, *Aggregatibacter kilianii*, has recently been named (Figure 1) [24].

**Figure 1.** Comparison of *Aggregatibacter* strains by whole genome sequences; distinct species are separated by dotted lines (strain PN\_491 is unclustered). A total of 3261 positions with single nucleotide polymorphism (SNP) are included in the dataset. Values at nodes are percentages of bootstrap replications supporting the node (500 replicates). Bar represents 200 SNPs. Reprinted from *Journal of Clinical Microbiology* [24] with permission.

In the early 1980s, three distinct surface antigens of *A. actinomycetemcomitans* were identified [9], while six serotypes (a through f) were recognised by 2001. The serological specificity is defined by structurally and antigenically distinct O-polysaccharide components of their respective lipopolysaccharide molecules. A seventh St, designated St(g), with a 1:1 ratio of 2,4-di-O-methyl-rhamnose and 2,3,6-tri-O-methyl-glucose, was recently added [25]. St(a), St(b), and St(c) are globally dominant [26], but the distribution may vary according to ethnicity and geography. In Scandinavia, the three dominant serotypes are equally prevalent, while predominance of St(c) is observed in Chinese, Japanese, Korean, Thai and Vietnamese populations [27–31]; a noticeable high prevalence of St(e) has been reported among Japanese periodontitis patients [32]. Assessment of serotype-specific antibodies supports these findings, as all early-onset periodontitis patients from Turkey and Brazil had elevated antibody levels to St(c) and St(a), while St(b) levels were higher in the US [33,34].

An initial characterisation of the population structure of the species was published in 1994, using multi-locus enzyme electrophoresis [35]. Two large and four small divisions were identified, with division I (St(a) and St(d)) and III (St(b) and St(c)) encompassing 34% and 58% of the 97 strains analysed, respectively. Two St(e) strains occupied separate divisions (II and VI), one St(c) strain constituted electrophoretic division IV, while division V was composed of two St(a) and one non-serotypable strain. Sequencing of a 16S rRNA gene fragment from 35 strains suggested a different structure with three major clusters [36]. RNA cluster I included 12 strains of four serotypes (a, d, e, and f), all 10 St(b) strains belonged to RNA cluster II, while RNA cluster III only included St(c) strains (N = 10). Strains of particular serotypes were not exclusively confined to specific RNA clusters; one St(a) strain belonged to the St(b) cluster (II), and two divergent RNA clusters were composed of single strains, namely a St(c) (RNA cluster IV) and a St(e) strain (RNA cluster V), respectively [36].

One study attempted to establish a multi-locus sequence typing (MLST) scheme for *A. actinomycetemcomitans* [37]. Six gene fragments from the *Haemophilus influenzae* MLST scheme were used. The investigation focused on the JP2 clone, which contributed 66 of 82 strains. MLST has insufficient power to reveal dissemination patterns of clonally related strains, and point mutations of two pseudogenes present in the JP2 clone were more versatile in this respect [37]. Moreover, a MLST web site was not organised and, therefore, the benefits of a portable typing scheme were not corroborated. But MLST of 16 non-JP2 strains carefully selected from the enzyme electrophoresis study [35] suggested the existence of four phylogenetic clusters, rooted by an outgroup consisting of an uncommon St(e) strain. Two related clusters were composed of St(b) and St(c) strains, respectively, while a more distinct cluster encompassed strains of St(a), St(d) and St(e) [37].

Restriction fragment length polymorphism using various restriction enzymes and arbitrarily-primed PCR has been used to differentiate types of *A. actinomycetemcomitans* cultured from patients with severe periodontitis and healthy controls [30,37–42]. The method is versatile and discriminative, but lacks portability and a common nomenclature; thus, it is of value for individual studies of specific strains, but lacks general applicability and descriptive significance.

Finally, whole genome sequencing has been introduced for characterisation of the species [43–45]. In the largest study, sequences from two human strains of *Aggregatibacter aphrophilus*, 30 human *A. actinomycetemcomitans* strains, and one St(b) strain isolated from a rhesus macaque Old World monkey were used for selection of 397 core genes which were concatenated and trimmed to produce a single alignment of 335,400 bp [45]. Five clades were recognised, designated clade b, clade c, clade e/f, clade a/d and clade e'. Although the analysis clearly separated six strains of serotype b from six strains of serotype c, a close similarity was observed between these two clades, as well as between clade a/d and e/f. In contrast, the clade designated e', encompassing four St(e) strains, was phylogenetically distinct. The open reading frames necessary for expression of St(e) antigen were highly conserved between clade e and clade e' strains, but e' strains were found to be missing the genomic island that carries genes encoding the cytolethal distending toxin. Moreover, the clade e' strains were more related to an Old World primate strain and carried the unusual 16S rRNA type V sequence (RNA types as defined by Kaplan et al. [36]). Although bacterial species are not defined by DNA sequence, average nucleotide identity (ANI) values locate whole genome sequences from this group/clade outside the species boundary [44]. Thus, strains belonging to the so-called clade e' (as well as the rhesus macaque

monkey strain) may possibly be transferred to new species, and *A. actinomycetemcomitans* may be restricted to strains with exclusive host specificity for humans.

A recent study compared whole genome sequences of strains from blood stream infections supplemented with oral reference strains [46]. Exclusion of so-called clade e' strains increased the number of core genes present in all strains from 1146 to 1357. Strains of *A. actinomycetemcomitans* are basically divided into three lineages (numbering of lineages differs from reference [44]). Lineage I encompasses the type strain and consists of two groups (St(b) and St(c), respectively). Lineage II consists of St(a) plus St(d)-(g). In contrast to lineage I, many strains of different serotypes from this lineage are competent for natural transformation, and the average size of genomes is approximately 10% larger than in lineage I. Lineage III also expresses St(a) membrane O polysaccharide, and the genome size is comparable to lineage II. However, all six investigated strains were incompetent for transformation due to inactivation of multiple competence genes [46].

In conclusion, St designations are valuable for initial typing of clinical strains, but insufficient for characterisation. Recognition of a general MLST scheme could be helpful, and whole genome sequences could be used for MLST and in silico serotyping, as well as further characterisation and epidemiologic investigations. The species description consisting of three separate lineages is figurative, but more knowledge on the new lineage III is needed to disclose the relevance for phenotype, host specificity and pathogenicity.

#### **3. General Characteristics**

*A. actinomycetemcomitans* is a fastidious, facultatively anaerobic, non-motile, small Gram-negative rod, 0.4–0.5 μm × 1.0–1.5 μm in size. Microscopically, the cells may appear as cocci in broth and in clinical samples. It grows poorly in ambient air, but well in 5% CO2 [47]. Colonies on chocolate agar are small, with a diameter of ≤0.5 mm after 24 h, but may exceed 1–2 mm after 48 h [48]. Primary colonies are rough-textured and adhere strongly to the surface of agar plates (Figure 2).

**Figure 2.** (**A**) Tenacious, rough-textured colonies of *A. actinomycetemcomitans* strain HK1651 on chocolate agar. Diameter of colonies did not reach 2 mm after 3 days incubation in 5% CO2. (**B**) Clinical isolate incubated on TSBV (tryptic soy-serum-bacitracin-vancomycin) agar for 4 days in 5% CO2. Expression of the distinctive "star-shaped" colony is facilitated by growth on TSBV agar. Pictures by courtesy of Jan Berg Gertsen and Rolf Claesson.

#### *3.1. Recovery, Phenotype, and Molecular Detection*

Relevant sites in the oral cavity for sampling of *A. actinomycetemcomitans* are periodontal pockets, the mucosa, and saliva. Sampling techniques include use of sterile paper points to be inserted in periodontal pockets, cotton swab for the mucosa, and chewing on a piece of paraffin for the collection of stimulated saliva. For transport of paper points, the VMGAIII-medium is recommended [49]; samples collected with cotton swap can be transported in a salt buffer or in TE-buffer [50]. Saliva can be transported in tubes without additives. For short-time transportation, saliva can be transported in tubes without additives. Otherwise, it can be frozen or stored at room temperature in a Saliva DNA Preservation Buffer. Proteomic analysis of gingival crevicular fluid and saliva is an expanding diagnostic field that may require improvements in standardised collection techniques and devices [51,52].

The selective medium TSBV (tryptic soy-serum-bacitracin-vancomycin) agar [53] is commonly used for culture. If *Enterobacterales* are present in significant amounts in the samples, a modified version of TSBV is recommended [54]. Detection of *A. actinomycetemcomitans* in clinical samples renders limited information on prediction, progression, and treatment planning of periodontal disease. For these purposes, the proportion of the bacterium at diseased sites is more relevant. This is in line with the ecological plaque hypothesis [55]. The detection level of *A. actinomycetemcomitans* is around 100 viable bacteria (colony-forming units) per mL. *Fusobacterium nucleatum* and other strict anaerobes will grow on TSBV in the absence of oxygen. The total concentration of viable bacteria is estimated by parallel cultivation on 5% blood agar plates, and the proportion of *A. actinomycetemcomitans* in the sample can be calculated.

*A. actinomycetemcomitans* is suspected when rough-textured, tenacious colonies appear on selective agar after one or two days (Figure 2). The species is distinguished from closely related bacteria by a positive catalase reaction and negative β-galactosidase reaction. Salient biochemical characters of *A. actinomycetemcomitans* have been published [56]. In addition, the bacterium is readily identified by MALDI-TOF mass spectrometry [57]; however, the current version of the Bruker database (v3.1) only includes mass spectra from a limited number of strains, and modest log-scores are not unusual when clinical strains are examined.

Leukotoxicity, i.e., the capacity of the bacterium to kill or inactivate immune cells, is properly determined in biological assays involving human cell lines [58], but a semi-quantitative method based on hemolysis on blood agar plates has been reported [42,59]. Quantification of the leukotoxin by enzyme-linked immunosorbent assay (ELISA) is also used; most studies have assessed the leukotoxin released from the surface of the bacterium, either during growth in broth [60], or by treatment of bacteria cultured in media that inhibit leukotoxin release with a hypertonic salt solution [42]. Leukotoxicity may also be estimated by determination of the total amount of leukotoxin produced by the strain. Bacterial suspensions are solubilized by SDS, and the leukotoxin is subsequently quantitated by Western blot–based methodology [60]. It is anticipated that the amount of leukotoxin released from the bacterial cell surface reflects the total amount of leukotoxin produced, but this relationship remains to be corroborated.

Polymerase chain reaction (PCR) is frequently used for identification and characterisation of *A. actinomycetemcomitans* in clinical samples. The leukotoxin promoter was an early focal point [59]. PCR amplification of the *ltx* promoter region and visualization on gel can discriminate the JP2 genotype from other strains of the species [61], but preferential amplification of smaller products characterised by a 530-bp deletion will overestimate the prevalence of the JP2 genotype. Recent improvements in PCR offer more precise quantification of periodontal pathogens in a complex plaque biofilm [62]. By real-time or quantitative PCR (qPCR), the instrument reports the cycling threshold (CT)-value, which can estimate the concentration of the target in the sample. qPCR has been used to separately quantitate JP2 and non-JP2 genotypes [63]. To approximate the total number of bacteria by qPCR, the 16S rRNA gene is generally targeted. The method can only provide a rough estimate, as primers and probes may preferentially bind to certain bacterial phyla, and because the number of copies of the gene varies substantially between different bacterial species [64].

Serotypes a through f can be identified by PCR as described [65,66]. A method for detecting St(g) has not yet been described.

#### *3.2. Aggregative Properties and the Leukotoxin Gene Operon*

*A. actinomycetemcomitans* expresses three potential toxins, fimbriae and a number of adhesins, plus a number of other gene products that may have significance for microbial interplay, persistence, transformation to planktonic state, and pathogenicity (Table 2).


**Table 2.** Genomic characteristics and putative virulence determinants of *A. actinomycetemcomitans.*

The distinct growth in broth as small granules adhering to the walls of the test tube was included in the initial description of [*Bacterium actinomycetem comitans*] [1]. Fresh isolates of *A. actinomycetemcomitans* invariably form colonies that are rough-textured with an opaque, star-shaped internal structure (Figure 2B). Subculture in broth yields clumps of autoaggregated cells that attach tightly to the glass, leaving a clear broth. *A. actinomycetemcomitans* possesses fimbriae, and these appendages can be irreversibly lost after prolonged subculture in the laboratory [77]. Antibodies to synthetic fimbrial peptide significantly reduce the binding of *A. actinomycetemcomitans* to saliva-coated hydroxyapatite beads, buccal epithelial cells and a fibroblast cell line, indicating a decisive role of these structures for adherence to multiple surfaces [78]. Moreover, autoaggregation (spontaneous formation of aggregates with rapid settling in un-agitated suspensions) was completely lost by a smooth-colony, isogenic variant [79]. Fimbriae are assembled as bundles of 5- to 7-nm-diameter pili composed of a 6.5 kDa protein designated Flp (fimbrial low-molecular-weight protein) [80,81]. The RcpA/B (rough colony proteins) were the first outer membrane proteins identified that were associated with rough colony variants [82], and they are encoded by a 14-gene locus designated the *tad* locus. The Tad (tight adherence) macromolecular transport system is a subtype of type II secretion. The *tad* locus is composed of nine *tad*, three *rcp* and two *flp* genes [67]. Mutation analysis of the naturally competent strain D7S revealed *flp-1*, *rcpA*, *rcpB*, *tadB*, *tadD*, *tadE* and *tadF* to be indispensable for expression of fimbriae, while mutants of five other genes expressed reduced levels of fimbriae, or fimbriae that had different gross appearance [83,84]. In a rat model, the *tad* locus was critical for colonizing the oral cavity and for pathogenesis, measured as maxillae bone loss and *A. actinomycetemcomitans*-specific IgG levels [85].

Many pathogenic bacteria can undergo phase variation, but smooth-to-rough conversion has not been substantiated for *A. actinomycetemcomitans*. Rather, the rough-to-smooth conversion is typically caused by mutations in the *flp* promoter region, and replacement with wild-type promoter can restore the rough phenotype [86]. However, one study indicated that smooth strains could re-express the fimbriae in low humidity environments [87].

In addition to expression of fimbriae decisive for autoaggregation and adherence to a wide range of solid surfaces (biofilm formation), *A. actinomycetemcomitans* encodes a spectrum of autotransporter adhesins, proteins that promote their own transport from the periplasm to the exterior surface, where they may be decisive for adhesion to specific human cellular epitopes. A homologue with similarity to the monomeric *H. influenzae* autotransporter, Hap, was designated Aae. Inactivation of *aae* in two naturally transformable strains caused a 70% reduction in adhesion to cultured epithelial cells [68]. Aae exhibits specificity for buccal epithelial cells from humans and Old World primates, and does not bind to human pharyngeal or cervical epithelial cells [88]. Two trimeric autotransporters with homology to the YadA adhesin/invasin family were identified. Omp100 has also been designated Api (*Aggregatibacter* putative invasin). *Escherichia coli* expressing ApiA bound to various types of human collagen plus fibronectin. Adhesion to human cells was specific to buccal epithelial cells from humans and Old-World primates, although the specificity was not as prominent as observed for AaE [70,89]. Screening of a large number of insertion transposon mutants identified the extracellular matrix adhesin A encoded by *emaA*, which is involved in collagen adhesion [90]. Collagen prevail in the supporting tissue of cardiac valves, and EmaA (extracellular matrix adhesin) may play a role in the pathogenesis of infective endocarditis [91].

Iron is an essential transition metal for nearly all forms of life. The host limits the availability of iron through a process termed nutritional immunity [92]. Haemolysis can be an initial step for release of iron from heme by Gram-negative bacteria. The RTX (repeats in toxin) family is an important group of toxins, whose name refers to glycine- and apartate-rich, calcium-binding repeats in the carboxy terminus of the toxin proteins [93]. RTX toxins are produced by many Gram-negative bacteria including members of family *Pasteurellaceae* – it has, indeed, been proposed that these toxins may originate in *Pasteurellaceae* [94].

