The novel cytokine interleukin (IL)-17 has been implicated in many infectious and autoimmune settings, especially rheumatoid arthritis. Consistent with its proinflammatory effects on bone, osteoblast cells are highly responsive to IL-17, particularly in combination with other inflammatory cytokines. To better understand the spectrum of activities controlled by IL-17, we globally profiled genes regulated by IL-17 and tumor necrosis factor alpha (TNF-alpha) in the preosteoblast cell line MC3T3-E1. Using Affymetrix microarrays, 80-90 genes were up-regulated, and 19-50 genes were down-regulated with IL-17 and TNF-alpha as compared with TNF-alpha alone. These included proinflammatory chemokines and cytokines, inflammatory genes, transcriptional regulators, bone-remodeling genes, signal transducers, cytoskeletal genes, genes involved in apoptosis, and several unknown or unclassified genes. The CXC family chemokines were most dramatically induced by IL-17 and TNF-alpha, confirming the role of IL-17 as a potent mediator of inflammation and neutrophil recruitment. Several transcription factor-related genes involved in inflammatory gene expression were also enhanced, including molecule possessing ankyrin repeats induced by lipopolysaccharide/inhibitor of kappaBzeta (MAIL/kappaBzeta), CCAAT/enhancer-binding protein delta (C/EBPdelta), and C/EBPbeta. We also identified the acute-phase gene lipocalin-2 (LCN2/24p3) as a novel IL-17 target, which is regulated synergistically by TNF-alpha and IL-17 at the level of its promoter. A similar but not identical pattern of genes was induced by IL-17 and TNF-alpha in ST2 bone marrow stromal cells and murine embryonic fibroblasts. This study provides a profile of genes regulated by IL-17 and TNF-alpha in osteoblasts and suggests that in bone, the major function of IL-17 is to cooperate and/or synergize with other cytokines to amplify inflammation.

Proteolytic enzymes produced by Porphyromonas gingivalis are thought to play critical roles in the pathogenesis of periodontitis. The aim of this study was to investigate the effect of gingipain cysteine proteinase gene inactivation on selected pathological and physiological functions of P. gingivalis. Our results showed that Argand Lys-gingipain activities are critical components for the efficient growth of P. gingivalis in human serum. However, when the serum was supplemented with peptides provided as pancreatic casein hydrolysate, the gingipains did not appear to be essential for growth. The effect of gingipain gene inactivation on the susceptibility of P. gingivalis to serum bactericidal activity was investigated using standardized human serum. The wild-type strain, P. gingivalis ATCC 33277, was largely unaffected by the bactericidal activity of human serum complement. On the other hand, mutants lacking Arg-gingipain A, Arg-gingipain B, or Lys-gingipain activity were susceptible to complement. Since gingipains are mostly located on the outer membrane of P. gingivalis, inactivation of the genes for these enzymes may modify cell surface properties. We showed that gingipaindeficient mutants differed in their capacities to assimilate radiolabeled amino acids, cause hemolysis, express adhesins, hemagglutinate, and form biofilms. Lastly, the gingipains, more specifically Arg-gingipains, were responsible for causing major cell damage to human gingival fibroblasts. In conclusion, our study indicated that, in addition to being critical in the pathogenic process, gingipains may play a variety of physiological roles in P. gingivalis, including controlling the expression and/or processing of virulence factors. Mutations in gingipain genes thus give rise to pleiotropic effects.Periodontitis is a group of inflammatory conditions with an infective etiology that leads to loss of tooth support. There is now a consensus that chronic periodontitis is initiated by several bacterial species that behave in a cooperative or synergistic fashion to produce the infection. A group of ϳ10 bacterial species, more particularly the highly proteolytic organisms Porphyromonas gingivalis, Treponema denticola, and Bacteroides forsythus, have been associated with chronic periodontitis (10,25,39,40). Much evidence points to P. gingivalis as the key pathogen in chronic periodontitis. For example, studies have shown that P. gingivalis is detected with greater frequency and at higher levels at periodontal sites that appear to be disease active (33, 41) and that certain periodontal health indicators in individuals are inversely correlated with the presence or level of P. gingivalis (15,16).Virulence factors produced by P. gingivalis include outer membrane vesicles, adhesins, lipopolysaccharides, hemolysins, and proteinases (9,13,17,18). Three different genes code for arginine-X (Arg-gingipain A and B [rgpA and rgpB])-and lysine-X (Lys-gingipain [kgp])-specific cysteine proteinases (31, 32), which occur in multiple molecular forms due to proteolytic...

