Up-regulation of sortase-dependent pili in Bifidobacterium longum BBMN68 in response to bile stress enhances its adhesion to HT-29 cells.

Adhesion to gastrointestinal tract is crucial for bifidobacteria to exert their probiotic effects. Our previous work found that bile salts significantly enhance the adhesion ability of Bifidobacterium longum BBMN68 to HT-29 cells. In this study, trypsin-shaving and LC-MS/MS-based surface proteomics were employed to identify surface proteins involved in bile stress response. Among the 829 differentially expressed proteins, 56 up-regulated proteins with a fold change >1.5 were subjected to further analysis. Notably, the minor pilin subunit FimB was 4.98-fold up-regulated in response to bile stress. In silico analysis and RT-PCR confirmed that gene fimB, fimA and srtC were co-transcribed and contributed to the biosynthesis of sortase-dependent pili Pil1. Moreover, scanning electron microscopy and immunogold electron microscopy assays showed increased abundance and length of Pil1 on BBMN68 under bile stress. As the major pilin subunit FimA serves as adhesion component of Pil1, an inhibition assay using anti-FimA antibodies further confirmed the critical role of Pil1 in mediating the adhesion of BBMN68 to HT-29 cells under bile stress. Our findings suggest that the up-regulation of Pil1 in response to bile stress enhances the adhesion of BBMN68 to intestinal epithelial cells, highlighting a novel mechanism of gut persistence in B. longum strains.

Safety, pharmacokinetic, and pharmacodynamic study of sibofimloc, a novel FimH blocker in patients with active Crohn's disease.

BACKGROUND AND AIM: Expression of FimH adhesin by invasive Escherichia coli in the gastrointestinal tract of patients with Crohn's disease (CD) facilitates binding to epithelial glycoproteins and release of pro-inflammatory cytokines. Sibofimloc is a first-in-class FimH blocker that showed little systemic absorption in healthy volunteers. The current study evaluated systemic absorption, safety, and effect on inflammatory biomarkers of sibofimloc in patients with CD. METHODS: This was an open-label, multicenter phase 1b study in adults with active CD. In part 1, two patients received a single oral dose of 3000-mg sibofimloc followed by 1500 mg b.i.d. for 13 days. In part 2, six patients received 1500-mg sibofimloc b.i.d. for 13 days. Blood was drawn for pharmacokinetic and biomarker analysis, and stool was collected for biomarker and microbiome analysis. RESULTS: Eight patients with active ileal or ileocolonic CD were enrolled into the study. Systemic sibofimloc exposure was low. Sibofimloc was well tolerated with only grade 1-2 events observed. Several pro-inflammatory biomarkers, including IL-1ß, IL-6, IL-8, TNF-α, IFN-γ, and calprotectin, were decreased in stool by end of study. CONCLUSIONS: This first study of the novel FimH blocker, sibofimloc, in patients with active CD demonstrated minimal systemic exposure with good tolerance, while decreasing several inflammatory biomarkers. EudraCT number: 2017-003279-70.

FimH as a mucosal adjuvant enhances persistent antibody response and protective efficacy of the anti-caries vaccine.

OBJECTIVE: To investigate whether the recombinant FimH-S.T protein could modulate immune response to anti-caries vaccine in vitro and in vivo. DESIGN: Recombinant FimH protein derived from Salmonella was constructed and purified. The expression of dendritic cell maturation markers and cytokines release were performed by flow cytometry, Real-time PCR and ELISA. In addition, BALB/c mice were administered with anti-caries PAc vaccine plus FimH-S.T, antibody responses were evaluated by ELISA. Splenocytes of immunized mice were detected for their proliferative ability in response to in vitro retreatment with PAc antigen by flow cytometry. Caries protection against dental caries formation was also investigated. RESULTS: The purified FimH-S.T induced phenotypic maturation of DC2.4 by up-regulating the expression of costimulatory molecules and MHC II, provoked the production and secretion of cytokines via TLR4-dependent signaling pathway in vitro. Furthermore, the mice immunized with the mixture of FimH-S.T and PAc significantly enhanced the PAc-specific antibodies in the serum along with saliva and promoted splenocyte proliferation. Our results also confirmed that PAc+FimH-S.T decreased the caries lesions formation which provided high protective efficacy against dental caries. CONCLUSION: Our study demonstrates that recombinant FimH-S.T could enhance specific IgA responses and protection of anti-caries vaccine, possessing mucosal adjuvant ability by activating DC2.4 via TLR4 signaling pathway.