In 1977, it was shown that polymorphonuclear leukocytes exposed to gingival bacterial plaque in vitro released lysosomal constituents [95], and the leukotoxin (Ltx) of *A. actinomycetemcomitans* was extracted and partially characterised in 1979 [6]. Ltx is a RTX cytolysin. By 1989, the gene was cloned and analysed [96,97], and the 530-bp deletion in the *ltx* promoter associated with enhanced expression of Ltx characterising the JP2 genotype was subsequently described [10]. The difference between minimally toxic and highly toxic strains were convincingly illustrated in clinical studies from Northern Africa [12]. The significance of the 530-bp deletion may reside in a potential transcriptional terminator spanning 100 bp [60]. The leukotoxin of *A. actinomycetemcomitans* is highly specific for human and primate white blood cells and is capable of neutralising local mucosal immune responses. However, purified leukotoxin can lyse sheep and human erythrocytes in vitro, and beta-haemolysis can be demonstrated on certain media [98].

In addition to the JP2 genotype characterised by the 530-bp promoter deletion, two other leukotoxin promoter variants have been reported. One genotype is characterised by a slightly enlarged (640-bp) deletion [99], while the other promoter variant carries an 886-bp insertion sequence [100]. Both these variants produce levels of leukotoxin similar to the JP2 genotype of *A. actinomycetemcomitans*.

#### *3.3. Geographic Dissemination of Specific Genotypes*

The JP2 clone of *A. actinomycetemcomitans* is suggested to have arisen 2400 years ago in the northern Mediterranean part of Africa [37]. The bacterial clone is endemically present in Moroccan and Ghanaian populations [12,101] and almost exclusively detected among individuals of African origin [37,102]. However, among 17 JP2 clone carriers, living in Sweden and identified during 2000–2014, ten were of Scandinavian heritage [31]. Among six of the identified JP2 clone carriers, three were of Swedish origin. Detection of the JP2 clone of *A. actinomycetemcomitans* has not been reported in Asian populations [30,100,103,104]. The occurrence of the JP2 clone of *A. actinomycetemcomitans* in Caucasians may be caused by horizontal transmission, and may weaken the theory of racial tropism of the clone [59]. More data and research are needed to explain the dissemination of the leukotoxic JP2 clone of *A. actinomycetemcomitans.*

Other genotypes characterised by an increased leukotoxic potential comprise a 640-bp deletion cultured from a host of Ethiopian origin [99], an 886-bp insertion sequence from a host of Japanese origin [100], and two strains of serotype c, originating from Thailand with a JP2-like deletion in the promoter region of *ltx*, and with virulence of similar magnitude to the JP2 genotype strains [105]. All these genotypes were collected from individuals with severe periodontitis.

#### **4. Prevalence and Clinical Significance**

Cultivable *A. actinomycetemcomitans* is present in at least 10% of periodontally healthy children with primary dentition [106]. An influential publication found carrier rates of 20% for normal juveniles, 36% for normal adults, 50% for adult periodontitis patients, and 90% for young periodontitis patients [107]. Early studies failed to culture the species from edentulous infants [108,109], but molecular studies using PCR on unstimulated saliva samples have challenged this association: 37 of 59 completely edentulous infants were positive for *A. actinomycetemcomitans*, reaching 100% at 12 months of age [110]. Vertical transmission is common. Two studies reported detection rates by culture of 30–60% in children of adult periodontitis patient, and the genotypes of the strains were always identical [111,112]. A smaller study from Brazil of women with severe chronic periodontitis did not corroborate this finding, as the

two culture-positive children carried genotypes that were different from those of their mothers [113]. Horizontal transmission of *A. actinomycetemcomitans* can occur, and transmission rates between 14% and 60% between spouses have been estimated [18,114]. However, members of most families with aggressive periodontitis also harbour additional clonal types of *A. actinomycetemcomitans* [115].

Once colonized, *A. actinomycetemcomitans* remains detectable in patients with periodontitis. Irrespective of periodontal treatment, colonisation by the same strain is remarkably stable within subjects for periods of 5 to 12 years, as revealed by restriction fragment length polymorphism [40], serotyping combined with arbitrarily primed PCR [116], or JP2 clone-specific PCR [117]. Genomic stability during persistent oral infections has been demonstrated by genome sequencing of strains cultured from the same individual 10 years later [118].

The natural habitat of *A. actinomycetemcomitans* is the oral cavity, but *A. actinomycetemcomitans* can be isolated from a variety of oral as well as non-oral infectious diseases, including arthritis, bacteraemia, endocarditis, osteomyelitis, skin infections, urinary tract infections and various types of abscesses [119]. Characterisation of 52 non-oral strains showed similarity to oral strains [120], and the portal of entry for systemic infections is usually the oral cavity [121].

#### *4.1. Infective Endocarditis*

The oral cavity is the only known habitat of *A. actinomycetemcomitans*, but only a few layers of crevicular epithelial cells separate the gingival location from the parenteral space of the host. Entry into the blood stream has not been quantitated, but incidental introductions may occur during tooth brushing, injuries, chewing of granular matters etc., and this may be accelerated by the presence of periodontitis. *A. actinomycetemcomitans* was originally co-isolated with *Actinomyces* from actinomycotic lesions [1], and the association with *Actinomyces* has been confirmed by case reports of infections in a variety of anatomical localizations. Among *Actinomyces* species, co-isolation of *A. actinomycetemcomitans* appears restricted to *Actinomyces israelii* [122,123].

Infective endocarditis is an infection of the endocardium, the lining of the interior surfaces of the chambers of the heart. It usually affects the heart valves (Figure 3A), where corrosion and incidental exposure of sub-endothelium tissue during the extensive motion of the valves may serve as a starting point for bacterial adhesion.

**Figure 3.** Imaging signs of infections and inflammation that may be associated with *A. actinomycetemcomitans*. (**A**) Transesophageal echocardiography of a heart with mitral valve infective endocarditis. The arrow marks a large vegetation on the posterior leaflet between left atrium (LA) and left ventricle (LV); usually, vegetations caused by *A. actinomycetemcomitans* are of smaller size. (**B**) 14-year old girl of African ethnicity. The radiograph shows an extensive and apparently rapid loss of the periodontal support of the lower incisor 31. Pictures by courtesy of close clinical collaborators of the authors.

*A. actinomycetemcomitans* is part of the *Haemophilus, Aggregatibacter, Cardiobacterium, Eikenella*, and *Kingella* (HACEK) group of fastidious Gram-negative bacteria that is responsible for 1.4–3% of cases infective endocarditis [124,125]. The group originally included *Haemophilus* species, *Actinobacillus*

*actinomycetemcomitans*, *Cardiobacterium hominis*, *Eikenella corrodens*, and *Kingella kingae* [7]. The HACEK acronym is still valid, but currently denotes non-*influenzae Haemophilus* sp., *Aggregatibacter* sp., *Cardiobacterium* sp., *E. corrodens*, and *Kingella* sp. [126]. *A. actinomycetemcomitans* is the HACEK organism most strongly associated with infective endocarditis [121,125], and bacteraemia with *A. actinomycetemcomitans* necessitates clarification of this putative focus of infection. In a retrospective study of 87 cases of HACEK bacteraemia from New Zealand, the association between HACEK bacteraemia and infective endocarditis varied among bacterial species ranging from 0% (*E. corrodens*) to 100% (*A. actinomycetemcomitans*) [127]. Specific features of infective endocarditis caused by *A. actinomycetemcomitans* have been reviewed [121].

#### *4.2. The Complex Interplay with Periodontitis*

Periodontitis is an inflammatory disease associated with loss of connective tissue and bone around teeth (Figure 3B). The bacterial tooth biofilm initiates the gingival inflammation, and further progression of the periodontal lesion depends on dysbiotic ecological changes within the gingival sulcus area. Unfavourable lifestyles and hygiene contribute to the development and progression of periodontitis, which has been designated as one of mankind's most common chronic inflammatory diseases [48].

The complexity of the periodontal microbiota and the variety of clinical symptoms delayed the identification of specific microbial aetiological agents. In 1996, *A. actinomycetemcomitans*, *Porphyromonas gingivalis*, and *Tannerella forsythia* were officially designated as aetiological agents of periodontitis [128]. *A. actinomycetemcomitans* was targeted based on prevalence studies in health and disease, serum antibody levels, and presence of virulence determinants (Tables 1 and 2). More recently, attention has been directed to the complex interplay between other cultivable and other non-cultivable bacteria in the oral microbiota, as well as to the interplay with the host [16,48,129,130]. Indeed, it has been suggested that *A. actinomycetemcomitans* conducts its business by concealing itself from the scrutiny of the host immune system, or even being a community activist that suppresses host responses to allow overgrowth of its collaborators [19].

The earlier classification of aggressive periodontitis was based mainly on the clinical presentation and the rapid loss of periodontal tissue [131]. A new classification scheme has been adopted, in which chronic and aggressive forms of the disease are now merged into a single category, which is characterised by a multi-dimensional staging and grading system [132,133]. Staging assesses severity and extent of disease at presentation, and attempts to include the complexity of disease management. The grading provides an evaluation of the risk of progression, and attempts to predict response to standard periodontal therapy [132].

#### **5. Therapy**

Treatment of periodontitis aims to stop the progression of the periodontal lesion and to maximise periodontal health [134]. Mechanical debridement of biofilm is considered the most effective therapy, but must be combined with a detailed oral hygiene. If periodontal lesions persist after 3–6 months, a second phase of therapy is planned. A favourable healing potential has been documented for lesions associated with the rapidly progressive, localised periodontitis that affects adolescents [135]. Systemic antibiotics should only be administered as adjunctive therapy in selected cases.

Access surgery with regenerative techniques have been used for periodontitis stages III–IV [132,134]. Notable risk factors are non-compliance, smoking, elevated gingival bleeding index, and inadequate plaque control [136].

Very different amoxicillin resistance rates have been reported, ranging from 0% in Switzerland [137], over 33% in Spain [138] to 84% in the United Kingdom [139]. The mechanisms of resistance were not reported. Production of β-lactamase is the most common cause of β-lactam resistance in Gram-negative bacteria, but these enzymes have not been detected in *A. actinomycetemcomitans*. The fastidious nature of the bacterium is a challenge for antimicrobial susceptibility testing, and methodology as well as interpretative criteria must be addressed when reports of resistance are evaluated. A recent

investigation using different methods could not confirm the emergence of resistance to β-lactams in *A. actinomycetemcomitans*; the study included strains that had previously been reported as resistant [140]. Thus, there is currently no convincing evidence for replacement of oral amoxicillin when antimicrobial agents are indicated for treatment of *A. actinomycetemcomitans*-associated periodontitis.

Gram-negative bacteria are generally more susceptible to the cephalosporin-class than the penicillin-class of β-lactams. For infective endocarditis, an intravenous course of at least four weeks with a third-generation cephalosporin, or a combination of ampicillin and an aminoglycoside, is recommended [121]. Recently, a well-designed randomised study reported favourable outcomes for oral antimicrobial follow up regimens given to patients with infective endocarditis deemed clinically stable and without complications [141]. *A. actinomycetemcomitans* could be a candidate microorganism for use of partial oral antimicrobial treatment of infective endocarditis, but the relative rare occurrence of HACEK bacteraemia poses difficulties for additional clinical studies.

#### **6. Conclusions**

*A. actinomycetemcomitans* is part of the human microbiota. It can be cultured from one-third of healthy adults, while PCR-based methods suggest a more ubiquitous presence. The bacterium's tenacious, aggregative character is instrumental for the remarkable genotype stability in colonised hosts, and for progression to persistent, distant infections after incidental entry into the parental space. *A. actinomycetemcomitans* are commonly detected if adolescents present with periodontitis. Chronically inflamed gingival crevices may spark the repeated, intermittent entry into the blood stream. Its participation in the disease process of periodontitis is beyond reasonable doubt, but its orchestration of severe periodontitis continues to be fascinating and disputed. Adhesion and leukotoxic features are well-described, but interplay with other members of the oral microbiota is more difficult to elucidate, as is the interchangeable position of eliciting antibody response and "staying under the radar". The recent division into three subspecies or lineages has not been investigated by clinical studies linking disease and lineage. Disease-specific treatment options are currently widely accepted.

**Author Contributions:** All authors made a substantial, direct, and intellectual contribution to the work. N.N.-L. compiled the contributions and made the first draft of the manuscript. All authors approved it for publication.

**Funding:** This research received no external funding.

**Acknowledgments:** Mogens Kilian is thanked for profound inspiration. Furthermore, we thank the members of the European Network for *Aggregatibacter actinomycetemcomitans* Research (ENAaR; https://projects.au.dk/ aggregatibacter/) for valuable discussions.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**

*aae*, *Aggregatibacter* autotransporter adhesin; Api, *Aggregatibacter* putative invasion; CT, cycling threshold; *emaA*, extracellular matrix adhesin A; HACEK, *Haemophilus*, *Aggregatibacter*, *Cardiobacterium*, *Eikenella*, and *Kingella*; JP2 clone, a specific juvenile periodontitis-related bacterial clone; Ltx, leukotoxin; MLST, multilocus sequence type; Omp, outer membrane protein; PCR, polymerase chain reaction; RTX, repeats in toxin; St, serotype; SNP, single nucleotide polymorphism; YadA, *Yersinia* adhesin A.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Review* **Virulence and Pathogenicity Properties of** *Aggregatibacter actinomycetemcomitans*

#### **Georgios N. Belibasakis 1, Terhi Maula 2, Kai Bao 1, Mark Lindholm 3, Nagihan Bostanci 1, Jan Oscarsson 3, Riikka Ihalin <sup>2</sup> and Anders Johansson 3,\***


Received: 27 September 2019; Accepted: 4 November 2019; Published: 6 November 2019

**Abstract:** *Aggregatibacter actinomycetemcomitans* is a periodontal pathogen colonizing the oral cavity of a large proportion of the human population. It is equipped with several potent virulence factors that can cause cell death and induce or evade inflammation. Because of the large genetic diversity within the species, both harmless and highly virulent genotypes of the bacterium have emerged. The oral condition and age, as well as the geographic origin of the individual, influence the risk to be colonized by a virulent genotype of the bacterium. In the present review, the virulence and pathogenicity properties of *A. actinomycetemcomitans* will be addressed.

**Keywords:** *Aggregatibacter actinomycetemcomitans*; leukotoxin; cytolethal distending toxin; lipopolysaccharides; cytokine binding factors; horizontal gene transfer; outer membrane vesicles; biofilm; proteomic

#### **1. Introduction**

*Aggregatibacter actinomycetemcomitans* is a facultative anaerobic Gram-negative bacterium that expresses several virulence factors, which activates a host response that could be associated to the pathogenesis of periodontitis [1]. This review will elaborate in more detail the virulence properties of *A. actinomycetemcomitans* that contribute to the increased pathogenicity of this species, particularly with regard to early and rapidly progressive forms of periodontal disease [2,3], such as localized aggressive periodontitis, where it is frequently a predominant find (Figure 1). The "crown jewel" of the virulence factors of *A. actinomycetemcomitans* has long been its leukotoxin [4,5]. However, a cytolethal distending toxin (CDT) has also been identified, making this species the only member of the oral microbiome to produce these two, or any of the two, protein exotoxins [6]. Its lipopolysaccharide is quite special in that the immunological responses elicited by the host can be used in classifying (serotyping) the virulence identity of each one of its strains [7]. More recently identified cytokine-binding molecules add to its potential virulence factors, suggesting additional pathogenicity mechanisms by which it can manipulate the host [8]. *A. actinomycetemcomitans* is also equipped with a wealth of outer membrane vesicles, like all Gram-negative species, which might confer special virulence properties to this species [9]. There is a great genetic diversity within this species, with base composition biases in the genomic islands suggesting their acquisitions via horizontal gene transfer [10]. Recent advances in biofilm modeling and proteomic technologies have helped define the localization of *A. actinomycetemcomitans* within biofilms, characterize the full range of its protein components, and define how these are regulated by other species, and vice versa, when growing within complex polymicrobial communities [11]. Increased knowledge about bacterial virulence markers in periodontal disease may be important tools in future strategies for personalized dentistry [12].