Porphyromonas gingivalis, a bacterium associated with active chronic periodontitis lesions, produces several proteolytic enzymes that are thought to be involved in host colonization, perturbation of the immune system, and tissue destruction. The aim of the present study was to investigate the contribution of Arg-and Lysgingipains produced by P. gingivalis to its growth. Although all of the proteins studied were degraded by P. gingivalis, only human serum albumin and transferrin supported growth during serial transfers in a chemically defined medium (CDM). Growth studies with site-directed gingipain-deficient mutants of P. gingivalis revealed that inactivation of both gingipains prevents growth, whereas inactivation of either Arg-or Lys-gingipain activity extended the doubling times to 33 or 13 h, respectively, compared to 9 h for the parent strain. Growth of the mutants and the parent strain was similar when the CDM was supplemented with a protein hydrolysate instead of human serum albumin. Incubation of resting P. gingivalis ATCC 33277 cells with fluorophore-labeled albumin indicated that the proteolytic fragments generated by the gingipains were internalized by the bacterial cells. Internalization of fluorophore-labeled albumin fragments was reduced or completely inhibited in the proteinase-deficient mutants. Interestingly, gingival crevicular fluid samples from diseased periodontal sites contained low-molecular-mass albumin fragments, whereas samples from healthy sites did not. The critical role of proteinases in the growth of P. gingivalis was further investigated using specific Arg-and Lys-gingipain inhibitors. Adding the inhibitors to CDM containing albumin revealed that leupeptin (Arg-gingipain A and B inhibitor) was more efficient at inhibiting growth than cathepsin B inhibitor II (Lys-gingipain inhibitor). Our study suggests that Arg-gingipains and, to a lesser extent, Lys-gingipain play an important role in the growth of P. gingivalis in a defined medium containing a human protein as the sole carbon and nitrogen source.Gram-negative anaerobic bacteria play an important role in the initiation and progression of periodontitis. More particularly, Porphyromonas gingivalis has been strongly associated with active chronic periodontitis lesions (24). This bacterial species produces several proteinases that are thought to be involved in host colonization, perturbation of the immune system, and tissue destruction (10,12,15). Most of the proteolytic activity exhibited by P. gingivalis is due to Arg-and Lys-gingipain cysteine proteinases (3,12,15). Two different genes code for the arginine-X (Arg-gingipain A [rgpA] and B [rgpB])-specific cysteine proteinases, whereas one gene codes for the lysine-X (Lys-gingipain [kgp])-specific cysteine proteinase (3). By cleaving a variety of host proteins, these proteinases may provide small peptides and amino acids that meet the nutritional requirements of P. gingivalis (7,22) and may thus participate in the pathogenic process of periodontitis. The critical role of P. gingiva...

Prior tumor cell studies have shown that the drugs sorafenib (Nexavar) and regorafenib (Stivarga) reduce expression of the chaperone GRP78. Sorafenib/regorafenib and the multi-kinase inhibitor pazopanib (Votrient) interacted with sildenafil (Viagra) to further rapidly reduce GRP78 levels in eukaryotes and as single agents to reduce Dna K levels in prokaryotes. Similar data were obtained in tumor cells in vitro and in drug-treated mice for: HSP70, mitochondrial HSP70, HSP60, HSP56, HSP40, HSP10, and cyclophilin A. Prolonged ‘rafenib/sildenafil treatment killed tumor cells and also rapidly decreased the expression of: the drug efflux pumps ABCB1 and ABCG2; and NPC1 and NTCP, receptors for Ebola/Hepatitis A and B viruses, respectively. Pre-treatment with the ‘Rafenib/sildenafil combination reduced expression of the Coxsackie and Adenovirus receptor in parallel with it also reducing the ability of a serotype 5 Adenovirus or Coxsackie virus B4 to infect and to reproduce. Sorafenib/pazopanib and sildenafil was much more potent than sorafenib/pazopanib as single agents at preventing Adenovirus, Mumps, Chikungunya, Dengue, Rabies, West Nile, Yellow Fever, and Enterovirus 71 infection and reproduction. ‘Rafenib drugs/pazopanib as single agents killed laboratory generated antibiotic resistant E. coli which was associated with reduced Dna K and Rec A expression. Marginally toxic doses of ‘Rafenib drugs/pazopanib restored antibiotic sensitivity in pan-antibiotic resistant bacteria including multiple strains of blakpc Klebsiella pneumoniae. Thus, Dna K is an antibiotic target for sorafenib, and inhibition of GRP78/Dna K has therapeutic utility for cancer and for bacterial and viral infections.