Recombinant fimbriae protein of Porphyromonas gingivalis induces an inflammatory response via the TLR4/NF­κB signaling pathway in human peripheral blood mononuclear cells.

Porphyromonas gingivalis (P. gingivalis) is a periodontal pathogen that may accumulate with other organisms in subgingival plaque biofilms and is associated with periodontal disease. P. gingivalis fimbriae (FimA) is a filamentous structure on the surface of bacteria that is closely associated with bacterial adhesion to and colonization of host tissues, and serves an essential role in biofilm formation. The present study aimed to construct P. gingivalis FimA prokaryotic expression plasmids, purify a FimA fusion protein and explore the effect of a recombinant FimA protein on the inflammatory response in human peripheral blood mononuclear cells (PBMCs). P. gingivalis FimA prokaryotic expression plasmids were constructed by gene cloning and recombination technology. SDS­PAGE was used to evaluate the purified recombinant FimA protein. The cell proliferation rate and inflammatory cytokine expression of PBMCs treated with the FimA fusion protein with or without transfection with toll­like receptor 4 (TLR4) small interfering (si)RNA were detected by CCK­8 assays and ELISAs, respectively. The expression levels of TLR4, nuclear factor kappa­light­chain­enhancer of activated B cells (NF­κB) and myeloid differentiation primary response 88 (MyD88) in PBMCs were detected by western blot analysis and reverse transcription quantitative polymerase chain reaction. A FimA fusion protein with high purity was obtained. FimA fusion protein treatment significantly increased PBMC proliferation and promoted the release of tumor necrosis factor­α (TNF­α), interleukin (IL)­6, matrix metalloproteinase (MMP)­8 and MMP­9 in PBMCs. TLR4 interference reversed the effects of the FimA fusion protein on PBMC proliferation and inflammatory cytokine release. The expression levels of TLR4, NF­κB and MyD88 in PBMCs were significantly increased following treatment with the FimA fusion protein, while the expression levels of these genes at the mRNA and protein levels decreased significantly in PBMCs following FimA fusion protein treatment and TLR4 interference. The FimA fusion protein increased PBMC proliferation and promoted the release of the inflammatory cytokines TNF­α, IL­6, MMP­8 and MMP­9 via the TLR4/NF­κB signaling pathway. FimA may serve as a promising therapeutic strategy for periodontal disease.

Immunization with the MipA, Skp, or ETEC_2479 Antigens Confers Protection against Enterotoxigenic E. coli Strains Expressing Different Colonization Factors in a Mouse Pulmonary Challenge Model.