**Figure 1.** Subgingival biofilm samples from pathological periodontal pockets cultivated on blood agar plates for 48 h at 37 ◦C in an anaerobic atmosphere. (**A**) Sample obtained from a periodontal pocket of a middle-aged patient diagnosed with generalized chronic periodontitis. The macroscopical observation of the plated sample showed occurrence of a variety of colonies corresponding to different bacterial species. (**B**) Sample obtained from a periodontal pocket of a 25-year-old patient diagnosed with localized aggressive periodontitis. The macroscopical observation of the plated sample showed predominantly the occurrence a single colony type. (**C**) Microscopical (50× magnification) examination indicated that all colonies belonged to the *A. actinomycetemcomitans* species, which was confirmed in assays based on genetic characterization.

#### **2. Leukotoxin (LtxA)**

The leukotoxin (LtxA) of *A. actinomycetemcomitans* affects the different leukocyte populations with various death mechanisms [4]. It activates neutrophil degranulation causing a massive release of lysosomal enzymes, net-like structures, and matrix metallo proteinases (MMPs) and induces apoptosis in lymphocytes [13–15]. Interestingly, net-like structures can also be released from the LtxA-exposed neutrophils under anaerobic conditions and contain citrullinated proteins with sequence homology to proteins found in inflamed joints [16,17]. In the monocytes/macrophages, the toxin activates the inflammasome complex including the cysteine proteinase caspase-1, which induces an activation and secretion of the pro-inflammatory cytokines IL-1β and IL-18 [2,18,19]. These cellular and molecular mechanisms have been previously described in detail [20] (Figure 2). Taken together, several of the mechanisms by which LtxA affects leukocytes are also involved in the pathogenic mechanisms of many inflammatory disorders, such as periodontitis [21]. LtxA show a high target cell specificity expressed and affect only cells of hematopoetic origin from humans and some other primates [5]. This species-specificity of LtxA is reported to act through a unique target cell receptor and a specific region in the toxin that recognizes and interacts with this receptor [22,23]. A region of LtxA contains a series of 14 tandemly repeated amino acid sequences in the repeat region of the toxin and are shown to be responsible for the receptor binding to Lymphocyte function–associated antigen 1 (LFA-1) [4,23]. The LFA-1 molecule is a heterodimer consisting of the αL (CD11a) and β2 (CD18) subunits and is suggested to help the toxin to have a correct orientation on the target cell membrane [24–27].

**Figure 2.** Leukotoxin (LtxA) induces a rapid inflammatory cell death in human macrophages. Briefly, LtxA binds to the LFA-1 receptor (1) and induces an extracellular release of ATP (2), which act as a ligand for the P2X7-receptor and result in an efflux of potassium (3). These processes promote the formation of an inflammasome multimer (4) that activates the cysteine proteinase caspase-1, resulting in a rapid activation (5) and secretion of IL-1β (6). Courtesy of Haubek and Johansson [20].

#### *2.1. LtxA Production*

The LtxA operon belongs to the core genome of *A. actinomycetemcomitans* and is so far present in all examined strains [28]. The operon consists of four coding genes named *ltxC, ltxA, ltxB*, and *ltxD* and an upstream promoter region [29]. The gene *ltxA* encodes the LtxA protein, *ltxC* a protein required for the posttranslational acylation of LtxA, and *ltxB* and *ltxD* proteins needed for the transport of the LtxA to the bacterial outer membrane. For regulation of the LtxA expression, there is a promoter region located upstream of the *ltxC* gene, and genetic differences within this region result in different genotypes with various LtxA expression [10].

Zambon [30] reported that leukotoxicity of *A. actinomycetemcomitans* isolated from individuals with periodontitis was enhanced compared with isolates from individuals without periodontitis. Later, it was discovered that many of the isolates with enhanced leukotoxicity have been shown to have a different type of promoter in the leukotoxin operon [29]. A specific genotype (JP2) of *A. actinomycetemcomitans* with enhanced leukotoxicity has been shown to significantly correlate to disease onset in infected individuals [31,32]. The JP2 genotype was first identified by Brogan and co-workers [33] and was a serotype b isolate with a 530 base pair (bp) deletion in the *ltxCABD* promoter. Based on this finding, isolates with such a deletion in the promoter are named JP2 genotype, and those lacking this deletion are non-JP2 genotype [20,34]. The discovery of the JP2 genotype introduced a new terminology of high and low leukotoxic *A. actinomycetemcomitans* based only on the *ltx* promoter type [33,35]. The presence of the JP2 genotype is highly associated to adolescents of North and West African origin [31,36]. However, the JP2 genotype has recently been shown to also colonize individuals of other geographic origin, as confirmed by genotyping [37,38]. Alterations in the *ltx* promoter region are the most studied genotypes associated with enhanced LtxA expression. In addition to the JP2 genotype, an insertion of 886 bp, as well as a 640 bp deletion in the *ltx* promoter, has been discovered in *A. actinomycetemcomitans* isolates [39,40]. These three different genotypes are all associated with high virulence due to enhanced production of LtxA. Whether the deletions or insertions per se are causing the increased leukotoxin production is not entirely clear.

Enhanced leukotoxicity has been reported from serotype b isolates with an intact *ltx* promoter region, indicating a high production of the toxin [41]. These isolates represent a subgroup of serotype b with a high association with disease progression in the infected individual. Genetic characterization of this subgroup showed a genetic pattern with similarities to that of the JP2 genotype, and such strains are frequently carried by young individuals with periodontitis [2,38,42]. A specific genetic marker (*cagE*) correlates to *A. actinomycetemcomitans* with enhanced leukotoxicity, including the JP2 genotype and other virulent serotype b isolates [42,43]. Interestingly, the activity of LtxA has been reported to be involved in induction of systemic autoantibodies to citrulline, which are risk markers for rheumatoid arthritis [17,44]. In addition, LtxA has a crucial role in sepsis induced by bacteremia in an animal model [45].

#### *2.2. Leukotoxin Secretion*

The secretion of LtxA is mediated by a Type I secretion system in line with other proteins of the repeat in toxin (RTX family) expressed by several Gram-negative pathogens [46–48]. A unique property for LtxA among the RTX proteins is that the secreted toxin is reported to be associated to the bacterial outer membrane [47]. The export of the toxin to the bacterial outer membrane has been shown to require expression of three proteins—LtxB, LtxD, and TdeA—for export of the toxin to the bacterial outer membrane, and a fourth—LtxC—for the acylation [49]. In addition, the inner membrane protein MorC, which affects the outer membrane structure, has been reported to be necessary for efficient export of the toxin [50]. The localization of LtxA was found to be on the outside of the bacterial membrane and on vesicles associated to the outer membrane [51,52]. The responsible mechanisms for the association of LtxA to the membrane are still not fully clarified, and whether the secreted LtxA remains associated to the bacterial outer membrane or is released to the environment is a topic of controversy. A suggestion is that the hydrophobic domain of LtxA mediates the association to the bacterial membrane [27]. Ohta and co-workers [53,54] reported that LtxA could be released from the bacterial membrane by DNase or RNase treatment, which indicates involvement of electrostatic forces between the negatively charged nucleic acid and the positively charged LtxA. This phenomenon was later supported by the observation that LtxA on the bacterial membrane and on vesicles was released in hypertonic NaCl solutions [55]. LtxA was also released from the bacterial membrane in presence of serum proteins, which indicates involvement of competitive mechanisms [56]. Differences in the culture media have been shown to determine the distribution of the produced LtxA between the bacterial outer membrane and the culture supernatant [56–58]. Whether LtxA is associated with the bacterial membrane or released to the environment at the infected site in vivo is still not known. However, the serum mediated release of the LtxA [56], as well as the activation of a systemic immunogenic response [59,60], indicates a release of LtxA from bacteria growing in an in vivo oral biofilm.

The secreted LtxA has been shown to be efficiently inactivated by proteases and superoxide radicals [55,61,62]. In 1981, McArthur and co-workers reported that the activity of LtxA in interaction with polymorphonuclear leukocytes (PMNs) was enhanced in the presence of human serum [63]. This phenomenon could later be explained by the protective effect of the serum protease inhibitors on the proteolytic enzymes released from LtxA challenged PMNs that degrades the toxin [56,64].

#### *2.3. Quantification of LtxA Production*

The great genetic diversity of *A. actinomycetemcomitans* has resulted in various genotypes with substantially different virulence properties, i.e., LtxA production [10,28]. The expression of LtxA is also influenced by environmental factors, such as growth conditions and substrates [46]. Notably, an anaerobic culture condition enhances substantially the production of LtxA, which mirrors the condition in the periodontal pocket [65].

Due to the complex regulation of *ltxA* expression, the balance between membrane-associated and secreted toxin, as well as its sensitivity to proteolytic degradation, a gold standard for quantification of LtxA production, is still not available. The first attempt to quantify LtxA activity was to add bacteria to isolated leukocytes and determine cell death by trypan blue exclusion [66]. In a study by Zambon and co-workers [67], the leukocyte lysis method was used to discriminate between leukotoxic

and non-leukotoxic strains. This study showed that the prevalence of more leukotoxic variants of *A. actinomycetemcomitans* was higher in young individuals with periodontitis than in older individuals with the disease or in periodontally healthy individuals.

Except for examining the leukotoxicity of the isolates, methods targeting gene expression or immunodetection have been developed [58,68]. These methods have been employed on a limited number of *A. actinomycetemcomitans* isolates and support findings from previous leukotoxicity determinations, with enhanced expression in the JP2 genotype [69,70]. To obtain a gold standard for quantification of leukotoxicity of isolated *A. actinomycetemcomitans* is one of several challenges for future research.

#### **3. Cytolethal Distending Toxin (CDT)**

The CDT family comprises a number of bacterial protein exotoxins that is expressed by several Gram-negative species. Due to its deleterious effects on the host, as revealed in various experimental models, CDTs are likely to be involved in the etiopathogenesis of the associated human infections. They can be described as genotoxins, as their main action is to elicit DNA damage upon the intoxicated host cells [71]. The CDT holotoxin consists of subunits CdtA, CdtB, and CdtC. While CdtA and CdtC subunits mediate the internalization of the CdtB into the cell, the latter is translocated to the nucleus, causing its deleterious effects on the host cells. This subunit is functionally homologous to deoxyribonuclease I, hence it can cause DNA damage. It is postulated that CdtB internalization occurs via a mechanism involving the recognition of cell membrane cholesterol by both CdtC and CdtB [72,73].

*A. actinomycetemcomitans* expresses a CDT and is the only known oral species with this property. An estimated 66% to 86% of its strains express a CDT, and its presence has been associated with the occurrence of periodontal disease [74]. It is very plausible that its pathogenic effects are related to its capacity to cause DNA damage, cell cycle arrest, and eventually apoptosis to the intoxicated cells. This has been shown in structural cells like gingival fibroblasts and periodontal ligament cells [75,76], gingival epithelial cells [77–80], or gingival tissue explants [79], denoting that it can compromise the structural integrity and homeostatic capacity of the tissues. The capacity of CDT to affect the gingival epithelium has also been shown in human gingival explants [81], as well as in vivo upon inoculation of the toxin on rat gingival tissue [82]. Cells of the immune system are also highly susceptible to the cell cycle–arresting and apoptotic action of CDT, as has been demonstrated in human T cell [83], B-cells [84], and mononuclear cells [85]. CDT may also subvert the phagocytic capacity of macrophages and subvert their cytokine producing capacity [85]. The deleterious effects of CDT on cells of the immune system denote an impairment of the local immunity, which may compromise the capacity of the periodontium to recognize and eliminate the bacterial challenge, be it *A. actinomycetemcomitans* or other microbial constituents of the biofilm community.

Another potentially pathogenic mechanisms activated by CDT is the stimulation of pro-inflammatory and osteolytic cytokine production by the intoxicated host cells [86]. It has been shown that CDT can stimulate the production of pro-inflammatory cytokines by peripheral blood mononuclear cells, such as interferon (IFN)-γ, Interleukin (IL)-1β, IL-6, and IL-8, a virulence property potentially independent of the toxin's deoxyribonuclease I activity [85]. However, *A. actinomycetemcomitans* can stimulate the production of several pro-inflammatory cytokines and regulate inflammasome expression irrespective of its CDT, as demonstrated by the use of the *CDT*-deletion strains [87,88]. An important virulence property of CDT is revealed by its capacity to induce the major osteolytic factor receptor activator of nuclear factor kappa-B ligand (RANKL). This is a crucial molecule that stimulates the differentiation of osteoclasts and, consequently, bone resorption in periodontitis [89]. It has been shown that CDT induces RANKL expression and production in periodontal connective tissue cells, such as gingival fibroblasts and periodontal ligament cells [90,91], as well as T-cells [92]. This implies that the CDT may increase the levels RANKL in the periodontal tissues and therefore potentiate bone destruction by this action. The induction of inflammatory and bone-destructive molecular

cascades in the periodontium by CDT may well constitute an additional mechanism through which *A. actinomycetemcomitans* is involved in the etiopathogenesis of periodontitis. On the other side of the bone remodeling equilibrium, when CDT acts directly on pre-osteoclasts, it may also induce apoptosis and hinder their differentiation to osteoclastic cells, thereby contributing a dysbalanced bone remodeling equilibrium that leads to periodontal breakdown [93].

#### **4. Lipopolysaccharide and Cytokine-Binding Factors**

#### *4.1. The Virulence Properties of A. actinomycetemcomitans Lipopolysaccharide*

Like other Gram-negative species, *A. actinomycetemcomitans* surface is covered by lipopolysaccharide (LPS), a potent pro-inflammatory molecule. *A. actinomycetemcomitans* LPS comprises a group of structurally related molecules in which the O-specific polysaccharide chain (O-antigen), formed by oligosaccharide repeating units, is the most variable portion (Figure 3).

**Figure 3.** Schematic representation of a putative structure of the lipid A and the core oligosaccharides of *A. actinomycetemcomitans* lipopolysaccharide (LPS). The lipid A (black) of *A. actinomycetemcomitans* LPS is formed by four primary fatty acyl chains (myristic or 3-hydroxymyristic acid) linked by ester and amide linkages to a disaccharide of glucosamine. Two of the primary fatty acyl chains are esterified by secondary fatty acids. The acylation pattern of lipid A is asymmetric with four fatty acyl chains on the non-reducing glucosamine and two on the reducing glucosamine. The inner core (purple) is linked to lipid A by a ketosidic bond and is formed by 3-deoxy-D-*manno*-oct-2-ulosonic acid (Kdo) together with heptose residues such as *glycero*-*manno*-heptose. The outer core (green) usually consists of hexoses such as glucose and galactose. Functional groups such as hydroxyl and phosphate groups are common substituents in the lipid A and the core oligosaccharides. The O-specific polysaccharide chain (O-antigen) is the most variable portion in the LPS. The O-antigen consists of a large variety of sugar residues in many combinations and glycosidic linkages. For simplicity, substituents such as hydroxyl and phosphate groups (other than those in lipid A), conformational details of the monosaccharide residues, and the stereochemistry of the glycosidic bonds are not presented.

The serotyping of *A. actinomycetemcomitans* to seven different serotypes from a to f, as well as to non-serotypeable, is based on the structural differences in the O-antigen part of LPS. Commonly found monosaccharide residues in the *A. actinomycetemcomitans* O-antigen include the hexoses glucose, galactose, mannose, and talose; the hexosamines glucosamine and galactosamine; and the deoxyhexoses rhamnose and fucose. However, between different *A. actinomycetemcomitans* strains, there appears to

be significant variation in the final architecture of the oligosaccharide repeating units, which may be either di-, tri-, or tetrasaccharide moieties [94–97]. Thus, variation in the gene clusters involved in the synthesis of the highly variable polysaccharide moieties serves as the basis for PCR serotyping of *A. actinomycetemcomitans* strains [94,98].

