Pyocyanin, a contributory factor in haem acquisition and virulence enhancement of Porphyromonas gingivalis in the lung

Several recent studies show that the lungs infected with Pseudomonas aeruginosa are often co-colonised by oral bacteria including black-pigmenting anaerobic (BPA) Porphyromonas species. The BPAs have an absolute haem requirement and their presence in the infected lung indicates that sufficient haem, a virulence up-regulator in BPAs, must be present to support growth. Haemoglobin from micro-bleeds occurring during infection is the most likely source of haem in the lung. Porphyromonas gingivalis displays a novel haem acquisition paradigm whereby haemoglobin must be firstly oxidised to methaemoglobin, facilitating haem release, either by gingipain proteolysis or capture via the haem-binding haemophore HmuY. P. aeruginosa produces the blue phenazine redox compound, pyocyanin. Since phenazines can oxidise haemoglobin, it follows that pyocyanin may also facilitate haem acquisition by promoting methaemoglobin production. Here we show that pyocyanin at concentrations found in the CF lung during P. aeruginosa infections rapidly oxidises oxyhaemoglobin in a dose-dependent manner. We demonstrate that methaemoglobin formed by pyocyanin is also susceptible to proteolysis by P. gingivalis Kgp gingipain and neutrophil elastase, thus releasing haem. Importantly, co-incubation of oxyhaemoglobin with pyocyanin facilitates haem pickup from the resulting methemoglobin by the P. gingivalis HmuY haemophore. Mice intra-tracheally challenged with viable P. gingivalis cells plus pyocyanin displayed increased mortality compared to those administered P. gingivalis alone. Pyocyanin significantly elevated both methaemoglobin and total haem levels in homogenates of mouse lungs and increased the level of arginine-specific gingipain activity from mice inoculated with viable P. gingivalis cells plus pyocyanin compared with mice inoculated with P. gingivalis only. These findings indicate that pyocyanin, by promoting haem availability through methaemoglobin formation and stimulating of gingipain production, may contribute to virulence of P. gingivalis and disease severity when co-infecting with P. aeruginosa in the lung

Impact of Porphyromonas gingivalis peptidylarginine deiminase on bacterial biofilm formation, epithelial cell invasion, and epithelial cell transcriptional landscape

Abstract Peptidylarginine deiminase (PPAD) is a virulence factor unique to pathogenic Porphyromonas species, especially P. gingivalis. Mechanistically, PPAD activity, in conjunction with Arg-specific gingipains, generates protein fragments with citrullinated C-termini. Such polypeptides are potential de novo epitopes that are key drivers of rheumatoid arthritis. This process could underlie the observed clinical association between rheumatoid arthritis and periodontitis. However, the role of PPAD in host colonization by P. gingivalis and, subsequently, in triggering periodontitis is not known. Therefore, the aim of the current study was to delineate the role of PPAD in bacterial biofilm formation, and to define whether adherence to, invasion of, and host responses to bacteria of gingival keratinocytes depend on PPAD activity. We studied these aspects using PPAD-competent and PPAD-incompetent strains of P. gingivalis, and demonstrated that neither biofilm formation nor its composition was affected by PPAD activity. Similarly, flow cytometry revealed that PPAD did not impact the ability of P. gingivalis to adhere to and, subsequently, invade keratinocytes. Network analyses of gene expression patterns, however, revealed a group of host genes that were sensitive to PPAD activity (CXCL8, IL36G, CCL20, and IL1B). These genes can be categorized as potent immune modulators belonging to the interleukin 1 system, or chemoattractants of lymphocytes and neutrophils. Thus, we conclude that PPAD, although it is a potent modulator of the immune response, does not affect bacterial biofilm formation or the ability of P. gingivalis to adhere to and invade gingival epithelial cells

Discovery of novel potential reversible peptidyl arginine deiminase inhibitor

Citrullination, a posttranslational modification, is catalyzed by peptidylarginine deiminases (PADs), a unique family of enzymes that converts peptidyl-arginine to peptidyl-citrulline. Overexpression and/or increased PAD activity is observed in rheumatoid arthritis (RA), Alzheimer’s disease, multiple sclerosis, and cancer. Moreover, bacterial PADs, such as Porphyromonas gingivalis PAD (PPAD), may have a role in the pathogenesis of RA, indicating PADs as promising therapeutic targets. Herein, six novel compounds were examined as potential inhibitors of human PAD4 and PPAD, and compared to an irreversible PAD inhibitor, Cl-amidine. Four of the tested compounds (compounds 2, 3, 4, and 6) exhibited a micromolar-range inhibition potency against PAD4 and no effect against PPAD in the in vitro assays. Compound 4 was able to inhibit the PAD4-induced citrullination of H3 histone with higher efficiency than Cl-amidine. In conclusion, compound 4 was highly effective and presents a promising direction in the search for novel RA treatment strategies

Porphyromonas gingivalis in Alzheimer's disease brains : evidence for disease causation and treatment with small-molecule inhibitors

