Studies of Streptococcus anginosus Virulence in Dictyostelium discoideum and Galleria mellonella Models.

For many years, Streptococcus anginosus has been considered a commensal colonizing the oral cavity, as well as the gastrointestinal and genitourinary tracts. However, recent epidemiological and clinical data designate this bacterium as an emerging opportunistic pathogen. Despite the reported pathogenicity of S. anginosus, the molecular mechanism underpinning its virulence is poorly described. Therefore, our goal was to develop and optimize efficient and simple infection models that can be applied to examine the virulence of S. anginosus and to study host-pathogen interactions. Using 23 S. anginosus isolates collected from different infections, including severe and superficial infections, as well as an attenuated strain devoid of CppA, we demonstrate for the first time that Dictyostelium discoideum is a suitable model for initial, fast, and large-scale screening of virulence. Furthermore, we found that another nonvertebrate animal model, Galleria mellonella, can be used to study the pathogenesis of S. anginosus infection, with an emphasis on the interactions between the pathogen and host innate immunity. Examining the profile of immune defense genes, including antimicrobial peptides, opsonins, regulators of nodulation, and inhibitors of proteases, by quantitative PCR (qPCR) we identified different immune response profiles depending on the S. anginosus strain. Using these models, we show that S. anginosus is resistant to the bactericidal activity of phagocytes, a phenomenon confirmed using human neutrophils. Notably, since we found that the data from these models corresponded to the clinical severity of infection, we propose their further application to studies of the virulence of S. anginosus.

Proteolytic Activity-Independent Activation of the Immune Response by Gingipains from Porphyromonas gingivalis.

Porphyromonas gingivalis, a keystone pathogen in periodontitis (PD), produces cysteine proteases named gingipains (RgpA, RgpB, and Kgp), which strongly affect the host immune system. The range of action of gingipains is extended by their release as components of outer membrane vesicles, which efficiently diffuse into surrounding gingival tissues. However, away from the anaerobic environment of periodontal pockets, increased oxygen levels lead to oxidation of the catalytic cysteine residues of gingipains, inactivating their proteolytic activity. In this context, the influence of catalytically inactive gingipains on periodontal tissues is of significant interest. Here, we show that proteolytically inactive RgpA induced a proinflammatory response in both gingival keratinocytes and dendritic cells. Inactive RgpA is bound to the cell surface of gingival keratinocytes in the region of lipid rafts, and using affinity chromatography, we identified RgpA-interacting proteins, including epidermal growth factor receptor (EGFR). Next, we showed that EGFR interaction with inactive RgpA stimulated the expression of inflammatory cytokines. The response was mediated via the EGFR-phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT) signaling pathway, which when activated in the gingival tissue rich in dendritic cells in the proximity of the alveolar bone, may significantly contribute to bone resorption and the progress of PD. Taken together, these findings broaden our understanding of the biological role of gingipains, which in acting as proinflammatory factors in the gingival tissue, create a favorable milieu for the growth of inflammophilic pathobionts. IMPORTANCE Gingipain cysteine proteases are essential virulence factors of Porphyromonas gingivalis, an oral bacterium implicated in development of periodontitis. Gingipains diffusing from anaerobic periodontal pockets lose proteolytic activity in the oxygenated environment of gingival tissues. We found that despite the loss of activity, gingipains still elicit a strong inflammatory response, which may contribute to the progression of periodontitis and bone resorption. Moreover, we identified the host molecules utilized by the pathogen as receptors for proteolytically inactivated gingipains. The broad distribution of those receptors in human tissue suggests their involvement in systemic diseases associated with periodontal pathogens.

Citrullination-Resistant LL-37 Is a Potent Antimicrobial Agent in the Inflammatory Environment High in Arginine Deiminase Activity.

LL-37, the only member of the mammalian cathelicidin in humans, plays an essential role in innate immunity by killing pathogens and regulating the inflammatory response. However, at an inflammatory focus, arginine residues in LL-37 can be converted to citrulline via a reaction catalyzed by peptidyl-arginine deiminases (PAD2 and PAD4), which are expressed in neutrophils and are highly active during the formation of neutrophil extracellular traps (NETs). Citrullination impairs the bactericidal activity of LL-37 and abrogates its immunomodulatory functions. Therefore, we hypothesized that citrullination-resistant LL-37 variants would retain the functionality of the native peptide in the presence of PADs. To test this hypothesis, we synthetized LL-37 in which arginine residues were substituted by homoarginine (hArg-LL-37). Bactericidal activity of hArg-LL-37 was comparable with that of native LL-37, but neither treatment with PAD4 nor exposure to NETs affected the antibacterial and immunomodulatory activities of hArg-LL-37. Importantly, the susceptibilities of LL-37 and hArg-LL-37 to degradation by proteases did not significantly differ. Collectively, we demonstrated that citrullination-resistant hArg-LL-37 is an attractive lead compound for the generation of new agents to treat bacterial infections and other inflammatory diseases associated with enhanced PAD activity. Moreover, our results provide a proof-of-concept for synthesis of therapeutic peptides using homoarginine.