The structures of the core oligosaccharide and the lipid A in *A. actinomycetemcomitans* LPS have a lower degree of structural freedom than the O-antigenic polysaccharides. The core oligosaccharide consists of 3-deoxy-D-*manno*-oct-2-ulosonic acid (Kdo), *glycero*-*manno*-heptose, glucose, and galactose, and appears conserved among different serotypes [96,97]. The lipid A consists of two phosphorylated glucosamine residues [96,97] and primarily myristic and 3-hydroxymyristic acid as the fatty acyl chains [97,99–102]. Amino compounds such as ethanolamine and glycine are found associated with *A. actinomycetemcomitans* lipid A and core oligosaccharides but in fewer numbers than the commonly found hydroxyl and phosphate group substituents [97]. *A. actinomycetemcomitans* growth is favoured in slightly alkaline environments [103,104] in which the phosphate groups and the Kdo occur in deprotonated form. The phosphate groups and the Kdo interact with positively charged ions and participate in hydrogen bonding, and thus contribute to the stabilization of the bacterial outer membrane.

The fatty acyl composition of lipid A generally varies between different Gram-negative species. For example, in lipid A of *Escherichia coli* and *Salmonella typhimurium* LPS the relative amount of myristic acid is lower than in *A. actinomycetemcomitans* LPS, while lauric acid and palmitic acid are more frequently found [100]. The lipid A composition of closely related species may not always be specific enough to allow taxonomic differentiation, as demonstrated by the similar composition of *A. actinomycetemcomitans* and *Aggregatibacter aphrophilus* lipid A [100]. Closely related species may, however, be differentiated by the composition of their (core) oligosaccharides. A distinct feature of the oligosaccharides of *A. actinomycetemcomitans* is the presence of both Dand L-*glycero*-D-*manno*-heptose, whereas *A. aphrophilus* LPS only contains the L-enantiomer of this aldoheptose [105,106]. By contrast, galactose appeared twice as abundant in LPS from *A. aphrophilus* as in LPS from *A. actinomycetemcomitans* [99,105,106].

Although there are several studies that indicate a distinct effect of *A. actinomycetemcomitans* LPS on rodent cells [107–111], we will focus here on describing how *A. actinomycetemcomitans* LPS stimulates human cells. This outlining is done due to the known differences between the murine/rat and human immune systems [112]. The various virulence-related effects of *A. actinomycetemcomitans* LPS are summarized in Table 1. The first cells encountered by detached *A. actinomycetemcomitans* LPS in junctional epithelium (JE) of a tooth are the epithelial cells. These human epithelial cells have been shown to respond to *A. actinomycetemcomitans* LPS by expressing IL-15 which results in enhanced IFN-γ production and proliferation of human T cells [113]. Moreover, *A. actinomycetemcomitans* LPS causes widening of the intercellular spaces in primary tissue cultures mimicking the JE, a phenomenon not observed with *Porphyromonas gingivalis* LPS [113]. Various direct effects of *A. actinomycetemcomitans* LPS on the other main human gingival cell type, fibroblasts, have also been reported. Collagen is ingested and digested by gingival fibroblasts in balanced conditions of healthy gingiva. *A. actinomycetemcomitans* LPS is able to enhance the phagocytosis of collagen by fibroblasts which may result in imbalance in regeneration of the gingival tissue [114]. In addition to changes in cellular functions, *A. actinomycetemcomitans* LPS stimulate the production of IL-6 and IL-8 [114], tissue plasminogen activator (t-PA), and plasminogen activator inhibitor 2 (PAI-2) by human gingival fibroblasts [115]. The plasminogen/plasmin system is involved in the complex process of extracellular matrix degradation and renewal in the gingival tissue, a step that most likely precedes the collagen phagocytosis by the gingival fibroblasts.

Gingival tissue contains various resident immune cells, of which macrophages play a central role in periodontitis. *A. actinomycetemcomitans* LPS has shown to stimulate the expression of microRNA miR-146a [116], which downregulates the expression of TNF receptor–associated factor 6 and IL-1 receptor–associated kinase 1, which serves as a negative feedback loop in cytokine signaling [117]. On the contrary, high doses of *A. actinomycetemcomitans* LPS downregulated the expression of miR-32 and miR-29b, which target the pro-apoptotic factor Bim of acute myeloid leukemia cells [118] and IL-6 receptor α [117], respectively. Another human cell type that originates from mesenchymal stem cells is osteoblast which plays a significant role in bone formation. *A. actinomycetemcomitans* LPS has capacity to increase the inducible nitric oxide synthase (iNOS) activity and induce the nitric oxide (NO) production by human osteoblast-like cell line [119]. If osteoblasts produce NO rapidly, when responding to bacterial infection, it may lead to bone resorption [120].


**Table 1.** Virulence-related properties of *A. actinomycetemcomitans* LPS.

Another type of resident immune cells in periodontium is dendritic cells (DCs), which are involved in antigen presenting to T cells. Conventional DCs originate from monocytes, like macrophages, and are called mDCs. Monocytes originating from localized aggressive periodontitis (LagP) patients, recently renamed molar-incisor pattern periodontitis with rapid progression [131], spontaneously give rise to mDCs [132]. *A. actinomycetemcomitans* LPS enhances the IL-12 production by mDCs leading to stimulation of IFN-γ expression of natural killer cells and undetectable levels of IL-4, which together may cause the polarization of naïve T cells toward the Th1 type response [123]. There are differences of DCs stimulation potential of *A. actinomycetemcomitans* LPS originating from different serotypes, serotype b LPS inducing the strongest production of IL-12, IFN-γ, TNF-α, IL-1β, IL-6, and IL-23 [124]. The differences in the response of DCs to serotype b *A. actinomycetemcomitans* LPS compared to the response to other serotype LPS most likely causes Th1/Th17 type of T cell response in serotype b related infection [133].

PMNs are innate immune cells which may play both defensive and destructive role in periodontitis [134]. Neutrophils produce reactive oxygen species (ROS) when responding to whole bacteria or their components. *A. actinomycetemcomitans* LPS has been shown to be more potent inducer of neutrophil ROS production than, for example, *P. gingivalis* or *Prevotella intermedia* LPS [125]. Moreover, *A. actinomycetemcomitans* LPS stimulate the production of inflammatory cytokines IL-1β and TNF-α by PMN more efficiently than *P. gingivalis* LPS [126], and in human whole blood, *A. actinomycetemcomitans* LPS causes higher production of the above mentioned cytokines as well as IL-6 than *E. coli* LPS [127]. *A. actinomycetemcomitans* LPS can shift the movement of monocytes and granulocytes from rolling to the passaging through the vascular wall by downregulating the surface expression of L-selectin and increasing the expression of β2 integrins, respectively [128].

Despite the vast literature concerning the inflammatory related functions of *A. actinomycetemcomitans* LPS, this outer membrane linked polysaccharide has also functions related to bacterial physiology. The serotype specific O-antigen part of *A. actinomycetemcomitans* LPS takes part in the secretion of leukotoxin, since a serotype b mutant with inactive *rmlC* and altered O-antigen sugar composition, contained more cytoplasmic and membrane-bound leukotoxin, and secreted less leukotoxin than the wild-type serotype b strain [135]. Moreover, the O-antigen part of *A. actinomycetemcomitans* LPS may mediate direct adhesion to abiotic surfaces [7]. Besides abiotic surfaces, *A. actinomycetemcomitans* LPS interacts with host molecules, such as human hemoglobin [129] and IL-8 [136], which may facilitate the iron acquisition and disturbance of host defense, respectively.

#### *4.2. Sensing of Host Signal Molecules*

*A. actinomycetemcomitans* is one of the human pathogens that is able to bind host cytokines, such as IL-1β, IL-8, and IL-6 [137,138], and internalize them [138,139], which leads to changes in the properties of biofilm, decreasing the metabolic activity [139], and changing the composition of the extracellular matrix [136,138]. Several potential bacterial proteins that may interact with human cytokines have been identified in *A. actinomyctemcomitans*, including intracellular ATP synthase subunit β [139], histone like DNA binding protein HU [139], and outer membrane proteins bacterial interleukin receptor I (BilRI) [138,140] and secretin channel HofQ [136]. The majority of these proteins have other functions in the bacterial cell related to metabolism, gene regulation, and uptake of nutrients and DNA for horizontal gene transfer, a character of so called "moonlighting" bacterial proteins [1]. Interactions with the above mentioned proteins may result in the observed uptake of cytokines, decreased metabolic activity and potentially, although not yet proved, also changes in the gene expression profile of *A. actinomycetemcomitans*. Both Gram-positive *Staphylococcus aureus* [141] and Gram-negatives *Pseudomonas aeruginosa* [142] and *Neisseria meningitidis* [143] are able to respond to cytokines, such as IL-1β, IFN-γ, IL-8, and TNF-α, by changing their virulence gene expression pattern.

Human cytokines are not the only host signalling molecules that *A. actinomyctemcomitans* is able to bind and sense. *A. actinomyctemcomitans* harbours the two-component system QseCB, which has originally been detected from enterohemorrhagic *Escherichia coli* EHEC [144]. In EHEC, QseCB senses either endogenous autoinducer-3 or host catecholamine hormones epinephrine/norepinephrine and induces the expression of Locus of Enterocyte Effacement (LEE) vital for the virulence of EHEC [145]. However, *A. actinomycetemcomitans* needs also iron, in addition to catecholamine hormone, to activate the QseC sensor kinase [146]. The QseCB signalling changes the gene expression pattern of *A. actinomycetemcomitans*, and especially the genes needed for anaerobic metabolisms are upregulated [146]. Moreover, QseC plays role in the biofilm formation in a flow cell in vitro model, since Δ*qseC* mutant strain forms significantly less biofilm in flow cells than the corresponding *A. actinomyctemcomitans* wild type and the complemented strains [147]. It is not known whether this impaired capacity to form biofilms also affects the virulence potential of *A. actinomyctemcomitans* in vivo. Yet, Δ*qseC* mutant strain causes less bone loss in murine model of periodontitis than the wild-type strain, suggesting a strong link between *A. actinomycetemcomitans* QseC and virulence [147].

#### **5. Outer Membrane Vesicles**

During the latest decades it has become apparent that membrane vesicles (MVs) are naturally shed during growth by many bacteria, archaea, and eukaryotes. Membrane vesicles, also known as "Type Zero secretion", or referred to as outer membrane vesicles (OMVs) in Gram-negative organisms, serve as a general but relevant mechanism of antigen delivery, and are discharged by both commensal and pathogenic organisms in vivo and when infecting host cells in vitro [148,149]. Biologically active virulence factors such as CDT and OmpA can be transported into HeLa cells and human gingival fibroblasts via *A. actinomycetemcomitans* OMVs [150]. OMVs are also involved in the export of leukotoxin, peptidoglycan-associated lipoprotein (Pal), and the chaperonin GroEL to host cells [52,151–153]. Characterization of the OMV proteome of one *A. actinomycetemcomitans* clinical isolate using Matrix-Assisted Laser Desorption/Ionization Time of Flight-Mass Spectrometry (MALDI-TOF MS) revealed an array of additional tentative virulence-related proteins, including BilRI, Omp100, TdeA, and a ferritin-like protein [154,155]. This is in line with an OMV proteome exhibiting multiple offensive and defensive functions, such as drug targeting, iron acquisition, and immune

evasion. A role of *A. actinomycetemcomitans* OMVs in serum resistance can be hypothesized based on observations that the vesicles could bind to the complement system regulator C4-binding protein in an OmpA-dependent manner [156]. Moreover, it has been shown that *A. actinomycetemcomitans* OMVs can transport small molecules such as those contributing to bone resorption, including LPS [157] and lipid A-associated proteins [158]. *A. actinomycetemcomitans* OMVs carry NOD1- and NOD2-active peptidoglycan, and upon vesicle internalization into non-phagocytic human cells such as gingival fibroblasts, the OMVs can act as an innate immunity trigger [9]. *A. actinomycetemcomitans* OMVs contain in addition nucleic acids [53], and recent evidence supports the concept that the OMVs can carry microRNA-sized small RNAs (msRNAs). These small RNAs might represent novel bacterial signaling molecules, which by means of OMVs can be transported into host cells to modulate the immune response [159]. *A. actinomycetemcomitans* OMVs appear to mainly internalize into host cells via clathrin-dependent endocytosis [9,160] but can also fuse with host cell membranes in a cholesterol-dependent manner [150]. Toxins associated with OMVs can function as adhesins in receptor-mediated endocytosis of vesicles [161], but such a role of CDT or leukotoxin appears less likely, as neither of the toxins are required for the uptake of OMVs into host cells [150,151]. Additionally, despite the evident localization of leukotoxin on the surface of *A. actinomycetemcomitans* OMVs, there is no requirement of the toxin receptor LFA1 for vesicle-mediated trafficking of LtxA into host cells [162].

#### **6. Biofilm Interactions and Proteomic Regulations**

#### *6.1. Localization in Pocket and Tissue*

As a microaerophic organism, *A. actinomycetemcomitans* is able to grow both supragingivally and subgingivally, corresponding to aerobic and anaerobic growth, respectively. As it can be detected at both locations, it has been postulated that the environment of supragingival plaque harbouring *A. actinomycetemcomitans* can act as a reservoir for the spread or reinfection of this bacterium of subgingivally [163]. When growing subgingivally, *A. actinomycetemcomitans* is reported to be detected in the loosely attached unattached plaque area in the middle pocket zone [164]. Earlier histopathological studies determined the prevalence and gingival localization of *A. actinomycetemcomitans* in periodontal lesions of juvenile periodontitis patients (earlier classification), using culture techniques on disrupted tissue or immunofluorescence microscopy on intact tissue. The former demonstrated the presence of *A. actinomycetemcomitans* in almost all diseased tissues examined, with evidence of microcolonies and single bacterial cells within the gingival connective tissue, as well as inside the phagocytic cells within the tissue [165]. The latter demonstrated an increase in *A. actinomycetemcomitans* colony-forming units, which correlated with its presence in the tissue and in the periodontal pocket [165]. In situ hybridization studies have detected *A. actinomycetemcomitans* in epithelial cells from the lining gingival crevice [166] or in close relationship with the polymorphonuclear infiltrate of the pocket [167]. By quantitative real-time PCR of gingival tissue lysates, it was shown that *A. actinomycetemcomitans* is present at a higher prevalence in tissues of younger patients with aggressive periodontitis as compared to chronic periodontitis or health [168].

#### *6.2. Localization in Biofilms and Proteomic Interactions with Other Species*

Studies on the localization of *A. actimomycetemcomitans* in biofilms largely comes from in vitro models. In supragingival and subgingival biofilms, *A. actinomycetemcomitans* does not appear to affect the number of the other species present and appears to form small, dense, and secluded cell clusters of its own species throughout the biofilm mass [169,170]. Yet, the lack of numeric changes on other species of the biofilm does exclude the possibility that *A. actinomycetemcomitans* exerts regulatory proteomic and metabolic changes in the biofilm, as discussed further.

The development of mass spectrometry (MS) technology made it possible to identify multiple proteins in a single run. Therefore, it is a useful tool to study how *A. actinomycetemcomitans* orchestrates proteomic changes in the context of biofilms. Using 2D gels, Llama-Palacios et al. discovered 87 protein spots differently expressed (1.5-fold, *p* < 0.05) between planktonic and mono-species biofilm cultures of *A. actinomycetemcomitans* [171]. Then, using MALDI-TOF MS, 13 upregulated proteins (from 24 proteins spots) and 37 downregulated proteins (from 50 spots) were identified. The upregulated proteins were mainly outer membrane proteins involved the immunologic process, whereas downregulated proteins were related to the metabolism, biosynthesis, and transport. This is consistent with the finding that mature biofilms display increased virulence [172] despite lower metabolic activity [173] as compared to planktonic culture.