Achieving cross-protective efficacy against multiple bacterial strains or serotypes is an important goal of vaccine design. Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrheal disease in underdeveloped nations. We have been interested in identifying and characterizing ETEC antigens that generate protective immune responses independent of ETEC colonization factor (CF) expression. Our previous studies used proteomics to identify the ETEC MipA, Skp, and ETEC_2479 proteins as effective in protecting mice from homologous challenge with ETEC H10407 using a pulmonary inoculation model. This model permits analysis of mouse survival, bacterial clearance, and the production of secretory IgA (sIgA) and has been employed previously for studies of enteric pathogens for which robust oral challenge models do not exist. MipA belongs to a family of proteins involved in remodeling peptidoglycan. Skp rescues misdirected outer membrane proteins. ETEC_2479 is predicted to function as an outer membrane porin. These proteins are conserved in pathogenic ETEC strains as well as in commensal Proteobacteria. Antibodies produced against the ETEC MipA, Skp, and ETEC_2479 proteins also reduced the adherence of multiple ETEC strains differing in CF type to intestinal epithelial cells. Here we characterized the ability of 10 heterologous ETEC strains that differ in CF type to cause clinical signs of illness in mice after pulmonary challenge. ETEC strains C350C1A, E24377A, E7476A, WS2173A, and PE360 caused variable degrees of lethality in this mouse model, while ETEC strains B7A, WS6866B, 2230, ARG-2, and 8786 did not. Subsequent challenge experiments in which mice were first vaccinated intranasally with MipA, Skp, or ETEC_2479, when combined with cholera toxin, showed both that each antigen was protective and that protection was strongly correlated with fecal IgA concentrations. We conclude that the MipA, Skp, or ETEC_2479 antigens generate protection in the mouse pulmonary challenge model against ETEC strains that express different CFs.

PA-MSHA inhibits the growth of doxorubicin-resistant MCF-7/ADR human breast cancer cells by downregulating Nrf2/p62.

Acquired resistance to doxorubicin in breast cancer is a serious therapeutic problem. In this study, we investigated whether Pseudomonas aeruginosa mannose-sensitive hemagglutinin (PA-MSHA) could inhibit the growth of doxorubicin-resistant breast cancer cells. We found that the expressions of Nrf2 and p62 in breast cancer were higher than that in the corresponding adjacent normal tissues and benign breast epithelial cell. The expressions of Nrf2 and p62 in breast cancer doxorubicin-resistant cells MCF-7/ADR were higher than that in doxorubicin-sensitive cells MCF-7. Silencing of Nrf2 or p62 rendered breast cancer cells more susceptible to doxorubicin. We further demonstrated that PA-MSHA inhibited growth and induced apoptosis of MCF-7/ADR cells but not MCF-7 cells. Subcutaneous administration of PA-MSHA greatly inhibited the growth of xenograft tumors from MCF-7/ADR cells in nude mice. In addition, PA-MSHA could downregulate Nrf2 and p62 in vitro and in vivo. These results suggested that activation of Nrf2 and p62 was associated with doxorubicin resistance in breast cancer. PA-MSHA could inhibit the growth of doxorubicin-resistant MCF-7/ADR cells and its potential mechanism might be due to the suppression of Nrf2/p62. It indicated the possibility of using PA-MSHA in doxorubicin-resistant breast cancer.

Pseudomonas aeruginosa-mannose-sensitive hemagglutinin inhibits pancreatic cancer cell proliferation and induces apoptosis via the EGFR pathway and caspase signaling.

Pseudomonas aeruginosa-mannose-sensitive hemagglutinin (PA-MSHA) has demonstrated efficacy against several solid tumors. In this study, we found that PA-MSHA inhibited the proliferation of PANC-1 and SW1990 pancreatic cancer cells, but had no obvious effects on HPDE6-C7 normal human pancreatic duct epithelial cells. Electron microscopy revealed the presence of apoptotic bodies and intracellular vacuole formation in PA-MSHA-treated pancreatic cancer cells. Flow cytometric analysis indicated the rate of apoptosis correlated with the PA-MSHA concentration. We observed a decrease in cell fractions in G0/G1 and G2/M phases, and an increase in the fraction in S phase (p < 0.01). PA-MSHA thus caused cell cycle arrest. Increasing concentrations of PA-MSHA did not alter total levels of EGFR, AKT or ERK, but levels of the corresponding phosphoproteins decreased. PA-MSHA also reduced tumor volume in a xenograft mouse model of pancreatic cancer (p < 0.01). Furthermore, caspase-3 levels decreased while the levels of cleaved caspase-3 increased (p < 0.01). These data suggest that by blocking cell cycle progression, PA-MSHA induces apoptosis and inhibits tumor growth. PA-MSHA-mediated inhibition of EGFR signaling and activation of the caspase pathway may play an important role in the induction of apoptosis in pancreatic cancer cells.

