Longus, a type IV pilus of enterotoxigenic Escherichia coli, is involved in adherence to intestinal epithelial cells.

Enterotoxigenic Escherichia coli (ETEC) is the leading bacterial cause of diarrhea in the developing world, as well as the most common cause of traveler's diarrhea. The main hallmarks of this type of bacteria are the expression of one or more enterotoxins and fimbriae used for attachment to host intestinal cells. Longus is a pilus produced by ETEC. These bacteria grown in pleuropneumonia-like organism (PPLO) broth at 37 degrees C and in 5% CO(2) produced longus, showing that the assembly and expression of the pili depend on growth conditions and composition of the medium. To explore the role of longus in the adherence to epithelial cells, quantitative and qualitative analyses were done, and similar levels of adherence were observed, with values of 111.44 x 10(4) CFU/ml in HT-29, 101.33 x 10(4) CFU/ml in Caco-2, and 107.11 x 10(4) CFU/ml in T84 cells. In addition, the E9034A Delta lngA strain showed a significant reduction in longus adherence of 32% in HT-29, 22.28% in Caco-2, and 21.68% in T84 cells compared to the wild-type strain. In experiments performed with nonintestinal cells (HeLa and HEp-2 cells), significant differences were not observed in adherence between E9034A and derivative strains. Interestingly, the E9034A and E9034A Delta lngA(pLngA) strains were 30 to 35% more adherent in intestinal cells than in nonintestinal cells. Twitching motility experiments were performed, showing that ETEC strains E9034A and E9034A Delta lngA(pLngA) had the capacity to form spreading zones while ETEC E9034A Delta lngA does not. In addition, our data suggest that longus from ETEC participates in the colonization of human colonic cells.

Intestinal adherence associated with type IV pili of enterohemorrhagic Escherichia coli O157:H7.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes hemorrhagic colitis and hemolytic uremic syndrome (HUS) by colonizing the gut mucosa and producing Shiga toxins (Stx). The only factor clearly demonstrated to play a role in EHEC adherence to intestinal epithelial cells is intimin, which binds host cell integrins and nucleolin, as well as a receptor (Tir) that it injects into the host cell. Here we report that EHEC O157:H7 produces adhesive type IV pili, which we term hemorrhagic coli pilus (HCP), composed of a 19-kDa pilin subunit (HcpA) that is encoded by the hcpA chromosomal gene. HCP were observed as bundles of fibers greater than 10 microm in length that formed physical bridges between bacteria adhering to human and bovine host cells. Sera of HUS patients, but not healthy individuals, recognized HcpA, suggesting that the pili are produced in vivo during EHEC infections. Inactivation of the hcpA gene in EHEC EDL933 resulted in significantly reduced adherence to cultured human intestinal and bovine renal epithelial cells and to porcine and bovine gut explants. An escN mutant, which is unable to translocate Tir, adhered less than the hcpA mutant, suggesting that adherence mediated by intimin-Tir interactions is a prelude to HCP-mediated adherence. An hcpA and stx1,2 triple mutant and an hcpA mutant had similar levels of adherence to bovine and human epithelial cells while a stx1,2 double mutant had only a minor defect in adherence, indicating that HCP-mediated adherence and cytotoxicity are independent events. Our data establish that EHEC O157:H7 HCP are intestinal colonization factors that are likely to contribute to the pathogenic potential of this food-borne pathogen.

The type 4 pili of enterohemorrhagic Escherichia coli O157:H7 are multipurpose structures with pathogenic attributes.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 produces long bundles of polar type 4 pili (T4P) called HCP (for hemorrhagic coli pili) that form physical bridges between bacteria associating with human and animal epithelial cells. Here, we sought to further investigate whether HCP possessed other pathogenicity attributes associated with T4P production. Comparative studies performed with wild-type EHEC EDL933 and an isogenic hcpA mutant revealed that HCP play different roles in the biology of this organism. We found that in addition to promoting bacterial attachment to host cells, HCP mediate (i) invasion of epithelial cells, (ii) hemagglutination of rabbit erythrocytes, (iii) interbacterial connections conducive to biofilm formation, (iv) specific binding to host extracellular matrix proteins laminin and fibronectin but not collagen, and (v) twitching motility. Nonadherent laboratory E. coli strain HB101 complemented with hcpABC genes on plasmid pJX22, which specifies for HCP overproduction in EDL933, became hyperadherent and invasive and produced a thick biofilm, suggesting that the presence of HCP confers HB101(pJX22) new attributes otherwise not exhibited by HB101. Analogous to other bacteria in which T4P are involved in the pathogenesis of several infectious diseases, our data strongly suggest that HCP display multiple functions that may contribute to EHEC colonization of different hosts and to virulence, survival, and transmission of this food-borne pathogen.

Antimicrobial efficacy against Pseudomonas aeruginosa biofilm formation in a three-dimensional lung epithelial model and the influence of fetal bovine serum.