Because oral biofilms occur as complex polymicrobial communities, the study of *A. actinomycetemcomitans* within multispecies biofilms is highly relevant. When integrated within a 10-species subgingival biofilm model, *A. actinomycetemcomitans* did not significantly impact the abundance of the other bacterial species, nor did it affect the biofilm structure, which is consistent with findings in a supragingival biofilm model. Using liquid chromatography–tandem mass spectrometry (LC-MS/MS), 3225 and 3352 microbial proteins were identified in multi-species biofilms in the absence or presence of *A. actinomycetemcomitans*, respectively [170]. Further investigations with label-free quantification (LFQ) method displayed 728 bacterial proteins and found 483 of them to be differentially regulated (2-fold, *p* < 0.05) among these two kinds of biofilms. Interestingly, the regulation trend for individual species was highly individual. For *Prevotella intermedia*, all quantified proteins were upregulated in the presence of *A. actinomycetemcomitans*, whereas the majority of the proteins were downregulated for *Campylobacter rectus*, *Streptococcus anginosus*, and *P. gingivalis*. These findings are well in line with the competing growth between *P. gingivalis* and *A. actinomycetemcomitans*, shown in a dual-species biofilm [174]. Furthermore, based on the GO analysis, *A. actinomycetemcomitans* appears to downregulate proteins with ferric iron binding functions and alter the metabolic rate for the overall biofilm.

To understand the effects of this *A. actinomycetemcomitans*-containing biofilms on host tissues, the biofilm model was introduced into a bioreactor-supervised 3D cell culture system, which consisted of epithelial and connective tissue structures to mimic the periodontium, as well as monocytes to stimulate the immune response [173]. As a result, *S. anginosus*, *A. oris*, *V. dispar*, *C. rectus*, and *P. gingivalis* were suppressed when present with the host tissue, while the tissue itself exhibited morphological, immunological, and proteomic changes. The numbers of *A. actinomycetemcomitans* in the biofilm were not reduced, but more of its proteins were expressed when co-cultured with the 3D (21 proteins) than the biofilm stand-alone (15 proteins) [173]. Yet, *A. actinomycetemcomitans* proteins only comprised a small fraction of all identified proteins in biofilm lysates (21 of 3363) and supernatants (one of 896).

Attachment to biotic surfaces or other biofilms enhances *A. actinomyctemcomitans* virulence properties. We have found that deletion of the gene *hns*, encoding a histone-like family of DNA-binding, nucleoid-structuring protein (H-NS), a global gene silencer [179], leads to a less piliated phenotype of *A. actinomycetemcomitans* and decreases its biofilm formation ability when cultured as mono-species biofilms [11]. LFQ showed that the majority (29) of the differentially expressed proteins (2-fold, *p* < 0.05) were upregulated in the *hns* mutant biofilm, supporting the role of H-NS as gene suppressor in *A. actinomyctemcomitans*. Notably, the affected proteins included virulence factors such as leukotoxin A and D (LtxA and LtxD). A similar repression activity of H-NS for virulence factors from other microbes was observed on hemolysin operons *hlyCABD* [180] and *ehxCABD* [181] in *E. coli* and *rtxACBD* in *Vibrio cholerae* [182]. Within multi-species biofilms, using the *hns* mutant there was a significant reduction of *A. actinomycetemcomitans* numbers, without affecting the number of other species. On the protein level, LFQ data suggested that many *Fusobacterium nucleatum* and *Streptococcus oralis* proteins were downregulated in biofilm harbouring the wild-type *A. actinomyctemcomitans* strain, as compared to its *hns* mutant counterpart, and these proteomic regulations may occur long before the corresponding bacterial growth is affected. Most of the regulated proteins were associated with peptide metabolic process and regulation of translation, supporting a protein-orchestrating role of H-NS in *A. actinomycetemcomitans*.

A literature summary of the proteomic findings on *A. actinomycetemcomitans* in single-species biofilms and multi-species biofilms is provided in Tables 2 and 3, respectively.


**Table 2.** Proteomic studies on single species *A. actinomycetemcomitans.*

Ion trap: IT, False positive: FP, False-discovery rate: FDR, Matrix assisted laser desorption ionization: MALDI, Time of flight mass spectrometry: TOF MS, Mass spectrometer: MS. \* Maximum identified/quantified proteins were report base on the following rules: (1) Only maximum identified protein number was reported if the experiment was done under different conditions. (2) Total protein numbers were reported if the experiment has replicates. (3) The number of identified and quantified proteins were reported if only regulated protein were reported.



*A.a*: *A. actinomycetemcomitans*, Label free quantification: LFQ, Matrix-assisted laser desorption ionization- time of flight mass spectrometry: MALDI-TOF MS, Two-Dimensional Differential Gel Electrophoresis: 2DE, \* Maxium identified/quantified proteins were report base on the following rules: (1) Only maximum identified protein number was reported if the experiment was done under different conditions. (2) Total protein numbers were reported if the experiment has replicates. (3) The number of identified and quantified proteins were reported if only regulated protein were reported. \*\* 10-species biofilm model (consisting of *Campylobacter rectus*, *Fusobacterium nucleatum*, *Porphyromonas gingivalis*, *Prevotella intermedia*, *Tannerella forsythia*, *Treponema denticola*, *Veillonella dispar*, *Actinomyces oris*, *Streptococcus anginosous*, *Streptococcus oralis*).

#### **7. Horizontal Gene Transfer**

*A. actinomycetemcomitans* strains can be divided into competent and non-competent ones, which refer to their ability to acquire new genes from up-taken extracellular DNA (eDNA) using homologous recombination. Competent strains may have some advantage of being able to uptake eDNA, which could be related to additional means to repair DNA damage, obtain nucleotides and possibly also novel genes [184]. Approximately 30% of clinical *A. actinomycetemcomitans* strains are naturally competent [185], leading to greater genetic diversity, whereas noncompetent strains are genetically stable and need to use other mechanisms, such as conjugative plasmids, for horizontal gene transfer (HGT) [186]. It is thought that the ancestral Pasteurellacean was naturally competent and that

noncompetent lineages lost their ability to take up DNA due to various mutations in the competence gene locus [187]. This may explain the findings that noncompetent strains are more common among some serotype groups, such as serotype b and c [185]. The competence gene locus consists of regulatory *sxy*, DNA uptake–related *comABCDE*, *pilABCD*, *comEAFE1*, *rec2*, and transformation-related *comM* and *urpA* [186,188,189]. Noncompetent strains may contain nonsense mutations, or insertions in one or several of these genes, which makes the strain either unable to uptake eDNA and/or to incorporate it into the genome [186]. In addition to the above described gene locus, the CRISPR-*cas* system is also closely involved in natural competence, since the loss of CRISPR-*cas* system is connected to the loss of competence [186]. Thus, non-competent *A. actinomycetemcomitans* strains are more prone to HGT caused by plasmids and phages. However, in the non-competent strains, the maintained CRISPRs contains few spacers that most likely are used for chromosomal gene regulation, since they possess specificity for endogenous genes [186].

The expression on genes of competence locus are regulated by *sxy* (*tfoX*), of which levels in the cell is affected by various environmental factors, such as extracellular calcium ions [190] and the biofilm mode of growth [190]. Moreover, in addition to competence locus and CRISPR-*cas* system, the development of competence and expression levels of *sxy* are stimulated by cyclic AMP [191] and affected by the *pga* gene cluster [190], respectively. The genes in *pga* cluster are needed in the production of most abundant extracellular polysaccharide, N-acetyl-D-glucosamine, in *A. actinomycetemcomitans* biofilms.

Naturally competent *A. actinomycetemcomitans* strains recognize related eDNA, which is suitable to be taken up, using uptake signal sequence (USS). The *Pasteurellaceae* family has two types of USS, *Hin* and *ApI*, which contain distinctive consensus sequences of nine base pairs. *A. actinomycetemcomitans* has *Hin* type USS, consisting of *A. actinomycetemcomitans* GTGCGGT consensus sequence followed by AT-rich repeats [187,191]. This sequence is most likely recognized by the outer membrane proteins involved in the eDNA uptake. However, the recognizing protein has not yet been identified.

#### **8. Conclusions**

*Aggregatibacter actinomycetemcomitans* is a facultative anaerobic Gram-negative bacterium with the capacity to employ many virulence mechanisms closely associated to the pathogenesis of periodontitis. The variety of virulence properties of this bacterium contribute to the pathogenicity of this species, particularly with regard to early and rapidly progressive forms of periodontal disease. *A. actinomycetemcomitans* can be found in a large proportion of the human population, and due to the large genetic diversity of this species, several different genotypes or phenotypes with various virulence properties have emerged. Without doubt, individuals carrying *A. actinomycetemcomitans* genotypes with a proven enhanced leukotoxin production have a significantly increased risk to develop disease. More recent studies have identified suitable genes of this species, which can potentially be traced as markers for epidemiological population monitoring and utilization in individual risk assessment programs. Such bacterial virulence markers in periodontal disease may prove to be important tools in future strategies for personalized dentistry.

**Author Contributions:** Conceptualization, A.J., G.N.B., N.B., J.O., K.B., M.L., R.I., and T.M; Leukotoxin, A.J; Cytolethal distending toxin, G.N.B.; Outer membrane vesicles, M.L. and J.O.; Lipopolysaccharides, cytokine binding and horizontal gene transfer, R.I. and T.M.; Proteomic N.B. and K.B.; Data Curation, A.J., R.I., and J.O.; Writing—Original Draft Preparation, A.J. and G.N.B.; Writing—Review & Editing, A.J., G.N.B., N.B., J.O., K.B., M.L., R.I., and T.M.

**Funding:** This work was supported by institutional funds and by funds from the Swedish Research Council (N.B.; 2017-01198), TUA grants from the County Council of Västerbotten, Sweden (A.J.; 7003193), and the Academy of Finland (R.I.; 265609, 303781).

**Acknowledgments:** We thank the European Network for *A. actinomycetemcomitans* Research (https://projects.au. dk/aggregatibacter/) for encouraging us to prepare this review. In addition, we thank Rolf Claesson for the nice photos for Figure 1 and Sotirios Kalfas for excellent artwork in Figure 2.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Case Report* **JP2 Genotype of** *Aggregatibacter actinomycetemcomitans* **in Caucasian Patients: A Presentation of Two Cases**

#### **Alexandra Stähli \*, Anton Sculean and Sigrun Eick**

Department of Periodontology, School of Dental Medicine, University of Bern, Freiburgstrasse 7, 3010 Bern, Switzerland; anton.sculean@zmk.unibe.ch (A.S.); sigrun.eick@zmk.unibe.ch (S.E.)

**\*** Correspondence: alexandra.staehli@zmk.unibe.ch

Received: 31 January 2020; Accepted: 25 February 2020; Published: 1 March 2020

**Abstract:** *Aggregatibacter actinomycetemcomitans* is a key pathogen that has been associated with periodontal disease. Its most important virulence factor is a leukotoxin capable of inactivating immune cells. The JP2 genotype of *Aggregatibacter actinomycetemcomitans* shows enhanced leukotoxic activity and is mostly present in individuals of North and West African origin with severe periodontitis. In this paper, two cases of Caucasians diagnosed with the JP2 genotype are presented. A 50-year-old female patient had three approximal sites with ≥ 6 mm clinical attachment loss (CAL) and eight sites with probing depth (PD) ≥ 5 mm. Microbiological diagnostics revealed *A. actinomycetemcomitans* JP2 genotype, but not *Porphyromonas gingivalis*. This JP2 genotype was highly leukotoxic to monocytic cells. The second case was a 55-year-old female patient with CAL of > 5 mm at all molars and PD of up to 12 mm. *A. actinomycetemcomitans* JP2 was identified, but not *P. gingivalis*. Her husband originated from North-Africa. In him, no *A. actinomycetemcomitans* was detected, but their 17-year-old daughter was diagnosed with periodontitis and was found to be positive for the JP2 genotype. Both patients were successfully treated with adjunctive antibiotics and the JP2 genotype was eliminated. In summary, here, the microbiological diagnosis was key for the treatment with adjunctive antibiotics.

**Keywords:** JP2 clone of *Aggregatibacter actinomycetemcomitans*; periodontitis; JP2 in Caucasian; microbiological diagnosis; adjunctive antibiotics

#### **1. Introduction**

Bacterial biofilm causes destruction of the periodontium in two ways: through direct action of bacteria and their products on the host-tissue and by activating the immune host response [1]. *Aggregatibacter actinomycetemcomitans* is one of the key pathogens in the course of periodontal disease. *A. actinomycetemcomitans* has been strongly associated with localized aggressive periodontitis [2], however, its mere presence could not be used to distinguish between chronic (CP) and aggressive forms of periodontitis (AP) [3]. In the Department of Periodontology, School of Dental Medicine, University of Bern, patients diagnosed with AP generally received antibiotics during nonsurgical periodontal therapy (i.e., hygienic phase). Retrospective analysis of our patients revealed that the prevalence of *A. actinomycetemcomitans* was higher in patients diagnosed with AP than in those diagnosed with CP [4]. Following periodontal therapy, especially surgical treatment, *A. actinomycetemcomitans* was less frequently detected in patients with AP than in those with CP [4]. *A. actinomycetemcomitans* possesses several virulence factors, that is, lipopolysaccharides that induce pro-inflammatory cytokines, a cytolethal distending toxin causing cell cycle arrest in T-cells, macrophages and epithelial cells, and a leukotoxin [5]. The leukotoxin produced by the bacterium is capable of killing or inactivating immune cells and of inducing the release of interleukin (IL)-1β [6].

Here, we focus on a subtype of *A. actinomycetemcomitans*, the highly leukotoxic JP2 genotype, which was first isolated from a child of African American origin with prepubertal periodontitis [7]. Later, it was found as a common isolate in individuals of North and West African descent with aggressive forms of periodontitis [8–12]. With respect to the JP2 genotype, a 530 base pair deletion in the promoter region of the leukotoxin gene is responsible for a 10- to 20-fold increased production of leukotoxin [13,14]. The JP2 clone is a subpopulation of the serotype b strains [15].

To date, seven serotypes of *A. actinomycetemcomitans*, designated from a to g, have been identified [16]. Among them, serotypes a, b, and c are globally dominant, whereby type c is the most prevalent [17]. Interestingly, they show different associations with disease depending on ethnicity, geographical localization, or periodontal status. For example, in the United States, serotype c was mostly associated with AP, but also other strains, and the JP2 genotype was found in patients suffering from periodontitis [18]. In Brazil, serotype c was found to be the most prevalent one and associated with both AP and CP. On the other hand, serotype b was also detected in periodontally healthy individuals [19]. Conversely, others found a connection between serotype b and aggressive periodontitis [20]. In Japan, serotype c was predominantly isolated from patients with AP, while the occurrence of serotype b was rare [21]. The specific JP2 genotype of *A. actinomycetemcomitans* was found to be strongly associated with severe periodontitis, particularly in Northern and Western Africa [22,23]. In Asia, the occurrence of the JP2 genotype has not been reported so far [23], and in Germany, it was detected in immigrants from North Africa living for more than 10 years in Germany, but not in Caucasians [24]. Dissemination of the JP2 genotype to non-African populations was only very rarely described [22]. Nevertheless, recent data obtained from nearly 3500 subgingival plaque samples of 1445 periodontitis patients in Sweden showed that the JP2 genotype was found in 1.2% of patients and most of them were of non-African descent [25]. Furthermore, serotype b was more often found in younger patients with periodontitis than in older cohorts [25].

In our department, microbiological diagnostics of subgingival biofilm samples is routinely performed. This includes subtyping of *A. actinomycetemcomitans* strains. After identifying the JP2 genotype in an immigrant from Morocco in 2013, such a clone was detected in two periodontitis patients of non-African origin. Here, the two cases starting with the diagnosis together with all steps of periodontal treatment are presented.