PA-MSHA inhibits proliferation and induces apoptosis in human non-small cell lung cancer cell lines with different genotypes.

The present study examined the potential of Pseudomonas aeruginosa-mannose sensitive hemagglutinin (PA-MSHA) to inhibit proliferation and induce apoptosis in non­small­cell lung cancer (NSCLC) cell lines. It also investigated its mechanisms of action in different genotypes of human NSCLC. A total of three NSCLC cell lines, PC­9, A549, and NCI­H1975, were treated with PA­MSHA at different concentrations. The anti­proliferative effect of PA­MSHA was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell cycle distribution and apoptosis induced by the treatment were measured by flow cytometry (FCM) with Annexin V/propidium iodide staining. Western blotting was conducted to determine the expression level of apoptosis­associated proteins. PA­MSHA was demonstrated to exert a time­ and concentration­dependent cytotoxic effect in PC­9, A549, and NCI­H1975 cells. The FCM indicated that all the different concentrations of PA­MSHA used in the present study induce apoptosis and cell cycle arrest of NSCLC cells. Treatment with PA­MSHA may exert anti­proliferative effects on NSCLC cells by affecting regulation of the cell cycle and inducing apoptosis that is mediated in part by an intrinsic apoptosis signaling pathway. These data suggest that PA­MSHA has the potential to inhibit proliferation and induce apoptosis in NSCLC cells. Furthermore, these data provide mechanistic details for the potential application of PA­MSHA­based therapeutic strategies for the treatment of different NSCLC genotypes. This present study suggests potential novel strategies to maximize effective therapeutic strategies targeting anti­epidermal growth factor receptor for future clinical trials.

Protective effect of pilin protein with alum+naloxone adjuvant against acute pulmonary Pseudomonas aeruginosa infection.

Pseudomonas aeruginosa is an important opportunistic human pathogen that causes a wide variety of severe nosocomial infections. Type IV pili of P. aeruginosa are made up of polymerized pilin that aids in bacterial adhesion, biofilm formation and twitching motility. The aim of this study was to evaluate the efficacy of alum and naloxone (alum+NLX) as an adjuvant for P. aeruginosa recombinant PilA (r-PilA) as a vaccine candidate in the improvement of humoral and cellular immunity. Primary immunization with r-PilA in combination with alum+NLX followed by two booster shots was sufficient to generate robust cellular and humoral responses, which were Th1 and Th2 type responses consisting of IgG1 and IgG2a subtypes. Analysis of the cytokine response among immunized mice showed an increased production of IL-4, INF-γ and IL-17 by splenocytes upon stimulation by r-PilA. These sera were also able to reduce bacterial load in the lung tissue of challenged mice. The reduction of systemic bacterial spread resulted in increased survival rates in challenged immunized mice. In conclusion, immunization with r-PilA combined with alum+NLX evokes cellular and humoral immune responses, which play an important role in providing protection against acute P. aeruginosa lung infection among immunized mice.

Use of flagellin and cholera toxin as adjuvants in intranasal vaccination of mice to enhance protective immune responses against uropathogenic Escherichia coli antigens.

Urinary tract infections (UTIs) caused by Uropathogenic Escherichia coli (UPEC) are among the most common infections in human. Antibiotics are common therapy for UTIs, but increase in antibiotic resistance will complicate future treatment of the infections, making the development of an efficacious UTI vaccine more urgent. In this study, we have evaluated intranasally the efficacy of FliC and FimH antigens of UPEC in different vaccine formulations with and without cholera toxin (CT) adjuvant. Immunization of mice with FliC in fusion form or admixed with FimH elicited higher levels of serum, mucosal and cell-mediated responses than FimH alone. Furthermore, the use of CT in synergism with FliC resulted in the stimulation of a mixed Th1 and Th2 responses against FimH and FliC as antigen and maintained the antibody responses for at least 24 weeks following the last vaccine dose. Of the vaccine preparations, Fusion, Fusion + CT, and FimH admixed with FliC and CT showed the best protection against UPEC. These data indicated that intranasal administration of a FliC and CT adjuvant-based vaccine has the potential to provide protective responses against UPEC strains.

