**1. Introduction**

The human oral microbiome is comprised of over 700 microbial species, as characterized by both cultivation and culture-independent molecular approaches such as the 16S rRNA gene-based method [1]. Almost all oral microorganisms are non-pathogenic opportunistic commensals to maintain oral health condition and defend against pathogenic microorganisms [2]. The most remarkable feature of oral microflora is that numerous oral bacteria form a biofilm, so called dental plaque, which is defined as microbial communities embedded in a self-produced matrix of extracellular polymeric substances and a dynamic metabolically structure, on tooth surface and oral mucosa [3,4]. Within biofilm formed in oral niche, the polymicrobial interactions between interspecies, such as recombination and horizontal gene transfer, are caused and then specialized clones are selected [5]. Several pressures by bacterial

communities and the host as well as the change in oral environment caused by the administration of antibiotics also a ffect the selection of bacterial species and the characterizations of their virulence. Once this homeostasis of oral microflora is disturbed by changing the local environments as well as the metabolic and physiological activities of bacteria in biofilm, oral infectious diseases such as dental caries and periodontitis, which are major two prevalent chronic diseases in oral cavity, are caused. Moreover, in the last two decades, numerous studies regarding the association between oral biofilm infectious diseases and various systemic diseases, such as cardiovascular diseases, atherosclerosis, diabetes mellitus, aspiration pneumonia, and autoimmune diseases, have been reported. To date, it has been considered that there are two major pathways by which oral bacterial infectious diseases a ffect systemic diseases (Figure 1). Bacteremia as direct pathway, oral bacteria residing in the oral cavity invade blood vessels in dental pulp and periodontal tissues, and then reach not only the heart but also the large blood vessels and various organs to cause systemic diseases. Another direct pathway involving aspiration, which often occurs in elderly people, involves oral bacteria reaching a respiratory organ, such as the lung via a pharynx and airway route, and causing respiratory diseases. Several bacterial products, such as endotoxin (lipopolysaccharide: LPS) and heat shock protein (HSP), as well as antigens, also involved in various systemic diseases due to the indirect causes of triggering immune responses. Therefore, the oral cavity is recognized as a source or reservoir of microbial infection and the establishment of methods to prevent oral infections is one of the most important and urgen<sup>t</sup> issues in dentistry. From the viewpoint of oral biofilm infection, understanding the roles of oral microorganisms and their pathogenic factors and elucidating various systemic diseases and their onset mechanisms related to oral infections would lead to the development of novel preventive measures.

**Figure 1.** Life style of biofilm and the conceptual pathogenic mechanisms of oral bacterial infection leading to various systemic diseases.

Among oral bacterial species, over 100 identified oral Streptococci, which can colonize shortly after birth and play important roles in the formation of oral physiological microflora, are the predominant commensal and opportunistic inhabitants in the oral cavity and upper respiratory tract in humans, and cause opportunistic infections at sites distant from the oral cavity as well as oral infections, especially in immunocompromised patients and elderly people [6]. Pathogenic Streptococci are identified as sources of invasive infections in humans and their infections are still one of the most serious diseases in modern medical world [7]. Table 1 shows systemic diseases affected and caused by oral Streptococcal infections. Therefore, it has been recently considered that the genus Streptococci severely impacts on human health by carrying a significant number of worldwide human infections, and has been separated into following 8 distinct groups: mitis, sanguinis, anginosus, salivalius, downei, mutans, pyogenic, and bovis using gene clustering, as well as phylogenetic and gene gain/loss analyses (Figure 2) [8]. The distribution of oral Streptococcal species in the oral cavity has been also reported [9,10]. The mitis and sanguinis groups, such as *S*. *sanguinis*, *S*. *mitis*, *S*. *gordonii*, and *S*. *oralis* are common commensals, primarily involved in initial dental plaque formation as the first colonizers of the tooth surface, but are also associated with an increased risk of systemic diseases and invasive infections, including infective endocarditis, by entering the bloodstream through transient bacteremia after daily activities such as brushing and flossing, as well as invasive dental procedures such as tooth extraction. A recent descriptive epidemiological study has reported the distribution of Streptococci causing infective endocarditis [11]. Figure 3 shows one clinical case of infective endocarditis mainly caused by oral *S*. *sanguinis*. We encountered the patient with infective endocarditis caused by oral Streptococci, who had severe systemic conditions such as mitral and tricuspid regurgitations and a continuous fever over 37 ◦C, and who was urgently hospitalized in our university hospital. This case was rigorously diagnosed by the detection of oral *Streptococcus sanguinis*, as well as the examination of chest radiograph and echocardiogram at the time of the onset of infective endocarditis. Therefore, as the presentative case of infective endocarditis, the detailed therapeutic course following the guideline is described below and in Figure 3. The guideline for systemic complications, such as infective endocarditis, shows that bactericidal antibiotics are selected based on the results from microbiological examination, such as blood culture, and long-term antibiotic treatment is performed at high doses in order to kill the causative bacteria and to prevent recurrence. It is also very important to identify the causative bacteria in order to suppress side effects as much as possible. Following this guideline, the patient received viccilin (ampicillin sodium: 6000 mg/day) and gentamicin (a type of aminoglycoside: 60 mg/day) after microbiological examination. Afterwards, the symptoms were improved and no bacteria were detected by blood culture. It has been recognized that *S*. *mutans* playing important roles in the initiation and progression of dental caries is inversely associated with oral health [12]. The *S*. *anginosus* group is detected as part of the oropharyngeal microflora and is commonly associated with a variety of purulent infections and abscess formations in the brain, meninx, heart, liver, lung, and spleen. It is caused by bacteremia, as well as periapical odontogenic lesions [13–17]. In particular, *S*. *intermedius* and *S*. *constellatus* found in dental plaque has been associated with the development of periodontal diseases [18]. In contrast, *S*. *salivarius* group, in which *S*. *salivarius* is predominant in the saliva and on the surface of oral mucosa, is related to oral health rather than disease by producing bacteriocins targeting cariogenic *S*. *mutans* in addition to some enzymes such as dextranase and urease, which can inhibit the accumulation of dental plaque and acidification, respectively. A previous probiotic study reported that *S*. *salivarius* provides potential oral benefits to children [19].