#### **2. Results**

#### *2.1. Case 1*

A 50-year-old female patient presented with localized CP according to the Classification System for Periodontal Diseases and Conditions set in 1999 [26]. At the initial examination, the patient was diagnosed with severe CP with three approximal sites with clinical attachment loss (CAL) ≥ 6 mm and eight sites with probing depth (PD) ≥ 5 mm (Table 1), as defined by the Centre for Disease Control and Prevention and the American Academy of Periodontology (CDC–AAP) [27,28]. No furcation involvement was detected. The patient reported to smoke occasionally. She was healthy and took no medications. Besides the third molars, teeth 16 and 27 were missing. The endodontically treated tooth 47 was scheduled for extraction because of an apical osteolysis. Besides this, the radiographs showed no further pathologies. No angular bony defects were visible and slight horizontal bone loss was noted. Microbiological diagnostics revealed high counts (more than 105) for *A. actinomycetemcomitans*, low counts (about 104) each for *Tannerella forsythia* and *Treponema denticola*, but no *Porphyromonas gingivalis*. Subtyping of *A. actinomycetemcomitans* showed a serotype b strain being positive for the deletion in the promotor region of in the leukotoxin operon (JP2 genotype). Another *A. actinomycetemcomitans* (without deletion in the promoter region of the leukotoxin operon) was not detected. At the next visit, she was asked for contact with people from North and Western Africa, but she had never been abroad before, nor had she closer contact to Africans. Further, additional biofilm was sampled to confirm the microbiological analysis and to culture the strain. Cultivation

confirmed the high counts (105) and identification *of A. actinomycetemcomitans*. Determination of antibiotic resistance found a minimal inhibitory concentration (MIC) of ≤ 0.5 μg/mL for amoxicillin and 4 μg/mL for metronidazole. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium assay confirmed a very high toxicity of that strain, being remarkably higher than those of the control JP2 genotype reference strain (HK1651) (Figure 1). Meanwhile, the patient improved her oral hygiene, and no *T. forsythia* and *T. denticola* were found anymore, but *A. actinomycetemcomitans* was still present in high counts. In order to eradicate *A. actinomycetemcomitans*, the further treatment plan entailed a hygienic phase with antibiotics (amoxicillin 375 mg and metronidazole 500 mg each tid for seven days). After the initial oral hygiene instruction and supragingival scaling, the patient showed good oral hygiene with a plaque index (O'Leary [29]) of < 20% of all tooth surfaces. Thereafter, subgingival scaling was performed in one session by hand curettes and an ultrasonic device with local anesthesia. Antibiotics as mentioned before and 0.02% chlorhexidine were given for 7 and 14 days, respectively. Tooth 47 was extracted. Three months after nonsurgical therapy, the patient was re-evaluated. The goals of periodontal therapy were achieved in all quadrants. There was no site with PD ≥ 5 mm and, therefore, no further surgical periodontal therapy was needed. Microbiological testing revealed an absence of *A. actinomycetemcomitans*, *P. gingivalis*, *T. forsythia*, and *T. denticola*. For supportive periodontal therapy, the patient was sent back to her dentist in private practice.


**Figure 1.** MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium) assay assessing the vitality of the MONO-MAC-6 cells after contact with the clinical *Aggregatibacter actinomycetemcomitans* JP2 genotype strains (case 1 and case 2) in comparison with *Aggregatibacter actinomycetemcomitans* leukotoxin, a reference JP2 genotype strain (HK1651) and Y4 strain.

#### *2.2. Case 2*

A 55-year-old female patient was referred to the clinic for periodontal treatment after having been diagnosed with chronic generalized periodontitis. The periodontal screening index [30] was 4 for each sextant and the periodontal chart showed CAL of > 5 mm at all molars. At teeth 11 and 22, PDs up to 12 mm were detected. In the maxilla, all molars showed a furcation involvement degree II at least at one side. Teeth numbers 18, 38, and 48 were missing. In the mandible, all molars showed degree I furcation involvement. The patient was systemically healthy and a non-smoker. On the radiographs, horizontal bone loss was detected at the distal aspects of teeth 16, 15, 25, and 26. Angular bony defects were observed mesially of all first molars as well as distally of tooth 11. Microbiological analysis revealed high counts (more than 105) for *A. actinomycetemcomitans*, low counts (about 104) each for *T. forsythia*, and moderate counts (about 105) for *T. denticola*, but no *P. gingivalis*. Subtyping of *A. actinomycetemcomitans* showed a serotype b strain being positive for the deletion in the promotor region of the leukotoxin operon (JP2 genotype). No other *A. actinomycetemcomitans* strain was identified. Her strain could be cultured (about 105 per sample) and showed low MIC values to amoxicillin (≤ 0.5 μg/mL) and a resistance to metronidazole (32 μg/mL). The toxicity of the strain to the MONO-MAC-6 cells was similar to that of the JP2 genotype reference strain (HK1651), but also higher than those of the Y4 strain (serotype b strain without deletion in the promotor region) (Figure 1).

At the next visit, she was asked for contacts with Africans and, indeed, she was married to a man from North Africa. Her husband agreed to a periodontal clinical diagnosis including a microbiological analysis. However, he was periodontally healthy and no *A. actinomycetemcomitans* was detected. In the following, her children also agreed to a periodontal clinical and microbiological diagnosis. The 17-year-old daughter was diagnosed with aggressive periodontitis together with a positive detection for *A. actinomycetemcomitans*, but no *P. gingivalis*. After this accidental diagnosis, the daughter also received periodontal therapy, including adjunctive antibiotics.

After a thorough oral hygiene instruction and supragingival scaling, the patient showed an excellent oral hygiene and a plaque index (O'Leary [29]) of less than 15%, and subgingival scaling was performed in two sessions within one week using hand curettes and local anesthesia. Upon the second session, antibiotics (amoxicillin 375 mg and metronidazole 500 mg each tid for seven days) were given because of the JP2 genotype detection and the severity of the tissue destruction. The reevaluation showed a conspicuous improvement with residual PD > 5 mm at teeth 11, 46, and 36. The latter further improved until the first recall so that surgical therapy was needed only for tooth 11 distally with PD of 9 mm and 46 mesially with PD of 7 mm. Now, the JP2 genotype of *A. actinomycetemcomitans* could not be detected anymore. For both teeth, a flap was raised using the simplified papilla preservation technique. After removal of granulation tissue, scaling and root planing was performed. Tooth 11 exhibited a three-wall defect with an intraosseous depth of 3.5 mm. Given the defect configuration, enamel matrix derivative and bone graft material were administered into the defect. Tooth 46 mesially exhibited a narrow angular bony defect of 3 mm depth, which was treated by means of an access flap surgery and application of an enamel matrix derivative. The patient was enrolled into a three-month recall at the Department of Periodontology. After one year, the recall interval was reduced to six months. A stable periodontal situation was noted with no PD > 4 mm. After another year, the patient was sent back to her dentist in private practice.

#### **3. Discussion**

In the present paper, we reported on two rare cases of *A. actinomycetemcomitans* JP2 genotype infection in Caucasians, highlighting the treatment sequences, the clinical outcomes, and the potential value of microbiological testing for the early detection of periodontal disease. Although, over the last 10 years, each *A. actinomycetemcomitans* positive sample has been screened for JP2 genotype presence, no further cases have been detected in our clinic up to now. This is in line with the findings of others who have only sporadically reported on the detection of JP2 genotype in non-African populations [31]. Conversely, the JP2 genotype is widespread and highly present in Northwest African populations. The reason this genotype of *A. actinomycetemcomitans* has remained geographically restricted despite globalization is still an unanswered question. However, it cannot be excluded that a specific host tropism exists that favors the colonization among these populations.

The highly leukotoxic JP2 genotype is strongly associated with AP. In Northwest African countries, there is a higher prevalence of AP reported among the young population than in other parts of the world, where it is a rare disease with a prevalence of less than 1% [32,33]. In contrast, both patients presented here were, at the time of the baseline examination, between 50 and 55 years of age and diagnosed with CP. An association between young age and the presence of JP2 genotype has been observed, however, with the increasing age of the host, these strains seem to disappear [12]. A prospective longitudinal cohort study has demonstrated that, initially, periodontally healthy subjects harboring the JP2 clone are more likely to develop periodontal attachment loss; a much less pronounced disease risk was found for those not carrying the JP2 genotype [33]. In our cases, it is unclear at what age the patients were infected by the JP2 genotype and how fast the periodontal defects evolved. The 50-year-old patient had no association with North or West African countries. The 55-year-old patient was married to a North African man.

It is of interest to note that there was no detection of *P. gingivalis* in any of these two cases. The fact that *P. gingivalis* was not detected could be correlated with the ability of certain subgingival bacteria to modulate the leukotoxicity of *A*. *actinomycetemcomitans.* Antibodies raised against *A*. *actinomycetemcomitans* and its leukotoxin may be inactivated by proteases of other bacteria such as *P. gingivalis* [34]. Gingipains are the primary virulence factor of *P. gingivalis*, showing a proteolytic activity against a broad spectrum of proteins [35]. Further, it has been shown that leukotoxin is proteolytically degraded by the action of gingipains [34]. *P. gingivalis* was able to completely destroy the leukotoxin of *A*. *actinomycetemcomitans* within an hour [34].

*A*. *actinomycetemcomitans* leukotoxin affects immune cells to release IL-1β [6]. Here, we tested the toxicity of the JP2 genotype strains of the two cases on MONO-MAC-6 cells. The JP2 genotype reference strain (HK1651) was more toxic than the Y4 strain. The difference might be not very high, but can be related to the experimental conditions using a lower bacterial concentration and a different cell line than that reported before [36]. One JP2 genotype strain showed a similar cell toxicity to the JP2 genotype reference strain on MONO-MAC-6 cells. The patient with that strain showed periodontal defects at the molar region and at the maxillary incisors, reflecting the typical localization pattern of AP [37].

However, MTT cytotoxicity assay revealed strong cytotoxicity of the other strain, interestingly, the case with no contact to Africans. Here, we can only speculate if there is a difference in production of leukotoxin or in retaining it at the cell surface. Leukotoxin is enriched in outer-membrane-like vesicles [36]. The obviously very high toxicity may contribute to the infection of a person with no genetic predisposition. Here, it has to be pointed out that leukotoxicity may be different depending on the test method that was used [38]. Variation of leukotoxicity was not only observed among JP2- and non-JP2 genotype of *A. actinomycetemcomitans,* but also among the methods of Western blotting, ELISA, cell lysis assay, and mRNA expression assay [38].

After diagnosis, the patients received two sessions of oral hygiene instructions and supragingival scaling by means of ultrasonic and hand instruments. Thereafter, subgingival non-surgical instrumentation was performed with adjunctive antibiotic therapy (amoxicillin and metronidazole). Microbiological testing after the hygienic phase showed that *A. actinomycetemcomitans* was no longer detected. In these cases, the microbiological characterization of *A. actinomycetemcomitans* strains influenced the therapeutic approach, namely to administer or not adjunctive systemic antibiotics. Otherwise, in view of the increasing bacterial resistance, patients diagnosed with CP are not treated with adjunctive amoxicillin and metronidazole during non-surgical mechanical therapy. The in vitro resistance of the strains to amoxicillin and metronidazole was determined. These data cannot be transferred directly to the clinic. A synergism between metronidazole and amoxicillin is well known, as amoxicillin increases the uptake of metronidazole in the bacterial cells [39]. First, this combination was successfully used to treat patients with *A. actinomycetemcomitans* associated periodontitis [40]. Nonetheless, it is well documented that, in patients diagnosed with CP or AP, better clinical outcomes can be obtained if systemic antibiotics are administered in conjunction with subgingival mechanical debridement, irrespective of their microbiological profile [41]. Therefore, in general, microbiological testing was found to be clinically irrelevant for the treatment strategy. It was demonstrated that the presence of putative periodontal pathogens quantified before the treatment was not key for the outcome of scaling and root planing (SRP) with or without amoxicillin and metronidazole [42].

A study evaluating the treatment response of patients infected with JP2 or non-JP2 genotype of *A. actinomycetemcomitans* has shown that patients infected with JP2 genotype had higher PD, CAL, and gingival inflammation than those infected with non-JP2 genotype at baseline. Patients with persisting JP2 genotype after full-mouth SRP and adjunctive administration of amoxicillin and metronidazole had increased gingival inflammation compared with patients where the JP2 strain was eliminated [43]. In the non-JP2 genotype-infected group, the clinical improvements in terms of PD reduction and CAL gain were statistically significantly higher compared with patients infected with the JP2 genotype. These data appear to suggest that the persistence of JP2 genotype in periodontal pocket diminishes the treatment response, which in turn may favor the progression of periodontitis.

In our material, we presented two cases of Caucasians infected with JP2 genotype who were successfully treated with full-mouth SRP and amoxicillin and metronidazole. The microbiological diagnosis was the key decision making factor for selecting the treatment strategy, including the use of amoxicillin and metronidazole. Additionally, it is important to point out that the microbiological diagnosis has finally led to a screening of the patients' family members and the diagnosis of an AP in the teenage daughter of one patient.

#### **4. Materials and Methods**

The two patients of non-African origin with detection of the JP2 genotype underwent active periodontal therapy at the Department of Periodontology during the years 2014 and 2015. Both patients were diagnosed with CP according to the classification set in 1999 [26]. The severity and extent of periodontal destruction varied considerably. For each patient, pooled samples of the deepest pockets of each quadrant were analyzed for the major bacteria associated with periodontal diseases using nucleic acid-based strip technology (micro-IDent®plus11, Hain Lifescience, Nehren, Germany) [4]. Identification of the serotype b strains and JP2 genotype strains was performed using the PCR technique [24].

Then, after asking for an additional biofilm sample, cultivation and isolation of the *A. actinomycetemcomitans* strain were performed. After confirming the identification (JP2 genotype), determination of antibiotic resistance to amoxicillin and metronidazole was done using the microbroth-dilution technique. Further, the toxicity to monocytic cells of human origin (MONO-MAC-6; DSMZ no. ACC 124) was assessed. Those were maintained in RPMI 1640 medium containing 10% fetale bovine serum (FBS) and, after washing, adjusted to 106/mL in RPMI 1640. Forty hour cultures of *A. actinomycetemcomitans* strains on agar plates were adjusted to 2 <sup>×</sup> 107/mL in RPMI 1640. Both suspension were mixed 1:1 and the vitality of MONO-MAC-6 cells was determined after 6 h of incubation at 37 ◦C with 5% of CO2 using the MTT assay, according to Mosmann [44]. As controls, *A. actinomycetemcomitans* HK1651 (JP2 genotype) and *A. actinomycetemcomitans* Y4 (both strains obtained from ATCC, #ATCC 700685, and ATCC 43718), as well as leukotoxin (2.5 μg/mL; purified as described by Kachlany et al. [45] from culture supernatant of a control *A. actinomycetemcomitans* HK1651 strain added by a final centrifugation using a 10 kDa centrifugal filter to remove proteins of lower weights), were used.

#### **5. Conclusions**

Colonization of Caucasians by the JP2 genotype of *A. actinomycetemcomitans* is rare. In the present study, the microbiological diagnosis played the key role for selecting the use of adjunctive systemic antibiotics, as well as for the ensuring an accurate periodontal diagnosis and adequate treatment for the patient's teenage daughter.

