Porphyromonas gingivalis is a leading pathogen in chronic periodontitis, a disease process involving progressive destruction of the tissues that support the teeth. Recently, the organism has been reported to produce a unique bacterial enzyme, P. gingivalis peptidyl-arginine deiminase (PPAD), which has the ability to convert arginine residues in proteins to citrulline. Protein citrullination alters protein structure and function; hence, PPAD may be involved in deregulation of the host’s signalling network and immune evasion. Further, accumulating evidence suggests a role for autoimmunity against citrullinated proteins in the development of rheumatoid arthritis (RA). As inflammatory conditions in the lungs of cigarette smokers contribute to the breakdown of immune tolerance to citrullinated epitopes, chronic exposure to citrullinated proteins at periodontitis sites may also predispose susceptible individuals to the development of autoantibodies and the initiation of RA. In this review, we discuss evidence that PPAD may represent a mechanistic link between periodontitis and RA, diseases that are known to be significantly associated at the epidemiological level.

Three distinct streptococcal species:Streptococcus anginosus, Streptococcus intermedius, andStreptococcus constellatus, belonging to theStreptococcus anginosusgroup (SAG), also known asStreptococcus millerigroup, have been attracting clinicians and microbiologists, not only as oral commensals but also as opportunistic pathogens. For years they have been simply classified as so called viridans streptococci, and distinct species were not associated with particular clinical manifestations. Therefore, description of SAG members are clearly underrepresented in the literature, compared to other medically relevant streptococci. However, the increasing number of reports of life-threatening infections caused by SAG indicates their emerging pathogenicity. The improved clinical data generated with the application of modern molecular diagnostic techniques allow for precise identification of individual species belonging to SAG. This review summarizes clinical reports on SAG infections and systematizes data on the occurrence of individual species at the site of infection. We also discuss the issue of proper microbiological diagnostics, which is crucial for further clinical treatment.

Neutrophil-derived networks of DNA-composed extracellular fibers covered with antimicrobial molecules, referred to as neutrophil extracellular traps (NETs), are recognized as a physiological microbicidal mechanism of innate immunity. The formation of NETs is also classified as a model of a cell death called NETosis. Despite intensive research on the NETs formation in response to pathogens, the role of specific bacteria-derived virulence factors in this process, although postulated, is still poorly understood. The aim of our study was to determine the role of gingipains, cysteine proteases responsible for the virulence of P. gingivalis, on the NETosis process induced by this major periodontopathogen. We showed that NETosis triggered by P. gingivalis is gingipain dependent since in the stark contrast to the wild-type strain (W83) the gingipain-null mutant strain only slightly induced the NETs formation. Furthermore, the direct effect of proteases on NETosis was documented using purified gingipains. Notably, the induction of NETosis was dependent on the catalytic activity of gingipains, since proteolytically inactive forms of enzymes showed reduced ability to trigger the NETs formation. Mechanistically, gingipain-induced NETosis was dependent on proteolytic activation of protease-activated receptor-2 (PAR-2). Intriguingly, both P. gingivalis and purified Arg-specific gingipains (Rgp) induced NETs that not only lacked bactericidal activity but instead stimulated the growth of bacteria species otherwise susceptible to killing in NETs. This protection was executed by proteolysis of bactericidal components of NETs. Taken together, gingipains play a dual role in NETosis: they are the potent direct inducers of NETs formation but in the same time, their activity prevents P. gingivalis entrapment and subsequent killing. This may explain a paradox that despite the massive accumulation of neutrophils and NETs formation in periodontal pockets periodontal pathogens and associated pathobionts thrive in this environment. Periodontitis, or gum disease, is characterized by chronic inflammation and erosion of the tooth-supporting tissues. The condition is fuelled by bacterial accumulation on the tooth surface below the gum line that resists the host innate immune response, including massive accumulation of neutrophils. Despite possessing a formidable array of bactericidal machineries, including neutrophil extracellular traps (NETs) formation whereby neutrophils release DNA-composed fibers decorated with bactericidal proteins and peptides to efficiently trap and kill bacteria. Nevertheless, neutrophils in periodontitis are unable to clear the infection due to the presence of key periodontal pathogens, including Porphyromonas gingivalis. This bacterium secretes a variety of virulence factors, including proteases (gingipains) that allow the organism to manipulate the host immune response to benefit the entire dysbiotic microbial community. Here, we describe a unique strategy whereby P. gingivalis trigger NET formation through gingipain-dependent cleavage of Protease Activated Receptor (PAR)-2 on the neutrophil surface. Importantly, NETs formed in this way are deficient in antibacterial activity but instead, supports bacterial growth due to degradation of bactericidal components by gingipains. This finding may explain a paradox that dysbiotic bacteria flourished in periodontal pockets in spite of massive accumulation of neutrophils and abundant NETs formation.

