*2.3. Proteases*

Some proteases secreted from Streptococci have associated with their virulence. In addition to the role of adhesin to glycoprotein and laminin, Streptococcal pyrogenic exotoxin B (SpeB), predominant cysteine protease, has relatively indiscriminant specificity to degrade the extracellular matrix proteins, including fibronectin, cytokines, chemokines, compliment components, immunoglobulins, immune system components such as the antimicrobial peptide cathelicidin LL-37, and serum protease inhibitors. It also activates interleukin-1β [44,45]. This protease also degrades some proteins targeted by autophagy in the host cell cytosol, which is an important innate immune defense, and this proteolytic activity helps Streptococci to evade autophagy, to replicate in the cytoplasm of host cells, to colonize deep-seated tissues, and finally to lead to tissue destruction [46]. Moreover, SpeB increases the production of proapoptotic molecules, such as tumor necrosis factor (TNF)- α and Fas ligand, by activation of matrix metalloproteinase (MMP)- 9 and -2 and then induces apoptosis of host cells [45]. Based on the significant roles of SpeB as critical virulence factor, SpeB combined with inactive SpeA, Streptococcal pyrogenic endotoxin, has been considered as a potential vaccine candidate, which can produce neutralizing antibodies to prevent Streptococcal infection [47].

C5a peptidase, also called SCPA, is a cell wall-anchored immunogenic 125-kDa protein and a well-conserved antigen in Streptococci, and enzymatically cleaves the compliment component C5a to specifically inactivate [48–50]. As an adhesin, C5a peptidase binds directly integrin by the Arg-Gly-Asp (RGD) motifs and the extracellular matrix, fibronectin, with high a ffinity as well to epithelial cells [51]. This peptidase also inhibits neutrophil chemotaxis and the recruitment of phagocytes to the site of Streptococcal infection by cleavage of C5a and promotes invasion and colonization on damaged epithelium as invasin [26,51]. Therefore, C5a peptidase plays roles as a virulence factor through its multifunctional activities and is considered to be a promising vaccine candidate.

### **3. Pathogenic Factors Associated with Biofilm Formation**

The characteristics of biofilm include high resistance to antibiotics and host immunity, as described above. Therefore, the Center for Disease Control (CDC) has warned that biofilm is involved in over 65% of human bacterial infections which are di fficult to prevent, and that the emergence of multidrug-resistant bacteria and delays in biofilm measures is a serious problem in the entire medical field. Biofilm formed in the microbial immediate environment after their colonization creates a self-produced matrix consisting of extracellular polymeric substances (EPS), which are composed of polysaccharides, proteins, nucleic acids, and lipids [52]. EPS confers the adhesion ability and mechanical stability of biofilm, as well as embedded bacterial cells. Regarding the roles of biofilm in the etiology of systemic infectious diseases, the characteristic of resistance against abuse of a wider spectrum of antibiotics for biofilm infections has been focused on, and it has been considered that the ine ffectiveness of the antimicrobial agen<sup>t</sup> as a major feature of biofilm is greatly involved in the emergences of multidrug-resistant bacteria and higher toxic pathogens [7]. It has been also reported that the transformation is caused by frequent horizontal gene transfer, which occurs between bacteria in dental plaque biofilm. This leads to the acquisition of new resistance genes and high antibiotic resistance [53]. Moreover, microorganisms in biofilm share their metabolites and have an intercellular communication (cell-cell interaction) mechanism called quorum sensing (QS) that senses the cell density showing numbers of self and di fferent species and synchronously regulates the expression of specific genes encoding virulent factors, such as enzymes and toxins. Therefore, solving these antibiotics-dependent problems requires the development of novel therapeutic methods to e ffectively suppress the biofilm formation without selective pressure, not using selective microorganisms based on the conventional antimicrobial sensitivity or the mechanism of antimicrobial action. Biofilm forms and matures through several stages (Figure 1). At each stage in the life style of biofilm, focusing on molecules common in bacteria involved in biofilm formation may lead to develop novel therapeutic agents. The first step of biofilm formation is that floating bacteria attaching to the biological surfaces, and this adhesion process is involved in various bacterial products and adhesins, including pili and surface proteins as described in the previous section.