PA-MSHA in combination with EGFR tyrosine kinase inhibitor: A new strategy to overcome the drug resistance of non-small cell lung cancer cells.

The inhibition of epidermal growth factor receptor (EGFR) signaling by Gefitinib provides a promising treatment strategy for non-small cell lung cancer (NSCLC); however, drug resistance to Gefitinib and other tyrosine kinase inhibitors presents a major issue. Using NSCLC cell lines with differential EGFR status, we examined the potency of PA-MSHA (Pseudomonas aeruginosa-mannose-sensitive hemagglutinin) in combination with Gefitinib on proliferation, apoptosis, cell cycle arrest, EGFR signaling and tumor growth. PC-9, A549, and NCI-H1975 cells were treated with PA-MSHA, Gefetinib, or PA-MSHA plus Gefetinib at different concentrations and times. The effects of the drugs on proliferation, cell cycle distribution and apoptosis were evaluated. The activation of EGFR and apoptotic signaling-related molecules was evaluated by Western blotting in the presence or absence of EGFR siRNA. Tumor growth and pathway signaling activation was assessed by xenografts in nude mice. A time-dependent and concentration-dependent cytotoxic effect of PA-MSHA was observed in all NSCLC cells tested. The combination of PA-MSHA plus Gefitinib enhanced the growth inhibition, sub-G1 content and apoptosis over that observed with either agent alone. Furthermore, the combination of PA-MSHA plus Gefitinib resulted in caspase-3/caspase-9 cleavage and increased inhibition of EGFR-dependent activation of AKT and ERK phosphorylation. Combination treatment was more effective in reducing tumor size and EGFR activation than either agent alone. These data suggest that PA-MSHA and Gefitinib function additively to suppress the proliferative effects of NSCLC cells of differential EGFR status. The combination of PA-MSHA and Gefitinib provides a potential new strategy to conquer drug resistance for anti-EGFR-targeted therapy of NSCLC.

Pseudomonas aeruginosa mannose-sensitive hemagglutinin inhibits proliferation and invasion via the PTEN/AKT pathway in HeLa cells.

We investigated the effects of Pseudomonas aeruginosa mannose-sensitive hemagglutinin (PA-MSHA) on the proliferation and invasion of human cervical cancer cell lines, as well as the molecular pathways underlying these effects. MTT cell proliferation assays revealed a time- and concentration-dependent cytotoxic effect of PA-MSHA on HeLa cells but not H8 cells. Flow cytometry with propidium iodide and annexin-V-fluorescein isothiocyanate labeling (FITC) indicated that various concentrations of PA-MSHA could induce apoptosis and G2-M cell cycle arrest in HeLa cells. PA-MSHA also impaired the migration and invasion abilities of HeLa cells in Wound healing and Transwell invasion assays. Western blot results demonstrated that PA-MSHA reduced the expression of p-AKT, p-GSK3ß, BCL-2, Vimentin and ß-catenin, but increased the levels of PTEN, BAD, BAX and E-cadherin in HeLa cells. Importantly, PTEN siRNA induced the activity of p-AKT, while PA-MSHA partly inhibited this induction, indicating that PA-MSHA may reduce the cell proliferation and invasion potential by activating PTEN and thus inhibiting the AKT pathway in vitro. These data suggest the potential application of PA-MSHA to the treatment of human cervical cancer.

Reduction of Salmonella Enteritidis in the spleens of hens by bacterins that vary in fimbrial protein SefD.