**Author Contributions:** Conceptualization, S.E. and A.S. (Alexandra Stähli); methodology, S.E.; treatment, A.S. (Alexandra Stähli); writing—original draft preparation, A.S. (Alexandra Stähli) and S.E.; writing—review and editing, A.S. (Anton Sculean). All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** Anna Magdo ´n (Laboratory of Oral Microbiology, Department of Periodontology, School of Dental Medicine, University of Bern) is acknowledged for identification and characterization of *A. actinomycetemcomitans* strains.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Article* **Distinct Signaling Pathways Between Human Macrophages and Primary Gingival Epithelial Cells by** *Aggregatibacter actinomycetemcomitans*

#### **Ellen S. Ando-Suguimoto 1,\*, Manjunatha R. Benakanakere 2,\*, Marcia P.A. Mayer <sup>1</sup> and Denis F. Kinane <sup>3</sup>**


Received: 28 November 2019; Accepted: 23 March 2020; Published: 27 March 2020

**Abstract:** In aggressive periodontitis, the dysbiotic microbial community in the subgingival crevice, which is abundant in Aggregatibacter actinomycetemcomitans, interacts with extra- and intracellular receptors of host cells, leading to exacerbated inflammation and subsequent tissue destruction. Our goal was to understand the innate immune interactions of A. actinomycetemcomitans with macrophages and human gingival epithelial cells (HGECs) on the signaling cascade involved in inflammasome and inflammatory responses. U937 macrophages and HGECs were co-cultured with A. actinomycetemcomitans strain Y4 and key signaling pathways were analyzed using real-time PCR, Western blotting and cytokine production by ELISA. A. actinomycetemcomitans infection upregulated the transcription of TLR2, TLR4, NOD2 and NLRP3 in U937 macrophages, but not in HGECs. Transcription of IL-1β and IL-18 was upregulated in macrophages and HGECs after 1 h interaction with A. actinomycetemcomitans, but positive regulation persisted only in macrophages, resulting in the presence of IL-1β in macrophage supernatant. Immunoblot data revealed that A. actinomycetemcomitans induced the phosphorylation of AKT and ERK1/2, possibly leading to activation of the NF-κB pathway in macrophages. On the other hand, HGEC signaling induced by A. actinomycetemcomitans was distinct, since AKT and 4EBP1 were phosphorylated after stimulation with A. actinomycetemcomitans, whereas ERK1/2 was not. Furthermore, A. actinomycetemcomitans was able to induce the cleavage of caspase-1 in U937 macrophages in an NRLP3-dependent pathway. Differences in host cell responses, such as those seen between HGECs and macrophages, suggested that survival of A. actinomycetemcomitans in periodontal tissues may be favored by its ability to differentially activate host cells.

**Keywords:** *A. actinomycetemcomitans*; inflammasome; immune response; periodontal disease

#### **1. Introduction**

Periodontitis is an infectious inflammatory disease that leads to the destruction of tooth-supporting tissues by an imbalanced immune response [1–5]. The inflammatory process is induced by a dysbiotic microbial community, with the Gram-negative facultative species *Aggregatibacter actinomycetemcomitans* associated the rapid progression rate of periodontitis, which was previously denominated localized aggressive periodontitis and is now classified as molar/incisor pattern

periodontitis [6,7]. *A. actinomycetemcomitans* is also associated with endocarditis [8] and may play a role in cardiovascular disease and arthritis [9,10]. The dysbiosis induced by *A. actinomycetemcomitans* may be the result of an immunological palsy induced by its virulence factors, such as leukotoxin (Ltx) and cytolethal distending toxin (Cdt) [11,12], which are associated with its ability to invade non-phagocytic cells [13].

During infection, the immune response is induced by microbial-associated molecular patterns (MAMPS), which are recognized by pattern-recognition receptors (PRRs) in eukaryotic cells. PRRs are found extracellularly as Toll-Like Receptors (TLRs) and are expressed on the cell surface, with their stimulation by MAMPS resulting from the activation of NF-κB, MAPK and IRF signaling pathways, culminating in the production of a number of cytokines, chemokines and immunomodulatory factors [14]. PRRs are also present in the cytoplasm and are here called Nod-Like Receptors (NLRs), with nucleotide binding domain/leucine rich repeats, including NLRP1, NLRP3, NLRC4, NOD1, NOD2, and AIM2 receptors; these detect intracellular microorganisms and their products [15–17]. Although both pathogenic and commensal microbes are recognized by PPRs, pathogens often induce the production of endogenous danger signals (danger-associated molecular patterns (DAMPs)) [18]. The cytosol senses DAMPS via NLRs, leading to the formation of multiprotein cytoplasmic complexes called inflammasomes [19]. These complexes activate caspase-1, which results in the release of mature interleukin-1β (IL-1β) and interleukin-18 (IL-18), thereby inducing pyroptosis and apoptosis [20].

*A. actinomycetemcomitans* is recognized by Toll-Like Receptor 4 (TLR4) and TLR2 [21]. Gingival epithelial cells (GECs) are the first defense barrier against pathogens in periodontal tissues; *A. actinomycetemcomitans* adheres to and invades epithelial cells [22]. The interaction of *A. actinomycetemcomitans* with GECs induces the expression of ICAM-1, TNF, GM-CSF, IL-6 and IL-8 and causes apoptosis in monocytes mediated by interaction with TLR2 [22–24]. Furthermore, macrophages infected with *A. actinomycetemcomitans* secrete IL-1β [25,26] and IL-18, a response which is associated with the purinergic receptor P2X7, an endogenous danger signal receptor [27].

Inflammasome activation may play a key role in periodontitis. The expression of NLRP3, which is involved in inflammasomes, is higher in chronic and aggressive periodontitis gingival tissues than in healthy tissues, especially at the periodontal epithelium layer [28]. In periodontal disease, NLRP3 salivary levels are higher in aggressive periodontitis cases compared to periodontally healthy subjects [29].

*A. actinomycetemcomitans* cytolethal distending toxin (AaCdt) was shown to be involved in NLRP3 activation in THP-1 monocytes and release of mature IL-1β [30], but other bacterial factors may be also associated with this response to *A. actinomycetemcomitans,* since infection of human monocytes with leukotoxin- and Cdt-deficient strains still resulted in upregulation of NLRP3, IL-1β and IL-18 expression [31]. *A. actinomycetemcomitans* was also shown to activate the inflammasome pathway in nonimmune cells. NRLP3 upregulation and secretion of mature IL-1β and IL-18 were observed in human osteoblastic MG63 cells upon exposure to *A. actinomycetemcomitans*, leading to apoptosis [32]. Furthermore, NOD1 and NOD2 were activated in human embryonic kidney cells in the presence of *A. actinomycetemcomitans* [33].

The response to a pathogen depends not only on the stimulus, but also on the cell type; however, there are no data regarding whether *A. actinomycetemcomitans* is able to activate inflammasomes in epithelial cells, as reviewed previously [34]. Given the significance of gingival epithelial cells and macrophages in aggressive periodontitis, the present study evaluated the signaling network initiated by *A. actinomycetemcomitans* in gingival epithelial cells and macrophages and, consequently, the induction of immune and inflammasome responses.

#### **2. Results**

*A. actinomycetemcomitans* is associated with localized aggressive periodontitis, however, the molecular mechanisms of the innate immune response in distinct myeloid and nonmyeloid cells of the oral cavity are unknown. We set out to understand the differential activation immune

vs. nonimmune cells by *A. actinomycetemcomitans*. We stimulated the U937 cell line to differentiate into macrophages and primary human gingival epithelial cells (HGECs) via *A. actinomycetemcomitans* Y4 at different time points to determine the innate immune responses. The innate immune genes, in particular *TLR2, TLR4* and *NLRP3,* were upregulated in U937 macrophages co-cultured with *A. actinomycetemcomitans* after two and three hours of incubation, where *NLRP3* doubled its expression compared to the control after two hours of co-culture and was downregulated after four and eight hours, whereas *NOD2* mRNA reached maximum levels after 8 h of stimulation, increasing expression by two times. On the other hand, infection of HGECs with *A. actinomycetemcomitans* did not result in altered transcription profile of genes encoding these receptors (Tables 1 and 2).

**Table 1.** Effect of co-culture of *A. actinomycetemcomitans* strain Y4 (Aa- MOI 1:100) with human gingival epithelial cells (HGECs) for 1, 2, 3, 4 and 8 h on the relative transcription of *TLR4, TLR2, NLRP3, NOD1, NOD2, IL-1*β*, IL-18* and *TNF-*α detected by real-time PCR and expressed in terms of fold-change in comparison with control. Control consisted of infected cells at 0 h. Transcription of the target gene was normalized according to mRNA levels of GAPDH; = data are shown as fold-change ± standard deviation (SD) representative of three independent experiments.


\* Statistically significant difference in comparison with control (ANOVA–Tukey's, p < 0.05).

**Table 2.** Effect of co-culture of *A. actinomycetemcomitans* strain Y4 (Aa- MOI 1:100) with U937 macrophages for 1, 2, 3, 4 and 8 h on the relative transcription of *TLR4, TLR2, NLRP3, NOD1, NOD2 IL-1*β*, IL-18* and *TNF-*α detected by real-time PCR and expressed in fold-change in comparison with control. Control consisted of infected cells at 0 h. Transcription of target gene was normalized according to mRNA levels of GAPDH; data are shown as fold-change ± SD representative of three independent experiments.


\* Statistically significant difference in comparison with control (ANOVA–Tukey's, p < 0.05).

Cytokine gene expression, *IL-1*β, *IL-18* and *TNF* mRNA levels, increased after co-culture of macrophages with *A. actinomycetemcomitans* (Table 2). On the other hand, the interaction of *A. actinomycetemcomitans* with HGECs resulted in a small increase in *IL-1*β and *IL-18* mRNA levels after one hour of co-culture, and decreased at later time points (Table 1). *TNF* mRNA levels increased after two hours of co-culture of *A. actinomycetemcomitans,* with U937 macrophages reaching the peak after three hours of co-culture (134 times increase), and after three and four hours with HGECs (2 and 3.72 times increase, respectively). Production of IL-1β increased after *A. actinomycetemcomitans* challenge in U937 macrophages but not in HGECs. The *A. actinomycetemcomitans* challenge induced production of TNF in U937 macrophages after two hours, whereas in HGECs, TNF was induced after a prolonged period (Figure 1).

**Figure 1.** Effect of co-culture of *A. actinomycetemcomitans* strain Y4 (Multiplicity of infection (MOI) 1:100) with U937 macrophages (**A**) or HGECs (**B**) for 1, 2, 3, 4 and 8 h on the levels of IL-1β and TNF-α levels in cell supernatants. Control consisted of infected cells at 0 h. Data (pg cytokine/mL) are presented as mean ± SD representative of three independent experiments. \* Statistically significant difference in comparison with control (ANOVA–Tukey's, p < 0.05).

Since transcription of *TLR2, TLR4 and NLRP3* was upregulated in U937 macrophages, we determined the activation of signaling molecules downstream of TLRs. Signaling pathway analysis indicated that NF-κB was activated (phosphorylation of serine 32 in IκB-α) and pro caspase-1 was induced and cleaved in *A. actinomycetemcomitans* infected-macrophages (Figure 2A). Infection with *A. actinomycetemcomitans*induced activation of ERK1/2 (phosphorylation of T 202/Y204 residues in ERK1) after 15 min of co-culture and AKT (phosphorylation serine 473 in AKT) after 120 min of co-culture. However, p4EBP1 and pcFos levels did not increase in *A. actinomycetemcomitans*-infected macrophages.

Interestingly, signaling induced by *A. actinomycetemcomitans* was distinct in HGECs. In these epithelial cells, AKT and 4EBP1were phosphorylated after stimulation with *A. actinomycetemcomitans,* whereas ERK1/2 was not phosphorylated (Figure 2B). On the other hand, the phosphorylation of Serine 473 (Ser 473), indicative of AKT activation, was not observed until 60 min of stimulation with *A. actinomycetemcomitans*; these data indicated that AKT activation was under the levels observed in noninfected cells at the early stages of co-culture.

**Figure 2.** Different pathways are activated by *A. actinomycetemcomitans* in macrophages and HGECs. Western blot was used to evaluate the phosphorylation of ERK1/2, 4EBP-1, cFos and AKT in U937 macrophages (**A**) and in HGECs (**B**) after infection with *A. actinomycetemcomitans* strain Y4 (MOI 1:100) at different time points. GAPDH was used as the control. The data shown are representative of three independent experiments.

IL-1β and TNF-α transcription and protein levels (Table 2 and Figure 1) were increased in *A. actinomycetemcomitans*-infected macrophages within a few hours of incubation, mainly because of the activation of NF-κB in these cells (Figure 3). These results suggested that, in macrophages, NF-κB activation by *A. actinomycetemcomitans* is dependent on AKT–ERK1/2 activation, although more profound studies should still be performed. NF-κB activation induced the release of pro-IL-1β and pro-IL-18, which, in the context of the inflammasome, are processed to their mature forms by caspase-1 in macrophages, as shown by the increased production of active caspase-1 in cells co-cultured with *A. actinomycetemcomitans* (Figure 3). The data indicated that infected macrophages exhibited *NLRP3* upregulation and release of IL-1.

**Figure 3.** Western blot image showing increased levels of phosphorylated IκB (plkB-α), (indicative of NF-κB activation), decreased levels of procaspase-1 and increased levels of cleaved caspase-1 (indicative of inflammasome activation in U937 macrophages) after co-culture of *A. actinomycetemcomitans* strain Y4 (MOI 1:100) at different time points. GAPDH was used as the control. The data shown are representative of three independent experiments.

The inflammasome response was analyzed in cells by silencing NLRP3. siNLRP3 macrophages infected with *A. actinomycetemcomitans* exhibited decreased *IL-1*β and *IL-18* mRNA levels. but not *TNF-*α (Figure 4). Taken together, *A. actinomycetemcomitans* distinctly activated innate immune and inflammasomes in myeloid cells (Figure 5) and nonmyeloid cells.

**Figure 4.** Effect of siNLRP3 silencing in *A. actinomycetemcomitans* Y4-infected U937 macrophages (MOI 1:100, 24 h of co-culture) on the relative expression of *NLRP3* (**A**), *IL-1*β (**B**)*, IL-18* (**C**) and *TNF-*α (**D**) detected by real time PCR. Scramble: Scramble control without *A. actinomycetemcomitans* (pool of nontargeting Sirna)*;* negative control: Cells without *A. actinomycetemcomitans*; control: Positive control: Cells with *A. actinomycetemcomitans* without *NLRP3* silencing; siNLRP3: Cells with silencing of *NLRP3* and co-culture with *A. actinomycetemcomitans*. \* Statistically significant difference in comparison with electroporated cells with *A. actinomycetemcomitans* and scramble control (ANOVA–Tukey's, p < 0.05). Data shown in fold-change relative to the scramble are presented as mean ± SD representative of three independent experiments.

**Figure 5.** U937 macrophage response after co-culture with *A. actinomycetemcomitans* Y4. Arrows mean up- or downregulation of gene transcription for *TLR4, TLR2, NOD2, NLRP3, Pro*-*IL-1*β*, Pro-IL-18* and *TNF-*α*,* and for phosphorylation of 4EBP1, AKT, pERK1/2, IκB-α and caspase-1. *A. actinomycetemcomitans* was able to induce an inflammatory response and inflammasome activation in macrophages. Adapted from Qiagen's website (https://www.qiagen.com/br/shop/genes-and-pathways/pathway-central/?q=).

#### **3. Discussion**

The immune response elicited by the dysbiotic community in aggressive periodontitis induced inflammation, resulting in tissue destruction and bone resorption [35]. In this study, we analyzed two important host cell defenses, namely, a first barrier of epithelial cells and a second specialized cell, the macrophages of myeloid origin.

Human gingival epithelial cells and macrophages displayed distinct responses after challenge with *A. actinomycetemcomitans.* The expression of innate immune receptors was not altered by *A. actinomycetemcomitans* infection in HGECs, suggesting that the epithelial barrier was a weak response, resulting in a discrete increase in IL-1β and IL-18 production after one hour of co-culture and decreased expression thereafter. These data were in accordance with others demonstrating that *A. actinomycetemcomitans* did not induce IL-1β production by gingival epithelial cells [36,37]. Moreover, the increase in mRNA expression of TNF after three hours indicated that the primary HGECs respond to the bacteria stimuli, as demonstrated in immortalized OBA-09 cells [22].

On the other hand, transcription of genes encoding TLR2, TLR4, NLRP3 and NOD2 was upregulated in *A. actinomycetemcomitans-*infected macrophages, suggesting a rapid innate immune response against the pathogen. Previous studies in macrophages [38,39] and dendritic cells [40] corroborated our observation that *A. actinomycetemcomitans* leads to increased expression of TLR2 and TLR4 between two and three hours and one and three hours, respectively. After this period of co-culture, there was a decrease in TLR2 and TLR4 expression, concomitant to an increase in the expression of internal receptors such as NLRP3 and NOD2, suggesting phagocytosis of *A. actinomycetemcomitans* and/or its products.