### *3.1. Bis-(3'-5')-Cyclic Dimeric Guanosine Monophosphate (Cyclic di-GMP) as a Bacterial Second Messenger*

The attached bacteria grow and increase their number to form a microcolony, and subsequently produce extracellular matrix components consisting of polysaccharides, DNA, and proteins which connect the bacterial cells and strengthen the adhesion to the biological surface. The extracellular matrix of mature biofilm protects bacterial cells from the stresses, such as phagocytosis by host cells and oxidation, and bacterial communication in biofilm is more highly activated by the accumulation of signal substances and metabolites involved in QS. Some dispersal bacteria detached from the mature biofilm attach themselves to the new biological surface and then cause the infection to spread. Recently, it has been shown that an intracellular second messenger called bis-(3'-5')-cyclic dimeric guanosine monophosphate (cyclic-di-GMP) plays an important role in the transition from reversible attachment to irreversible attachment and also regulates various genes expression through transcriptional factors [54]. With regard to the transition from the floating state to the biofilm state and vice versa, it has been reported that the change in the concentration of cyclic-di-GMP, the intracellular second messenger in bacterial cells, regulates the bacterial virulence, motility, the cell cycle and the synthesis of extracellular matrix, as well as biofilm formation [55]. Most cyclic-di-GMP-dependent signaling pathways also regulate the ability of bacteria to interact with other bacterial and eukaryotic cells. Therefore, cyclic di-GMP plays important roles in biofilm lifestyle including the multicellular bacterial biofilm development. Regarding these abilities, the modulation of bacterial cyclic-di-GMP signaling pathways might be a novel potential way to control biofilm formation in the medical area, and cyclic-di-GMP is considered to be a possible candidate for a vaccine adjuvant [56].

### *3.2. Extracellular DNA (eDNA)*

In addition to polysaccharides and proteins, the extracellular matrix components in biofilm contain not only bacteria but also DNA derived from the host, and the interaction of eDNA in biofilm with other extracellular matrix components is also considered in terms of pathogenic factors in biofilm (Table 3). The roles of these eDNAs in the formation of biofilm have been also focused on as a target to replace or complement the use of antibiotics [57]. A previous study reported that the addition of DNase I suppresses biofilm formation and also degrades the mature biofilm, suggesting that eDNA is essential for biofilm formation and maturation as well as for its structural maintenance properties [58]. Therefore, it has been considered that enzymatic degradation of eDNA can prevent biofilm formation or sensitize biofilm to antimicrobials. Regarding oral bacterial biofilm, evidence showing eDNA plays a number of important roles in biofilm formation and maturation on oral soft and hard tissues and in its structural integrity has been accumulated [59]. The concentration of eDNA in Streptococcal biofilm is also involved in strength and rigidity of biofilm structure as well as biofilm formation and maturation. When the concentration of eDNA in Streptococcal biofilm is extremely high, biofilm maturation is suppressed and the bacteria in biofilm tends to detach [60]. This suggests that a high concentration of eDNA in formed or matured biofilm makes its structure fragile and makes bacteria easily disperse. In other words, when the concentration of eDNA in biofilm is increased and its concentration reaches a certain level, some bacteria in biofilm are detached and then attach to new sites to form biofilm, resulting in the spread of infection. Intriguingly, DNA derived from di fferent bacteria, such as *Escherichia coli*, *Staphylococcus aureus*, and *Pseudomonas aeruginosa* in humans have also shown similar characteristics [60]. A recent interesting study has reported that calcium ion-regulated autolysin AtlA maturation mediates the release of eDNA by *S*. *mutans*, which contributes to its biofilm formation in infective endocarditis [61]. Therefore, all eDNAs present in biofilm, regardless of their origin, have been shown to be involved in biofilm formation, maturation, and structure, and an eDNA-targeting novel strategy may be applicable to novel treatments for bacterial biofilm-related infectious diseases.


**Table 3.** Interaction of eDNA with other pathogenic factors present in the extracellular matrix of biofilm.

### *3.3. DNA Binding Protein*

The eukaryotic cell has a protein called histone, which plays a role of compactly housing its chromosomal DNA in the nucleus. Prokaryotes, such as bacteria, also have histone-like DNA binding protein (HLP) to compactly house their chromosomal DNA in small bacterial cells. The bacterial HLP equivalent to eukaryotic histone goes beyond the concept of the nucleoid-related protein which forms a DNA-protein complex, and involves itself in various intracellular processes, including the binding ability to DNA and mRNA, regulation of gene transcription and translation, replication, and rearrangement. To clarify the pathogenicity and roles of HLP in biofilm, we cloned the *hlp* gene of *S*. *intermedius* (*Si*-*hlp*) and sequenced its DNA. Through the homology analysis of the amino acid sequence predicted from its DNA sequence, it has been revealed that HLP has high homology (89–94%) at an amino acid sequence level and is structurally highly conserved in Streptococci [62,63]. Further functional analysis showed that *Si*-HLP forms homodimers outside of the cells and co-stimulation of *Si*-HLP with pathogen-associated molecular patterns (PAMPs) produced by bacteria synergistically or additively induces pro-inflammatory cytokines production in human monocytes, indicating that HLP itself has a possible role in causing inflammation at the site of bacterial infection [62]. Moreover, the knockdown of HLP expression with the antisense RNA expression system inhibits the growth of *S*. *intermedius* and suppresses its biofilm formation, suggesting that HLP is an essential protein for the viability and growth of *S*. *intermedius* as well as biofilm formation [63]. The knockdown of HLP reduced the hydrophobicity of the cell surface and suppressed the expression of its cytolytic toxin, intermedilysin, which is the main pathogenic factor of *S*. *intermedius*, suggesting that HLP also affects the regulation of pathogenic factors expression in addition to bacterial adhesion and aggregation [63]. As a bacterial pathogenic factor, HLP is not only involved in bacterial survival, growth, biofilm formation, and maturation, but also has the ability to directly induce pro-inflammatory responses in host cells and therefore HLP has huge roles in bacterial infection. Interestingly, fluorescence microscopic observation showed that eDNA and *Si*-HLP in biofilm were co-localized and uniformly distributed in biofilm (Figure 4). These findings sugges<sup>t</sup> that HLP in addition to eDNA may also be a target as a novel treatment for biofilm infection control.