Porphyromonas gingivalis, the keystone pathogen in chronic periodontitis, was identified in the brain of Alzheimer's disease patients. Toxic proteases from the bacterium called gingipains were also identified in the brain of Alzheimer's patients, and levels correlated with tau and ubiquitin pathology. Oral P. gingivalis infection in mice resulted in brain colonization and increased production of Aβ1-42, a component of amyloid plaques. Further, gingipains were neurotoxic in vivo and in vitro, exerting detrimental effects on tau, a protein needed for normal neuronal function. To block this neurotoxicity, we designed and synthesized small-molecule inhibitors targeting gingipains. Gingipain inhibition reduced the bacterial load of an established P. gingivalis brain infection, blocked Aβ1-42 production, reduced neuroinflammation, and rescued neurons in the hippocampus. These data suggest that gingipain inhibitors could be valuable for treating P. gingivalis brain colonization and neurodegeneration in Alzheimer's disease

AXL targeting by a specific small molecule or monoclonal antibody inhibits renal cell carcinoma progression in an orthotopic mice model

AXL tyrosine kinase activation enhances cancer cell survival, migration, invasiveness, and promotes drug resistance. AXL overexpression is typically detected in a high percentage of renal cell carcinomas (RCCs) and is strongly associated with poor prognosis. Therefore, AXL inhibition represents an attractive treatment option in these cancers. In this preclinical study, we investigated the antitumor role of a highly selective small molecule AXL inhibitor bemcentinib (BGB324, BerGenBio), and a newly developed humanized anti‐AXL monoclonal function blocking antibody tilvestamab, (BGB149, BerGenBio), in vitro and an orthotopic RCC mice model. The 786‐0‐Luc human RCC cells showed high AXL expression. Both bemcentinib and tilvestamab significantly inhibited AXL activation induced by Gas6 stimulation in vitro. Furthermore, tilvestamab inhibited the downstream AKT phosphorylation in these cells. The 786‐0‐Luc human RCC cells generated tumors with high Ki67 and vimentin expression upon orthotopic implantation in athymic BALB/c nude mice. Most importantly, both bemcentinib and tilvestamab inhibited the progression of tumors induced by the orthotopically implanted 786‐0 RCC cells. Remarkably, their in vivo antitumor effectiveness was not significantly enhanced by concomitant administration of a multi‐target tyrosine kinase inhibitor. Bemcentinib and tilvestamab qualify as compounds of potentially high clinical interest in AXL overexpressing RCC

Gingipains : critical factors in the development of aspiration pneumonia caused by Porphyromonas gingivalis

Aspiration pneumonia is a life-threatening infectious disease often caused by oral anaerobic and periodontal pathogens such as Porphyromonas gingivalis. This organism produces proteolytic enzymes, known as gingipains, which manipulate innate immune responses and promote chronic inflammation. Here, we challenged mice with P. gingivalis W83 and examined the role of gingipains in bronchopneumonia, lung abscess formation, and inflammatory responses. Although gingipains were not required for P. gingivalis colonization and survival in the lungs, they were essential for manifestation of clinical symptoms and infection-related mortality. Pathologies caused by wild-type (WT) P. gingivalis W83, including hemorrhage, necrosis, and neutrophil infiltration, were absent from lungs infected with gingipain-null isogenic strains or WT bacteria preincubated with gingipain-specific inhibitors. Damage to lung tissue correlated with systemic inflammatory responses, as manifested by elevated levels of TNF, IL-6, IL-17, and C-reactive protein. These effects were unequivocally dependent on gingipain activity. Gingipain activity was also implicated in the observed increase in IL-17 in lung tissues. Furthermore, gingipains increased platelet counts in the blood and activated platelets in the lungs. Arginine-specific gingipains made a greater contribution to P. gingivalis-related morbidity and mortality than lysine-specific gingipains. Thus, inhibiting gingipain may be a useful adjunct treatment for P. gingivalis-mediated aspiration pneumonia

Plasmin inhibition by bacterial serpin : implications in gum disease

Tannerella forsythia is a periodontopathogen that expresses miropin, a protease inhibitor in the serpin superfamily. In this study, we show that miropin is also a specific and efficient inhibitor of plasmin; thus it represents the first proteinaceous plasmin inhibitor of prokaryotic origin described to date. Miropin inhibits plasmin through the formation of a stable covalent complex triggered by cleavage of the Lys(368)-Thr(369) (P2-P1) reactive site bond with a stoichiometry of inhibition of 3.8 and an association rate constant (k(ass)) of 3.3×10(5) M(−1)s(-1). The inhibition of the fibrinolytic activity of plasmin was nearly as effective as that exerted by α(2)-antiplasmin. Miropin also acted in vivo by reducing blood loss in a mice tail bleeding assay. Importantly, intact T. forsythia cells or outer membrane vesicles, both of which carry surface-associated miropin, strongly inhibited plasmin. In intact bacterial cells, the antiplasmin activity of miropin protects envelope proteins from plasmin-mediated degradation. In summary, in the environment of periodontal pockets, which are bathed in gingival crevicular fluid consisting of 70% of blood plasma, an abundance of T. forsythia in the bacterial biofilm can cause local inhibition of fibrinolysis, which could have possible deleterious effects on the tooth-supporting structures of the periodontium