In vitro models that mimic in vivo host-pathogen interactions are needed to evaluate candidate drugs that inhibit bacterial virulence traits. We established a new approach to study Pseudomonas aeruginosa biofilm susceptibility on biotic surfaces, using a three-dimensional (3-D) lung epithelial cell model. P. aeruginosa formed antibiotic resistant biofilms on 3-D cells without affecting cell viability. The biofilm-inhibitory activity of antibiotics and/or the anti-biofilm peptide DJK-5 were evaluated on 3-D cells compared to a plastic surface, in medium with and without fetal bovine serum (FBS). In both media, aminoglycosides were more efficacious in the 3-D cell model. In serum-free medium, most antibiotics (except polymyxins) showed enhanced efficacy when 3-D cells were present. In medium with FBS, colistin was less efficacious in the 3-D cell model. DJK-5 exerted potent inhibition of P. aeruginosa association with both substrates, only in serum-free medium. DJK-5 showed stronger inhibitory activity against P. aeruginosa associated with plastic compared to 3-D cells. The combined addition of tobramycin and DJK-5 exhibited more potent ability to inhibit P. aeruginosa association with both substrates. In conclusion, lung epithelial cells influence the efficacy of most antimicrobials against P. aeruginosa biofilm formation, which in turn depends on the presence or absence of FBS.

Mimicking the host and its microenvironment in vitro for studying mucosal infections by Pseudomonas aeruginosa.

Why is a healthy person protected from Pseudomonas aeruginosa infections, while individuals with cystic fibrosis or damaged epithelium are particularly susceptible to this opportunistic pathogen? To address this question, it is essential to thoroughly understand the dynamic interplay between the host microenvironment and P. aeruginosa. Therefore, using model systems that represent key aspects of human mucosal tissues in health and disease allows recreating in vivo host-pathogen interactions in a physiologically relevant manner. In this review, we discuss how factors of mucosal tissues, such as apical-basolateral polarity, junctional complexes, extracellular matrix proteins, mucus, multicellular complexity (including indigenous microbiota), and other physicochemical factors affect P. aeruginosa pathogenesis and are thus important to mimic in vitro. We highlight in vitro cell and tissue culture model systems of increasing complexity that have been used over the past 35 years to study the infectious disease process of P. aeruginosa, mainly focusing on lung models, and their respective advantages and limitations. Continued improvements of in vitro models based on our expanding knowledge of host microenvironmental factors that participate in P. aeruginosa pathogenesis will help advance fundamental understanding of pathogenic mechanisms and increase the translational potential of research findings from bench to the patient's bedside.

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.

Genetic and transcriptional analysis of the siderophore malleobactin biosynthesis and transport genes in the human pathogen Burkholderia pseudomallei K96243.

Burkholderia pseudomallei is a gram-negative facultative intracellular pathogen that causes melioidosis, an invasive disease of humans and animals. To address the response of this bacterium to iron-limiting conditions, we first performed a global transcriptional analysis of RNA extracted from bacteria grown under iron-limiting and iron-rich conditions by microarrays. We focused our study on those open reading frames (ORFs) induced under iron limitation, which encoded predicted proteins that could be involved in the biosynthesis and uptake of the siderophore malleobactin. We purified this siderophore and determined that it consisted of at least three compounds with different molecular weights. We demonstrated that ORFs BPSL1776 and BPSL1774, designated mbaA and mbaF, respectively, are involved in the biosynthesis of malleobactin, while BPSL1775, named fmtA, is involved in its transport. These genes are in an operon with two other ORFs (mbaJ and mbaI) whose transcription is under the control of MbaS, a protein that belongs to the extracytoplasmic function sigma factors. Interestingly, the transcription of the mbaA, fmtA, and mbaS genes is not controlled by the availability of the siderophore malleobactin.

Identification of proinflammatory flagellin proteins in supernatants of Vibrio cholerae O1 by proteomics analysis.

The genome of Vibrio cholerae contains five flagellin genes that encode proteins (FlaA-E) of 39-41 kDa with 61-82% identity among them. Although the existing live oral attenuated vaccine strains against cholera are protective in humans, there is an intrinsic residual cytotoxic and inflammatory component associated with these candidate vaccine strains. Bacterial flagellins are known to be potent inducers of proinflammatory molecules via activation of Toll-like receptor 5. Here we found that purified flagella from wild type V. cholerae 395 induced significant release of interleukin (IL)-8 from cultured HT-29 human colonic epithelial cells. Furthermore we found that filtered supernatants of KKV90, a DeltaflaA isogenic strain unable to produce flagella, were still able to activate production of IL-8 albeit to significantly lower levels than the wild type, suggesting that other activators of proinflammatory molecules were still present in these supernatants. A comparative proteomics analysis of secreted proteins of V. cholerae 395 and KKV90 identified additional proteins with potential to induce IL-8 release in HT-29 cells. Secreted proteins in the range of 30-45 kDa identified by two-dimensional electrophoresis and mass spectrometry revealed the presence of two additional flagellins, FlaC and FlaD, that appeared to be secreted 3- and 6-fold more, respectively, in the mutant compared with the wild type. Double isogenic mutants flaAC and flaAD were unable to trigger IL-8 release from HT-29 cells. In sum, we have shown that purified flagella and secreted flagellin proteins (FlaC and FlaD) are inducers of IL-8 release from epithelial cells via Toll-like receptor 5. This observation may explain, in part, the observed reactogenicity of cholera vaccine strains in humans.