The objective of this research was to determine whether variation in the presence of fimbrial protein SefD would impact efficacy of bacterins as measured by recovery of Salmonella enterica serovar Enteritidis (Salmonella Enteritidis) from the spleens of hens. Two bacterins were prepared that varied in SefD content. Also, two adjuvants were tested, namely, water-in-oil and aluminum hydroxide gel (alum). Control groups for both adjuvant preparations included infected nonvaccinated hens and uninfected nonvaccinated hens. At 21 days postinfection, Salmonella Enteritidis was recovered from 69.7%, 53.1%, and 86.0% from the spleens of all hens vaccinated with bacterins lacking SefD, bacterins that included SefD, and infected nonvaccinated control hens, respectively. No Salmonella was recovered from uninfected nonvaccinates. Results from individual trials showed that both bacterins reduced positive spleens, but that the one with SefD was more efficacious. Alum adjuvant had fewer side effects on hens and egg production as compared to water-in-oil. However, adjuvant did not change the relative recovery of Salmonella Enteritidis from spleens. These results suggest that SefD is a promising target antigen for improving the efficacy of immunotherapy in hens, and is intended to reduce Salmonella Enteritidis in the food supply.

Mannose-mediated inhibitory effects of PA-MSHA on invasion and metastasis of hepatocellular carcinoma via EGFR/Akt/IκBß/NF-κB pathway.

BACKGROUND & AIMS: Elevation of high-mannose glycans is a common feature of malignant cells and has been suggested to be the basis for alternative cancer therapy for several years. Here we want to investigate the antitumour effect of pseudomonas aeruginosa-mannosesensitive haemagglutinin (PA-MSHA), a genetically engineered heat-inactivated PA strain with mannose-sensitive binding activity, on hepatocellular carcinoma (HCC). METHODS: Tumourigenicity and metastatic potentials of HCC were studied after PA-MSHA treatment by utilizing the in vitro/in vivo model of HCC. Expression of apoptosis-associated proteins and epithelial-mesenchymal transition (EMT) related genes were evaluated, and possible signalling pathways involved were investigated. RESULTS: PA-MSHA induced significant cell proliferation inhibition and cell cycle arrest of HCC through decreasing the levels of cyclins D1, cyclins E, CDK2, CDK4, proliferating cell nuclear antigen (PCNA), and increasing the level of p21 and p27. Moreover, PA-MSHA suppressed the invasion, migration and adhesion of HCC through inhibiting epithelial-mesenchymal transition (EMT). PA-MSHA also inhibited EGFR/Akt/IκBß/NF-κB pathway and overexpression of NF-κB significantly abrogated PA-MSHA induced EMT inhibition. In addition, competitive inhibition of the mannose binding activity of PA-MSHA by D-mannose significantly blocked its effect on cell cycle arrest and EMT. PA-MSHA also abrogated lung metastasis of HCC and significantly inhibited tumour growth in the in vivo study. CONCLUSIONS: Our study demonstrated the essential role of EGFR/Akt/IκBß/NF-κB pathway in the inhibitory effect of PA-MSHA on invasion and metastasis of HCC through suppressing EMT, and revealed an attractive prospect of PA-MSHA as a novel candidate agent in the treatment of HCC.

Cytotoxic effects of Kingella kingae outer membrane vesicles on human cells.