The engagement of microbial components with TLR2 and TLR4 mediates transcriptional responses through activation of NF-κB, leading to the production of pro-inflammatory cytokines, including TNF-α and inactive pro-IL-1β [39]. Furthermore, the response of macrophages to *A. actinomycetemcomitans* infection was obvious, with immediate and continuous upregulation of *TNF-*α, *IL-1*β and, to a lesser extent, *IL-18*.

*A. actinomycetemcomitans* phagocytosis by macrophages was impaired due to cytolethal distending toxin production, but the ability to produce nitric oxide (NO) and TNF-α was still functional in the intoxicated macrophages [41]. The upregulation of extra- and intracellular receptors for PAMPS and DAMPS in macrophages reinforced these observations. Binding of PAMPS to TLRs and NOD2 activates the NF-kB pathway, inducing the expression of inflammatory cytokines [42]. When the signaling transduction pathways were analyzed, as shown in Figure 4, the mitogen activated pathway (MAPK) ERK1/2 was activated in *A. actinomycetemcomitans-*infected macrophages, which was suggestive of NF-κB activation after 120 min of incubation, as indicated increased levels of pIkB-α at this time point (Figure 5). The increased level of phosphorylated serine–threonine kinase (pAKT), also observed after 120 min of macrophage interaction with *A. actinomycetemcomitans,* indicated activation of the PI3K/AKT kinase pathway, which indirectly activates NF-κB via proteasome degradation of IκB. The phosphorylation of 4EBP1 at Thr37/46 decreases its association with eIF4E and consequently inhibits the mRNA translation of growth, thereby promoting protein synthesis [43]. Our results demonstrated that in the late co-culture period, there were decreased levels of phosphorylated 4EBP1, suggesting that *A. actinomycetemcomitans* was not able to affect the viability of the macrophages.

Interestingly, the signaling pathway responses in HGECs were different from those of macrophages, with decreased levels of phosphorylated ERK1/2 and increased levels of 4EBP1 in the phosphorylated form. Additionally, AKT phosphorylation was not observed up to 30 min after interaction of *A. actinomycetemcomitans* with HGECs, which was in accordance with the pro-apoptotic phenotype of epithelial cells reported after 60 min of bacteria–HGEC interaction (Handfield et al., 2005). AKT is key in cellular survival [44] and was only activated when the infected cells were incubated for a prolonged period. Inhibition of the AKT/mTOR pathway in infected macrophages and at early stages of infection in HGECs may exert bacterial clearance effects. AKT inhibition promoted by *Streptococcus pneumoniae* was associated with infection progression and inhibition of autophagy [45,46]. In contrast, attenuation

of the AKT/mTor pathway enhanced autophagy and *Salmonella ssp.* clearance [47], indicating that subversion of this pathway may result in different outcomes according to the infecting agent.

Another studied protein was pc-FOS, which is one of the downstream factors induced by ERK1/2 pathway [48]. c-FOS phosphorylation is involved in osteoclastogenesis, leading to differentiation of precursor cells into osteoclasts due to the production of colony-stimulating factor 1 (CSF-1) and receptor activator of the NF-kB ligand (RANKL) [49]. Although previous data indicated that *A. actinomycetemcomitans* lipopolysaccharide (LPS) induced c-FOS phosphorylation in human gingival fibroblasts [50], our data indicated no increase in pc-FOS after co-culture with live *A. actinomycetemcomitans*, suggesting that ERK1/2 pathway activation did not induce c-FOS phosphorylation in the HGECs or U937 macrophages.

After *A. actinomycetemcomitans*infection, increased levels of IL-1β, TNF-α and IL-18 transcripts were observed in macrophages (Figure 1). Activation of NF-κB leads to pro-IL1 production, whereas pro-IL-18 is constitutively expressed but its expression increases after cellular activation [51,52]. The increase in IL-1β levels after one to three hours and in TNF-α levels after prolonged incubation with *A. actinomycetemcomitans-*infected macrophages possibly indicated that the binding of IL-1β to its receptor IL-1R1 led to a cascade of downstream events, eventually resulting in the expression of TNF-α. Silencing of NRLP3 indicated that activation of this intracellular receptor mediates pro-IL-1β and pro-IL-18 production in infected macrophages (Figure 5). However, the release of the mature and bioactive IL-1 family of cytokines, including IL-1β and IL-18 [51,52], is mediated by caspase-1 in inflammasomes [53]. Our data also indicated that *A. actinomycetemcomitans* Y4 infection in macrophages led to cleavage of procaspase-1 to caspase-1(Figures 4 and 5), resulting in the release of active IL-1β. This observation contradicted a previous study which reported that *A. actinomycetemcomitans* Y4 was not able to induce caspase-1 expression in monocytes, despite the increased expression of IL-1β [31].

On the other hand, the response of HGECs to *A. actinomycetemcomitans* resulted in a slight increase in transcript levels of *IL-1*β and *IL-18* after one hour of incubation, which returned to levels below those achieved in control cells thereafter (Table 1). Furthermore, the levels of TNF-α and IL-1β in cell supernatants were high in infected macrophages, but not in HGECS, shortly after incubation with *A. actinomycetemcomitans* (Figure 1). These data were in accordance with others reporting that HGEC interaction with *A. actinomycetemcomitans* extracts for eight hours did not result in IL-1β production, and increased levels of this cytokine were observed only after 12 h of incubation [54]. Other data indicated that *A. actinomycetemcomitans* infection leads to upregulation of *IL-1*β in HGECs after 60 min of incubation [55]. However, we showed that this upregulation was not maintained after longer interaction periods and did not result in the production of significant amounts of pro-IL-1β. When we analyzed the TNF levels, we observed a small increase of this cytokine in the presence of *A. actinomycetemcomitans*, however, in the presence of *Porphyromonas gingivalis,* the increase was higher after 24 h of co-culture with HGECs, showing that the cells were responsive to another periodontal pathogen [56]. Despite a limited number of samples of HGCEs from different donors, this study was in accordance with the low inflammatory response in the presence of *A. actinomycetemcomitans* when co-cultured with immortalized gingival cells OBA-9 (unpublished data). Furthermore, differences between this work and other studies regarding the tested *A. actinomycetemcomitans* strain, as well as differences in the origins of the monocytic cells and distinct macrophage phenotypes, may have contributed to conflicting data [31]. Overall, our results were in accordance with those that previously verified that *A. actinomycetemcomitans* and its product, Cdt, were able to induce inflammasome activation in macrophages [30,57].

Thus, our data indicated that, although macrophages responded to *A. actinomycetemcomitans* infection by upregulating the expression of intra- and extracellular receptors and production of cytokines (Figure 4), HGEC response to *A. actinomycetemcomitans* was mild, differing from that of other periodontopathogens, such as *P. gingivalis* [36].

Induction of inflammasomes in response to the microbial community was shown to control the microbiota in the gut, whereas its depletion induced dysbiosis [58]. Macrophages promptly recognize *A. actinomycetemcomitans* and its products, activating inflammasomes, which may be important in control of oral dysbiosis, but may also result in tissue destruction and perpetuation of inflammation. However, epithelial cells are the first barrier against pathogens and their response is important for the subsequent immune response. Epithelial cells internalize *A. actinomycetemcomitans* and a pro-apoptotic phenotype is induced by this species [55]. Thus, the low response to the pathogen by epithelial cells may be an additional factor of evasion of host defense mechanisms, facilitating colonization and dissemination to the underlying tissues in oral mucosa. This study demonstrated the differences between the responses of macrophages and gingival epithelial cells in the presence of *A. actinomycetemcomitans.*

#### **4. Material and Methods**

#### *4.1. Eukaryotic Cells and Bacteria Culture Conditions*

Gingival tissue was obtained from young healthy adult patients after third molar extractions (HGECs) by approval of the Institutional Review Board [5,56]. HGECs at the 3rd passage were harvested, seeded at a density of 0.5 <sup>×</sup> 105 cells/well on 6-well plates in KSFM (Keratinocyte Serum Free Medium) medium (Life Technologies, Carlsbad, CA, USA) supplemented with 10 mg/mL insulin, 5 μg/mL transferrin (Sigma-Aldrich, St Louis, MO, USA), 50 μg/mL bovine pituitary extract (BPE) (Life Technologies), 3-factor supplement (10 μM of 2-mercaptoethanol, 10 μM ethanolamine and 10 nM NA-Selenite- Sigma-Aldrich), 1% penicillin–streptomycin solution (Sigma-Aldrich) and 25 μg/L of fungizone (Life Technologies).

The human monocytic cell line U937 [59] was maintained in suspension culture in RPMI-1640 (Life Technologies, Carlsbad, CA, USA) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) (Hyclone, Logan, UT, USA), 1% penicillin–streptomycin solution (Sigma-Aldrich) and 25 μg/L of fungizone and amphotericin B solution (Gibco, Scotland, UK), at 37 ◦C in a humidified atmosphere of 5% CO2. U937 cells were differentiated into adherent macrophage-like cells by exposure of 4 <sup>×</sup> 10<sup>6</sup> cells to 20 nM phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich) for 24 h and left to differentiate for an additional 48 h in 5% CO2 at 37 ◦C [60].

*A. actinomycetemcomitans* strain Y4 serotype b was grown under microaerophilic conditions at 37 °C in tryptic soy broth (Sigma-Aldrich) supplemented with 0.6% weight/volume yeast extract.

#### *4.2. Co-culture Assay*

HGECs or differentiated U937 macrophages cultivated in media (5% fetal bovine serum) without antibiotics were infected with *A. actinomycetemcomitans* Y4 cells culture at mid log-phase at an multiplicity of infection (MOI) of 1:100 (eukaryotic cell:bacteria). After incubation for 1, 2, 3, 4 and 8 h for U937 macrophages and HGECs, the supernatants were collected and cells were washed twice with 1x phosphate saline buffer (PBS) prior to total RNA extraction. In order to verify the activation of signaling pathways in *A. actinomycetemcomitans-*infected cells, the co-cultures were obtained as described at time points 0, 15, 30, 60, 90 and 120 min, and the cell lysates were used in a Western blot assay. Noninfected HGECs and U937 macrophages were used as negative controls. The study was performed in three independent experiments and the HGECs were obtained from three different donors.

#### *4.3. Gene Expression*

Expression of inflammasome-related genes in infected HGECs and U973 macrophages was assessed by reverse transcription followed by real-time PCR (RT-qPCR). Total RNA was extracted from cultured U937 cells and HGECs using the RNeasy mini kit (Qiagen, Hilden, Germany), according to the manufacturer's instruction. Ten micrograms of RNA was used to obtain first strand cDNA synthesis using the High-Capacity cDNA Archive kit (Applied Biosystems, Foster City, CA, USA) in a total volume of 100 μL. Real-time PCR was performed using an ABI 7500 system (Applied Biosystem). The transcription of genes encoding the receptors NLRP3 (Hs00918082\_m1), NOD1 (Hs01036720\_m1), NOD2 (Hs01550753\_m1), TLR2 (Hs02621280\_s1) and TLR4 (Hs00152939\_m1) and cytokines IL-1β

(Hs01555410\_m1), IL-18 (Hs01038788\_m1) and TNF-α (Hs00174128\_m1) were evaluated using Taqman probes for human and theTaqMan Gene Expression Master Mix (Applied Biosystem). Transcription of human GAPDH (Hs02786624\_g1) was used as an endogenous control. Results were analyzed using 2−ΔΔCt, where the results were normalized using the housekeeping gene or gene of interest from the reference sample in the case of cells at 0 h and compared with genes of the other samples using the threshold cycle (Ct) values from the real-time reaction [61].

#### *4.4. Activation of Inflammasome-Related Signaling Pathways*

The amount of phosphorylated proteins indicative of activation of different pathways and other proteins involved in inflammasomes were determined in HGECs and U937 macrophages co-cultured with *A. actinomycetemcomitans* by Western blotting. The cells were washed twice with 1x PBS, suspended in 1x SDS-PAGE loading dye (BioRad, Hercules, CA, USA) and boiled. SDS-PAGE was carried out according to the Laemmli method [62]. The gel was transferred to the nitrocellulose membrane (Life Technologies) at 4 ◦C in a Mini-Trans-Blot cell (Life Technologies) apparatus for 2 h at 370 mA. The primary antibodies were for phosphorylated ERK1/2 (pERK1/2) at T202/Y204, pAKT (Ser 473), p4EBP-1 (Trr 37/46) and pc-Fos (Ser 32). In U937 cells, pIκB-α (Ser32) (Cell Signaling, Danvers, MA, USA), caspase-1 p10 (C20) and cleaved caspase p10 were also evaluated. As a control, the antibody anti-GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA) was used at 1:1000 dilution. The secondary antibody was anti-rabbit IgG, HPR-linked and diluted at 1:2000. Protein detection was performed using Amershan ECL Prime Western Blotting Detection reagent (GE Healthcare, Uppsala, Sweden). After detecting one protein, the primary and secondary antibody detected previously was removed using Restore™ Western Blot Stripping Buffer (Thermo-Scientific) and the same gel was used to detect other proteins. The results are representative of three independent experiments and cells at 0 h were used as the control.

#### *4.5. Cytokines Quantification*

Levels of secreted IL-1β and TNF-α were determined in the supernatants of U937 and HGEC that were co-cultured with *A. actinomycetemcomitans* by ELISA using an R&D systems kit (Minneapolis, MN, USA). The plates were read in a microplate reader at an optical density (OD) of 450 nm. The amounts of each cytokine were determined after comparison with the respective standard curve.

#### *4.6. Silencing of NLRP3*

In order to confirm inflammasome activation by *A. actinomycetemcomitans in* macrophages, silencing of NLRP3 was performed. U937 cells (2 <sup>×</sup> 106 cells) were transfected with siNLRP3 (Dharmacon-Thermo Scientific, Waltham, MA, USA) using the Amaxanucleofactor kit V (Lonza, Allendale, NJ, USA). Cell suspensions were centrifuged at 90× *g* for 10 min, the medium was removed and 1 μM of siNLRP3 in 100 μL of nucleofactor V buffer were added. As a negative control, scramble siRNA (siControl non-targeting siRNA Pool, Horizon Discovery, UK) was used. The samples were electroporated in nucleofactor program V-001 (Lonza), RPMI medium was added and the cells were transferred into 6-well plates with 2.5 mL of phorbol 12-myristate 13-acetate (PMA) in RPMI medium and incubated for 24 h. Co-cultures of transfected U937 macrophages with *A. actinomycetemcomitans* were performed as described. Transcription of genes encoding NRLP3, IL-1β, IL-18 and TNF-α was determined by RT-qPCR and the production of cytokines in the cell supernatants was measured by ELISA.

#### *4.7. Statistical Analysis*

Comparisons between samples were performed by one-way ANOVA followed by post-Tukey's test (Graphpad Prism version 4.0, La Jolla, CA, USA). Results were considered significant when p < 0.05.

#### **5. Conclusions**

Taken together, our data indicated that *A. actinomycetemcomitans* enhanced the expression of NLRP3, TLR4, TLR2 and NOD2 in macrophages but not in HGECs, consequently inducing distinct signaling pathways and cytokine production and demonstrating varied innate immune responses depending on the cell type.

**Author Contributions:** Conceptualization, E.A.S., M.R.B., M.P.A.M; methodology, E.A.S., M.R.B; software, E.A.S.; validation, E.A.S., M.R.B..; formal analysis, E.A.S.; investigation, E.A.S, M.R.B.; resources, M.R.B.; data curation, E.A.S.; writing—original draft preparation, E.A.S.; writing—review and editing, E.A.S, M.R.B., M.P.A.M., D.F.K..; visualization, E.A.S.; supervision, M.R.B., M.P.A.M.; project administration, M.R.B.; funding acquisition, E.A.S., M.R.B., D.F.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by FAPESP grants 2012/05887-0, 2015/18273-9 and the School of Dental Medicine, University of Pennsylvania.

**Acknowledgments:** We thank Joseph DiRienzo for providing *A. actinomycetemcomitans* strain Y4. E.A.S was supported by FAPESP grant 2012/05887-0 and 2015/18273-9. M.R.B was supported by internal funding by the School of Dental Medicine, University of Pennsylvania, Philadelphia.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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