The skin constitutes a formidable barrier against commensal and pathogenic bacteria, which permanently and transiently colonise the skin, respectively. Commensal and pathogenic species inhabiting skin both express proteases. Whereas proteases secreted by commensals contribute to homeostatic bacterial coexistence on skin, proteases from pathogenic bacteria are used as virulence factors, helping them colonise skin with breached integrity of the epithelial layer. From these initial sites of colonisation, pathogens can disseminate into deeper layers of skin, possibly leading to the spread of infection. Secreted bacterial proteases probably play an important role in this process and in the deterrence of innate defence mechanisms. For example, Staphylococcus aureus proteases are essential for changing the bacterial phenotype from adhesive to invasive by degrading adhesins on the bacterial cell surface. Secreted staphylococcal proteases mediate pathogen penetration by degrading collagen and elastin, essential components of connective tissue in the dermis. The activation of the contact system and kinin generation by Streptococcus pyogenes and S. aureus proteases contributes to an inflammatory reaction manifested by oedema, redness and pain. Kinin-enhanced vascular leakage might help bacteria escape into the circulation thereby causing possible systemic dissemination of the infection. The inflammatory reaction can also be fueled by the activation of protease-activated receptors on keratinocytes. Concomitantly, bacterial proteases are involved in degrading antimicrobial peptides, disarming the complement system and neutrophils and preventing the infiltration of the infected sites with immune cells by inactivation of chemoattractants. Together, this provides protection for colonising and/or invading pathogens from attack by antibacterial forces of the skin.

Cathelicidin LL-37 plays an essential role in innate immunity by killing invading microorganisms and regulating the inflammatory response. These activities depend on the cationic character of the peptide, which is conferred by arginine and lysine residues. At inflammatory foci in vivo, LL-37 is exposed to peptidyl arginine deiminase (PAD), an enzyme released by inflammatory cells. Therefore, we hypothesized that PAD-mediated citrullination of the arginine residues within LL-37 will abrogate its immunomodulatory functions. We found that, when citrullinated, LL-37 was at least 40 times less efficient at neutralizing the proinflammatory activity of LPS due to a marked decrease in its affinity for endotoxin. Also, the ability of citrullinated LL-37 to quench macrophage responses to lipoteichoic acid and poly(I:C) signaling via TLR2 and TLR3, respectively, was significantly reduced. Furthermore, in stark contrast to native LL-37, the modified peptide completely lost the ability to prevent morbidity and mortality in a mouse model of d-galactosamine–sensitized endotoxin shock. In fact, administration of citrullinated LL-37 plus endotoxin actually exacerbated sepsis due to the inability of LL-37 to neutralize LPS and the subsequent enhancement of systemic inflammation due to increased serum levels of IL-6. Importantly, serum from septic mice showed increased PAD activity, which strongly correlated with the level of citrullination, indicating that PAD-driven protein modification occurs in vivo. Because LL-37 is a potential treatment for sepsis, its administration should be preceded by a careful analysis to ensure that the citrullinated peptide is not generated in treated patients.

Candida albicans is a pathogenic fungus capable of switching its morphology between yeast-like cells and filamentous hyphae and can associate with bacteria to form mixed biofilms resistant to antibiotics. In these structures, the fungal milieu can play a protective function for bacteria as has recently been reported for C. albicans and a periodontal pathogen—Porphyromonas gingivalis. Our current study aimed to determine how this type of mutual microbe protection within the mixed biofilm affects the contacting host cells. To analyze C. albicans and P. gingivalis persistence and host infection, several models for host–biofilm interactions were developed, including microbial exposure to a representative monocyte cell line (THP1) and gingival fibroblasts isolated from periodontitis patients. For in vivo experiments, a mouse subcutaneous chamber model was utilized. The persistence of P. gingivalis cells was observed within mixed biofilm with C. albicans. This microbial co-existence influenced host immunity by attenuating macrophage and fibroblast responses. Cytokine and chemokine production decreased compared to pure bacterial infection. The fibroblasts isolated from patients with severe periodontitis were less susceptible to fungal colonization, indicating a modulation of the host environment by the dominating bacterial infection. The results obtained for the mouse model in which a sequential infection was initiated by the fungus showed that this host colonization induced a milder inflammation, leading to a significant reduction in mouse mortality. Moreover, high bacterial counts in animal organisms were noted on a longer time scale in the presence of C. albicans, suggesting the chronic nature of the dual-species infection.