> **Table 1.** Systemic diseases affected by oral Streptococcal infections.


**Table 1.** *Cont*.


**Figure 2.** The phylogenetic relationship among 8 major groups of human Streptococcal species.

**Figure 3.** A clinical case of infective endocarditis caused by oral *Streptococcus sanguinis*. A 72 years-old male patient with mitral and tricuspid regurgitations was urgently hospitalized for continuous fever over 37 ◦C and diagnosed as infective endocarditis by detection of oral Streptococcus, *S*. *sanguinis*. During the hospitalization for 1 month, patient received viccilin (ampicillin sodium: 6000 mg/day) and gentamicin (a type of aminoglycoside: 60 mg/day). After the improvement of symptoms and no bacterial detection by blood culture, patient underwent artificial valve replacement and tricuspid ring annuloplasty, and then was discharged from hospital due to the stabilization of symptoms. The patient came to our dental department for the prevention of recurrence with a referral from the medical doctor. (**a**) Chest radiograph and echocardiogram at the time of the onset of infective endocarditis. Cardiac hypertrophy (Cardio-thoracic Ratio: CTR: ≥ 50%) was observed due to abnormalities in the mitral valve, and the left atrium was enlarged markedly. Vegetation (green arrow) was observed in the mitral valve. (**b**) Oral and X-ray photographs of patient with infective endocarditis. Gingival redness and slight swelling were observed in full mouth, and dental calculus deposition were observed on mandibular anterior teeth and upper left molars. Mobility of upper anterior teeth and left premolars was also observed. From dental X-ray radiographs, root fractures of upper right central and lateral incisors, and a endodontic-periodontal combined lesion of the upper left incisor and canines were found. Severe alveolar bone loss around the upper anterior teeth and left premolars as well as root caries on the upper lateral incisor and 1st premolar was also observed. The number of total Streptococci in 10 μL of saliva was 1.0 × 10 7 copies and various periodontal pathogens, such as *Porphyromonas gingivalis*, *Aggregatibacter actinomycetemcomitans*, *Tannerella forsythia*, *Treponema denticola* and *Fusobacterium nucleatum*, were also detected at significantly high level.

Streptococci have an array of virulence factors, which include surface proteins for adhesion, invasion/internalization, extracellular enzymatic proteases, and toxins delivered to cell surface as well as extracellular environments, which are associated with their colonization at various sites in the human body, dissemination, evasion from immune system for survival, destruction of host tissues, and modulation of the host immune function [20,21]. Some vaccine candidates are currently being considered to protect against infectious diseases caused by SStreptococci. This review article focuses on the mechanistic links among crucial pathogenic factors from oral Streptococci, inflammation, and systemic diseases to provide the current understanding of oral infections and widespread systemic diseases. In particular, our recent findings regarding the roles of histone-like DNA binding protein and extracellular DNA in biofilm formation and systemic diseases are also summarized.

### **2. Pathogenic Factors Involved in Adhesion, Colonization, Internalization and Invasion**

In general, the first step on bacterial infections is adherence of bacterial cells with host tissues via the interaction between bacterial adhesion factor, adhesin, and its receptor. This step is extremely important for bacterial survival in bacterial multicellular communities and the establishment of infections. Streptococci express a wide range of adhesins that are specific for the surface of host tissues to colonize, grow, and form biofilm (Table 2). These adhesion and colonization factors of oral Streptococci also play pivotal roles in resistant to antimicrobial peptides and protection against host innate immune defense system. Among the numerous adhesion factors, representative molecules are described below.


**Table 2.** The pathogenic factors expressed in oral Streptococci for colonization, inflammation, infection causing systemic diseases.