**Figure 4.** Co-localization and distribution of eDNA and *Si*-HLP in *S*. *intermedius* biofilm. eDNA in the formed *S*. *intermedius* biofilm was stained with propidium iodide (PI; red fluorescence), and *Si*-HLP was stained with anti-*Si*-HLP antibody and Alexafluor 488 (green fluorescence). Fluorescence microscopic observation showed that eDNA and HLP are co-localized (yellow fluorescence) and uniformly distributed in biofilm.

### *3.4. Membrane Vesicle*

Membrane vesicles released from lots of bacterial species extracellularly contain proteins, nucleic acids such as DNA and RNA, and toxins. Lipoproteins, one of PAMPs, are also included as the cell membrane components of the surface of vesicles and released from the vesicles. Released PAMPs induce pro-inflammatory cytokines production after binding to the pattern recognition receptors (PRRs) expressed in host cells, suggesting that vesicles are involved in the exacerbation of inflammation [64]. Studies on membrane vesicles has been studied mainly using Gram-negative bacteria for a long time, but many research results on membrane vesicles of Gram-positive bacteria have been also shown increasingly in the last 10 years [65]. Membrane vesicles contained in the extracellular matrix of biofilm have various biological functions, such as intercellular communication, transport of toxins in vesicles, and horizontal gene transfer. Moreover, due to the similarity to liposomes, membrane vesicles is being tried in applications as drug delivery systems and vaccines using nanobiotechnology in the medical field [66].

As the second step following this bacterial adherence and biofilm formation, bacteria, which evaded antimicrobial peptides and host defense systems such as neutrophils and internalization by macrophages, invade the susceptible tissues to stimulate host cellular responses using capsule and PAMPs, such as lipoteichoic acid (LTA), in Streptococci. Neutrophils, key response cells recruited to the infectious site, release granule proteins and chromatin that together form extracellular fibers that bind bacteria. These neutrophil extracellular traps (NETs) are a form of innate response that binds microorganisms, prevents them from spreading, and degrade virulence factors and kill bacteria [67]. A recent intriguing study has shown that a nuclease, DeoC, in *S*. *mutans* degrades NETs and contributes to the escape of *S*. *mutans* from neutrophil killing and to the spread of *S*. *mutans* through biofilm dispersal [68]. After invasion into host cells and blood vessels of bacteria evaded from host innate defense system, bacteria are disseminated to tissues around the infection site and dispersed to colonize new sites through the blood stream.

### **4. E**ff**ects of Oral Streptococci on Systemic Diseases**

The Viridans Group Streptococci is one of the most predominant bacterial groups in the oral bacterial flora, and has long been considered to be pathogens of severe infections such as infective endocarditis, sepsis, and meningitis (Table 1) [69]. In recent years, among the pathogenic factors possessed by the cariogenic bacterium *S*. *mutans*, a collagen binding protein (CBP, coding gene; *cnm*) has been focused for being associated with various systemic diseases. *S*. *mutans* expressing a CBP invade blood vessels, damage vascular endothelial cells, bind to collagen in the vascular endothelium to suppress platelet aggregation, and induce the expression of MMP-9, finally leading to the exacerbation of cerebral hemorrhage [70]. The epidemiological research also showed that the correlation between the occurrence of brain microbleeding and the high detection rate of CBP-positive *S*. *mutans* strains, suggesting that CBP-positive *S*. *mutans* is an independent risk for the onset and progression of cerebrovascular diseases [71]. Moreover, *S*. *mutans* expressing a CBP invade blood vessels, reach the liver and are then taken up into hepatic parenchymal cells to induce the production of cytokines such as interferon (IFN)-γ in the liver. It has also been reported that the imbalance of immune reactions and immune mechanisms caused by the infection of *cnm*-positive *S*. *mutans* leads to aggravation and deterioration of enteritis and ulcerative colitis in the digestive tracts [72]. Furthermore, the relationship between high DMFT (decayed, missing, and filled teeth) index and high urinary protein levels in patients with *cnm*-positive *S*. *mutans* has been shown and also suggests the association of its infection with renal diseases such as IgA nephropathy [73]. Recent study has reported that a 190-kDa protein antigen (PA), known as SpaP, P1 and antigen 1/2, of *S*. *mutans* affects the interaction with human serum, and the heart valves extirpated from rat infected with CBP-positive/PA-negative *S*. *mutans* strain showed prominent bacterial mass formation using in vivo infective endocarditis model, suggesting that CBP-positive/PA-negative *S*. *mutans* strain contribute to the pathogenicity in infective endocarditis [74].