Data from this study suggest that Doxy and CMTs have the potential to inhibit the periodontopathogenic bacterial proteinases, which contribute to tissue destruction cascades during periodontitis directly and indirectly by triggering the host response.

Degradation of type I collagen by Porphyromonas gingivalis was monitored by fluorogenic, sodium dodecyl sulfate^polyacrylamide gel electrophoresis (SDS^PAGE), and growth assays. All three assays showed that inactivation of both the rgpA and rgpB genes was necessary to completely eliminate the capacity of P. gingivalis to cleave type I collagen. Leupeptin, an Arg-gingipain-specific protease inhibitor, almost completely inhibited collagen degradation by P. gingivalis cells whereas cathepsin B inhibitor II, a Lys-gingipain inhibitor, did not. A purified preparation of Arg-gingipains A and B hydrolyzed gelatin but did not cleave type I collagen, suggesting that the enzymes must be attached to the cell surface to exert collagenase activity. A number of substances used as adjuncts in periodontal therapy were also tested for their capacity to inhibit collagenase activity of P. gingivalis. Tetracycline, doxycycline, and chlorhexidine strongly inhibited collagenase activity.

Although Moraxella catarrhalis continues to be a significant cause of disease in both children and adults, the steps involved in pathogenesis remain poorly understood. We have identified three open reading frames in the M. catarrhalis genome that encode homologues of the two-partner secretion system (TPS) Moraxella catarrhalis is an important gram-negative human mucosal pathogen. It is one of the three major causes of acute otitis media, along with Streptococcus pneumoniae and nontypeable Haemophilus influenzae (10, 43). In adults with chronic bronchitis and chronic obstructive pulmonary disease (COPD), M. catarrhalis causes lower respiratory tract infections that often lead to acute exacerbations (33, 34). In addition, M. catarrhalis can cause sinusitis in infants and young children (23,43). Because 90% of M. catarrhalis clinical isolates are beta-lactamase positive (23,43) and no protective vaccine is available (29, 30), M. catarrhalis continues to be a major source of human disease..Most of the research involving M. catarrhalis pathogenesis has focused largely on the identification and characterization of outer membrane proteins (OMPs) on the bacterial surface, although most of these have an undefined role in virulence (23,32,43). M. catarrhalis expresses some OMPs, including the transferrin binding protein TbpB (28) and the hemin and hemoglobin utilization proteins HumA and MhuA (11,12), to obtain iron from the human host. In addition, other OMPs, such as the ubiquitous surface protein UspA2, can be involved in serum resistance (1) or in natural competence, as described for the type IV pilus (26). To date, only a few OMPs, including UspA1 and UspA2H (1, 24), the M. catarrhalis adherence protein McaP (41), and the hemagglutinating protein Hag/M. catarrhalis immunoglobulin D-binding protein (3,14,19), have been reported to directly mediate binding to cell lines in vitro. Therefore, it is clear that like many other gram-negative pathogens, M. catarrhalis has developed multiple virulence mechanisms to successfully colonize the human mucosal surface.In this report, we have identified a locus in M. catarrhalis 7169 containing three open reading frames (ORFs) that encode homologues of the previously described two-partner secretion systems (TPS) in various other pathogens, including Bordetella pertussis (21,22). The B. pertussis TPS pathway is composed of the filamentous hemagglutinin FhaB (generically named TpsA) and the transporter FhaC (TpsB) (25). FhaB is the major adhesin involved in bacterial attachment and colonization of the human upper respiratory tract, and this protein is also a component of the acellular diphtheria-pertussis vaccine (25, 36). The M. catarrhalis hemagglutinin-like locus described in this study contains three ORFs, termed mchA1, mchA2, and mchB. The TPS motif identified in MchA1 and MchA2 was found to be homologous to FhaB of B. pertussis. MchB has homology to FhaC of B. pertussis (22). This is the first report of a TPS in M. catarrhalis, and our data demonstrate that this system is likely...

In this study, we investigated the ability of Fusobacterium nucleatum subsp. nucleatum to increase its tissue-invasive potential by acquiring cell-associated human matrix metalloproteinase 9 (MMP-9) activity. Binding of pro-MMP-9 to fusobacteria was demonstrated by enzyme-linked immunosorbent assay. Zymography and a colorimetric assay showed that bound pro-MMP-9 can be converted into a proteolytically active form. The potential contribution of this acquired host activity in tissue invasion was demonstrated using a reconstituted basement membrane (Matrigel).