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 μm 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.

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.Burkholderia pseudomallei is a gram-negative facultative intracellular pathogen that is the causative agent of melioidosis. Although this invasive disease is endemic to southeast Asia and northern Australia, its worldwide availability, high rate of mortality, and aerosol infectivity resulted in its classification as a select agent (12, 65). B. pseudomallei is also a saprophytic organism that has developed high resistance to many hostile environmental conditions such as acidic environments, dehydration, prolonged nutrient starvation, and antiseptic and detergent solutions (12).Despite increasing knowledge on the epidemiology of this disease, very little is known on the molecular mechanisms of the infection; only a few virulence factors, i.e., the capsular polysaccharide, lipopolysaccharide, type IV pilus, and a type III secretion apparatus, have been described with animal models (55,64). This pathogen can invade phagocytic and nonphagocytic cells using the type III secretion apparatus to promote its escape into the cytoplasm, where the bacterium induces polar actin polymerization (56, 57). Intracellular B. pseudomallei cells can induce host cell membrane protrusions that may facilitate cell-to-cell spread of the bacteria and induce the fusion of adjacent cells forming giant multinucleated cells (24,25,54). Sequencing of the two B. pseudomallei chromosomes (22) has generated a significant amount of data expected to yield information on new virulence factors and putative targets for vaccine development.A potential virulence factor in B. pseudomallei that so far has not been studied is the ability to utilize iron, a feature that may play an important role in the pathogenesis of the disease caused by this bacterium. Iron is required for the growth of nearly all microorganisms in the environment, as well as in biologica...

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.

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.Type 4 pili (T4P) represent a unique class of adhesive pili described as long bundles of flexible and filamentous polymers, whose pilin subunits are mounted in a helical fashion and exposed on the surface of several of gram-negative bacteria of clinical, industrial, and environmental importance. These pili are defined on the basis of their structural, biochemical, morphological, and antigenic characteristics. Some are considerably highly conserved in different bacteria and share several major features, including a short conserved signal peptide, a hydrophobic amino-terminal domain, and a carboxy-terminal disulfide bond (57).T4P have been described in Escherichia coli pathogroups such as enteropathogenic E. coli (17), enterotoxigenic E. coli (18), and enterohemorrhagic E. coli (EHEC) (66), as well as in other gram-negative pathogenic bacteria, including Moraxella catarrhalis (30), species of Neisseria (36), Pseudomonas aeruginosa (32), and Vibrio cholerae (60). A number of cellular functions associated with pathogenicity have been attributed to T4P, such as adhesion to host cells, microcolony and biofilm formation, bacterial aggregation, receptors for phages, immune evasion, twitching motility, DNA uptake, and cell signaling (9). EHEC O157:H7 is an emerging and significant food-borne pathogen that has been implicated in many outbreaks in the United States and other countries (21, 50). The clinical manifestations of EHEC infections range from self-limiting diarrhea to hemorrhagic colitis, which can evolve to severe complications known as hemolytic urem...

4 CFU/ml in Caco-2, and 107.11 ؋ 10 4 CFU/ml in T84 cells. In addition, the E9034A⌬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⌬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⌬lngA(pLngA) had the capacity to form spreading zones while ETEC E9034A⌬lngA does not. In addition, our data suggest that longus from ETEC participates in the colonization of human colonic cells.