Kingella kingae is an emerging pathogen causing osteoarticular infections in pediatric patients. Electron microscopy of K. kingae clinical isolates revealed the heterogeneously-sized membranous structures blebbing from the outer membrane that were classified as outer membrane vesicles (OMVs). OMVs purified from the secreted fraction of a septic arthritis K. kingae isolate were characterized. Among several major proteins, K. kingae OMVs contained virulence factors RtxA toxin and PilC2 pilus adhesin. RtxA was also found secreted as a soluble protein in the extracellular environment indicating that the bacterium may utilize different mechanisms for the toxin delivery. OMVs were shown to be hemolytic and possess some leukotoxic activity while high leukotoxicity was detected in the non-hemolytic OMV-free component of the secreted fraction. OMVs were internalized by human osteoblasts and synovial cells. Upon interaction with OMVs, the cells produced increased levels of human granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 6 (IL-6) suggesting that these cytokines might be involved in the signaling response of infected joint and bone tissues during natural K. kingae infection. This study is the first report of OMV production by K. kingae and demonstrates that OMVs are a complex virulence factor of the organism causing cytolytic and inflammatory effects on host cells.

The hemorrhagic coli pilus (HCP) of Escherichia coli O157:H7 is an inducer of proinflammatory cytokine secretion in intestinal epithelial cells.

BACKGROUND: Enterohemorrhagic Escherichia coli (EHEC) O157:H7, the causative agent of hemorrhagic colitis and the hemolytic uremic syndrome (HUS), produces long bundles of type IV pili (TFP) called hemorrhagic coli pili (HCP). HCP are capable of mediating several phenomena associated with pathogenicity: i) adherence to human and bovine epithelial cells; ii) invasion of epithelial cells; iii) hemagglutination of rabbit erythrocytes; iv) biofilm formation; v) twitching motility; and vi) specific binding to laminin and fibronectin. HCP are composed of a 19 kDa pilin subunit (HcpA) encoded by the hcpA chromosomal gene (called prepilin peptidase-dependent gene [ppdD] in E. coli K-12). METHODOLOGY/PRINCIPAL FINDINGS: In this study we investigated the potential role of HCP of E. coli O157:H7 strain EDL933 in activating the release of pro- and anti-inflammatory cytokines from a variety of host epithelial cells. We found that purified HCP and a recombinant HcpA protein induced significant release of IL-8 and TNF-alpha, from cultured polarized intestinal cells (T84 and HT-29 cells) and non-intestinal HeLa cells. Levels of proinflammatory IL-8 and TNF-alpha, but not IL-2, IL6, or IL-10 cytokines, were increased in the presence of HCP and recombinant HcpA after 6 h of incubation with >or=50 ng/ml of protein, suggesting that stimulation of IL-8 and TNF-alpha are dose and time-dependent. In addition, we also demonstrated that flagella are potent inducers of cytokine production. Furthermore, MAPK activation kinetics studies showed that EHEC induces p38 phosphorylation under HCP-producing conditions, and ERK1/2 and JNK activation was detectable after 3 h of EHEC infection. HT-29 cells were stimulated with epidermal growth factor stimulation of HT-29 cells for 30 min leading to activation of three MAPKs. CONCLUSIONS/SIGNIFICANCE: The HcpA pilin monomer of the HCP produced by EHEC O157:H7 is a potent inducer of IL-8 and TNF-alpha release, an event which could play a significant role in the pathogenesis of hemorrhagic colitis caused by this pathogen.

FimH can directly activate human and murine natural killer cells via TLR4.

Although the importance of natural killer (NK) cells in innate immune responses against tumors or viral infections are well documented, their ability to directly recognize pathogens is less well defined. We have recently reported FimH, a bacterial fimbrial protein, as a novel Toll-like receptor (TLR)4 ligand that potently induces antiviral responses. Here, we investigated whether FimH either directly or indirectly can activate human and murine NK cells. We demonstrate that FimH potently activates both human and murine NK cells in vitro to induce cytokines [interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha] and cytotoxicity. Importantly, NK cells directly recognize FimH-expressing pathogens as FimH(+), but not FimH(-), bacteria were able to activate human NK cells. FimH activation of NK cells required TLR4 and MyD88 signaling, as NK cells from both TLR4(-/-) and MyD88(-/-) mice as well as human NK-92 cells, which lack TLR4, were all unresponsive to FimH. In addition, TLR4 neutralization significantly abrogated the response of human NK cells to FimH. Activation of purified NK cells by FimH was independent of lipopolysaccharide (LPS) or other bacterial contaminations. These data demonstrate for the first time that highly purified NK cells directly recognize and respond to FimH via TLR4-MyD88 pathways to aid innate protection against cancer or microbial infections.