### **5. Relationship Between Oral Streptococci and Autoimmune Diseases**

Autoimmune diseases are types of chronic inflammation that occur in target organs as a result of the failure of immune tolerance to self-antigens and cellular immune responses by antibodies produced against self-antigens. In recent years, in addition to the reaction to the microorganisms which caused some types of infections, "molecular mimic" which cross-reacts with self-antigens has been considered to play roles in the mechanisms of onset and progression of autoimmune diseases. From this point of view, since the oral cavity is inhabited by lots of bacteria, it is always exposed to antigens derived from various bacteria, suggesting an association between the sensitization to antigens from resident bacteria and the onset of autoimmune diseases.

Regarding the association between oral bacteria, especially Gram-positive bacteria, and autoimmune diseases, some studies have focused on primary biliary cirrhosis (PBC) as autoimmune diseases. PBC is an autoimmune disease of unknown pathogenesis that often occurs in postmenopausal middle-aged women and its lesion is mainly composed of non-suppurative inflammation (chronic non-suppurative destructive cholangitis) around the intrahepatic small bile ducts. With progression of PBC to liver failure from liver cirrhosis, liver transplantation is the only way to treat the disease, and therefore it has been considered that PBC is an intractable disease. Laboratory findings of patients with PBC show that elevated biliary tract enzymes and high levels of IgM, and positive results for many autoantibodies, such as anti-mitochondrial antibody and anti-gp210, nuclear membrane protein, at a high rate (> 90%). Previous reports showed that LTA, a cell wall component of Gram-positive bacteria, was detected in the cytoplasm of lymphocytes and plasma cells infiltrating the site of chronic non-suppurative inflammation around interlobular bile ducts and in the serum of PBC patients, and it has been also reported that the levels of anti-LTA antibodies of IgM and IgA classes in PBC patients are higher than compared to those in healthy subjects and in patients with chronic hepatitis C, indicating that some Gram-positive bacteria might be involved in the onset and progression of PBC [75,76]. Moreover, the results of ELISA using whole cells of several Gram-positive Streptococci showed that the sera of PBC patients are highly reactive with these Streptococcal bacteria, especially *S*. *intermedius* and *Si*-HLP, compared to those of healthy subjects and patients with chronic hepatitis C, and HLP was detected in the lesion of PBC by immunohistochemical staining [77]. These results sugges<sup>t</sup> that Streptococci and HLP may play important roles in the onset and progression of PBC. The administration of either live or heat-killed several Streptococci including *S*. *intermedius* twice a week for 8 weeks to the gingiva of BALB/c mice cause chronic non-suppurative inflammation around portal vein and

the liver small bile ducts closely resembling PBC. Moreover, PBC-like clinical condition is observed even 20 months after the last administration and immunohistochemical staining showed that HLP was also detected in the non-suppurative inflammation area around the small bile duct of the liver, and inflammation was observed in the renal tubules [78]. Interestingly, although no bacteria were detected in the infected focal area, the depositions of LTA and HLP were observed around the small bile ducts similar to tissues from PBC patients, and the transplantation with the splenocytes (T cells) of this mouse into RAG2-/- immunodeficient mice caused similar chronic non-suppurative inflammation around the small bile ducts [79]. These findings also sugges<sup>t</sup> the relationship between oral biofilm infection and autoimmune diseases. In patients with PBC, anti-gp210 autoantibodies are positive, and these positive patients progress to cirrhosis at a high rate compared to negative patients, and therefore anti-gp210 antibody levels are treated as a prognostic factor and are suggested to be deeply involved in the progression of PBC. More interestingly, a previous study reported that the epitope of gp210 was also found within the HLP sequence and the anti-HLP antibody cross-reacted with gp210 in mouse, indicating the sharing of the epitope [78]. Taken together, it has been suggested that Streptococci, especially dominant resident bacteria in the oral cavity and LTA, are strongly associated with the onset and progression of PBC.