### *2.1. Cell Wall-Anchored Polypeptides*

Among the cell wall-anchored polypeptides produced by oral Streptococci, antigen I/II acts as a mediator on the adherence of Streptococci to salivary glycoproteins called pellicles coated on the surfaces of teeth as well as collagen, fibronectin and laminin in the tissues, and is also engaged in biofilm formation by interacting with other oral microorganisms, such as *Actinomyces naeslundii*, *Porphyromonas gingivalis*, and *Candida albicans*, platelet aggregation, and tissue invasion [22,23]. Antigen I/II is conserved in oral Streptococcus species including *S*. *pyogenes*, *S*. *suis* and *S*. *agalactiae*. Spy1325, a member of the antigen I/II family and cell surface-anchored molecule produced by oral Streptococci, is very well conserved in group A Streptococcus (GAS) strains. Interestingly, the immunization of mice with recombinant Spy1325 fragments conferred protection against GAS-mediated mortality, suggesting that Spy1325 may represent a shared virulence factor among GAS, GBS, and oral Streptococci [24]. Therefore, these adhesion factors are considered as a candidate molecule for preventive and therapeutic measures against Streptococcal infections, including dental caries.

Fibronectin-binding protein expressed in all Streptococci plays the role of providing a bridge between Streptococci and host cells by attachment to the extracellular matrix, fibronectin [23,25]. Fibronectin binding proteins can be divided into two types: one type contains fibronectin binding repeats and another type has no repeats [26,27]. This kind of adhesion is di fferent between the binding activity and structure. Some can bind soluble fibronectin and others attach to immobilized fibronectin expressed on the surface of host cells. Moreover, most of these adhesins are anchored to the bacterial cell wall, but some are not. In addition to the role of adhesins mediating attachment, fibronectin-binding protein has been identified as invasins invading epithelial and endothelial cells, which have contributed to evasion from host's innate immune defense mechanisms, such as the complement system and phagocytosis [27].

As another cell wall-anchored protein, collagen binding proteins adhere to collagen-rich tissues for colonization of oral and extra-oral tissues [28] and also bind complement C1 recognition protein, C1q, as inhibitors of the classical complement defense. Therefore, this function confers the ability of immune evasion on oral Streptococci [20].

Serine-rich repeat glycoproteins expressed in wide range of oral Streptococci has multiple serine-rich repeats, which are estimated as an approximately 75% of this protein [23]. After invading into bloodstream, oral Streptococci can bind to human platelets through this adhesion and are disseminated systemically [29]. Binding to platelets leads to form a thrombus, by which oral Streptococci can evade the host immune defense and antibiotics circulated in blood, and then cause infective endocarditis [30]. Therefore, this adhesin is considered as a major virulence factor of endocarditis.

Pili are filamentous apparatus typically extending 1–3 mm from the bacterial cell surface, and the genes encoding pili are identified in discrete loci called pilus islands flanked by mobile genetic elements [20,23]. Pili as virulent factors adhere to various host epithelial cells as well as extracellular matrix proteins such as collagen and fibrinogen, and then promotes Streptococcal colonization and biofilm formation on various sites in the host as well as non-biological surfaces [31]. In addition to adhesion, pili can facilitate bacterial invasion into human epithelial and endothelial cells and lead to Streptococcal dissemination in the host during the critical infection steps [32,33]. Moreover, pili have immunomodulatory abilities to evoke inflammatory cytokine responses and thwart the host innate immune defenses of resist phagocyte killing [34–36]. Therefore, pili have gotten much attention as potential vaccine candidates because of animal studies showing conferred protective immunity against Streptococcal infection [37].

M proteins expressed on a bacterial cell surface, α-helical coiled-coil dimers extending as hair like projections, bind host proteins such as immunoglobulins, fibronectin, and fibrinogen, complement factors such as albumin, and adhere to epithelial cells [38]. Interestingly, antigenically variable M proteins are considered as major virulence factors and immunogens in Streptococci by inducing pro-inflammatory responses and inhibiting phagocytic activities to assist Streptococci in evading host innate immune defenses [39,40]. Soluble M1 protein secreted from Streptococci has been also considered as a novel Streptococcal superantigen because it contributes to excess T cell activation and inflammatory response, such as the induction of T-cell proliferation and Th1 type cytokines production, during invasive Streptococcal infections [41]. Therefore, M proteins may be promising vaccine immunogens [39].

### *2.2. Cell Wall-Anchorless Polypeptides*

Enolase, cell wall-anchorless adhesin and cytoplasmic glycolytic enzyme, is well conserved structurally in Streptococci [23,42]. α-enolase functionally binds to plasmin and plasminogen as well as laminin, fibronectin, and collagens, and also enhances plasminogen activation [43]. Plasmin cleaved from plasminogen by plasminogen activators can degrade extracellular matrix, in turn breakdown epithelial barriers and finally lead to bacterial invasion and infection. Therefore, this anchorless cell surface protein has been considered in promising vaccine candidates for the prevention of Streptococcal infection [42].