Proteomic mapping of stimulus-specific signaling pathways involved in THP-1 cells exposed to Porphyromonas gingivalis or its purified components.

Periodontitis is an inflammatory disease initiated by host-parasite interactions which contributes to connective tissue destruction and alveolar bone resorption. Porphyromonas gingivalis (P.g.), a black-pigmented Gram-negative anaerobic bacterium, is a major pathogen in the development and progression of periodontitis. To characterize the role that P. gingivalis and its cell surface components play in disease processes, we investigated the differential expression of proteins induced by live P.g., P.g. LPS, and P.g. FimA, using two-dimensional gel electrophoresis in combination with mass spectrometry. We have tested whether, at the level of protein expression, unique signaling pathways are differentially induced by the bacterial components P.g. LPS and P.g. FimA, as compared to live P.g. We found that P.g. LPS stimulation of THP-1 up-regulated the expression of a set of proteins compared to control: deoxyribonuclease, actin, carbonic anhydrase 2, alpha enolase, adenylyl cyclase-associated protein (CAP1), protein disulfide isomerase (PDI), glucose regulated protein (grp78), and 70-kDa heat shock protein (HSP70), whereas FimA treatment did not result in statistically significant changes to protein levels versus the control. Live P.g. stimulation resulted in 12 differentially expressed proteins: CAP1, tubulin beta-2 chain, ATP synthase beta chain, tubulin alpha-6 chain, PDI, vimentin, 60-kDa heat shock protein, and nucleolin were found to be up-regulated, while carbonic anhydrase II, beta-actin, and HSP70 were down-regulated relative to control. These differential changes by the bacteria and its components are interpreted as preferential signal pathway activation in host immune/inflammatory responses to P.g. infection.

Characterization of the type 3 fimbriae with different MrkD adhesins: possible role of the MrkD containing an RGD motif.

Four novel mrkD alleles namely mrkD(V1), mrkD(V2), mrkD(V3), and mrkD(V4) were identified in seventeen Klebsiella pneumoniae meningitis strains using PCR-RFLP and sequence determination. Comparative analysis revealed a most variable region containing an RGD motif in the receptor domain of MrkD(V3). In order to determine if the sequence confers the K. pneumoniae mrkD(V3) the highest level of the fimbrial activity, a type 3 fimbriae display system was constructed in Escherichia coli. The E. coli JM109[pmrkABCD(V3)F] displaying meshwork-like fimbriae also had the most fimbrial activity, supporting a possible role of the varied sequences. In a dose-dependent manner, the GRGDSP hexapeptide appeared to inhibit the adhesion of the E. coli JM109[pmrkABCD(V3)F] to HCT-8, an ileocecal epithelial cell line. In addition, the adhesion activity was reduced by the addition of anti-alpha5beta1 integrin monoclonal antibody, indicating that the RGD containing region in MrkD(V3) is responsible for the binding of type 3 fimbriae to integrin.

The cytotoxic fimbrial structural subunit of Xenorhabdus nematophila is a pore-forming toxin.

We have purified a fimbrial shaft protein (MrxA) of Xenorhabdus nematophila. The soluble monomeric protein lysed larval hemocytes of Helicoverpa armigera. Osmotic protection of the cells with polyethylene glycol suggested that the 17-kDa MrxA subunit makes pores in the target cell membrane. The internal diameter of the pores was estimated to be >2.9 nm. Electron microscopy confirmed the formation of pores by the fimbrial subunit. MrxA protein oligomerized in the presence of liposomes. Electrophysiological studies demonstrated that MrxA formed large, voltage-gated passive-diffusion channels in lipid bilayers.