Jorge A. Girón

Quorum Sensing Is a Global Regulatory Mechanism in Enterohemorrhagic Escherichia coli O157:H7

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is responsible for outbreaks of bloody diarrhea and hemolytic-uremic syndrome in many countries. EHEC virulence mechanisms include the production of Shiga toxins (Stx) and formation of attaching and effacing (AE) lesions on intestinal epithelial cells. We recently reported that genes involved in the formation of the AE lesion were regulated by quorum sensing through autoinducer-2, which is synthesized by the product of the luxS gene. In this study we hybridized an E. coli gene array with cDNA synthesized from RNA that was extracted from EHEC strain 86-24 and its isogenic luxS mutant. We observed that 404 genes were regulated by luxS at least fivefold, which comprises approximately 10% of the array genes; 235 of these genes were up-regulated and 169 were down-regulated in the wild-type strain compared to in the luxS mutant. Down-regulated genes included several involved in cell division, as well as ribosomal and tRNA genes. Consistent with this pattern of gene expression, the luxS mutant grows faster than the wild-type strain (generation times of 37.5 and 60 min, respectively, in Dulbecco modified Eagle medium). Up-regulated genes included several involved in the expression and assembly of flagella, motility, and chemotaxis. Using operon::lacZ fusions to class I, II, and III flagellar genes, we were able to confirm this transcriptional regulation. We also observed fewer flagella by Western blotting and electron microscopy and decreased motility halos in semisolid agar in the luxS mutant. The average swimming speeds for the wild-type strain and the luxS mutant are 12.5 and 6.6 microm/s, respectively. We also observed an increase in the production of Stx due to quorum sensing. Genes encoding Stx, which are transcribed along with lambda-like phage genes, are induced by an SOS response, and genes involved in the SOS response were also regulated by quorum sensing. These results indicate that quorum sensing is a global regulatory mechanism for basic physiological functions of E. coli as well as for virulence factors.

The Locus of Enterocyte Effacement (LEE)-Encoded Regulator Controls Expression of Both LEE- and Non-LEE-Encoded Virulence Factors in Enteropathogenic and Enterohemorrhagic Escherichia coli

ABSTRACT Regulation of virulence gene expression in enteropathogenicEscherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) is incompletely understood. In EPEC, the plasmid-encoded regulator Per is required for maximal expression of proteins encoded on the locus of enterocyte effacement (LEE), and a LEE-encoded regulator (Ler) is part of the Per-mediated regulatory cascade upregulating the LEE2, LEE3, andLEE4 promoters. We now report that Ler is essential for the expression of multiple LEE-located genes in both EPEC and EHEC, including those encoding the type III secretion pathway, the secreted Esp proteins, Tir, and intimin. Ler is therefore central to the process of attaching and effacing (AE) lesion formation. Ler also regulates the expression of LEE-located genes not required for AE-lesion formation, including rorf2, orf10,rorf10, orf19, and espF, indicating that Ler regulates additional virulence properties. In addition, Ler regulates the expression of proteins encoded outside the LEE that are not essential for AE lesion formation, including TagA in EHEC and EspC in EPEC. Δler mutants of both EPEC and EHEC show altered adherence to epithelial cells and express novel fimbriae. Ler is therefore a global regulator of virulence gene expression in EPEC and EHEC.

The flagella of enteropathogenic Escherichia coli mediate adherence to epithelial cells

Enteropathogenic Escherichia coli (EPEC) utilizes a type III protein secretion system to target effector molecules into the host cell leading to effacement of the intestinal mucosa. This secretion apparatus shares many structural features of the flagellar type III export system involved in flagella assembly and motility. We report here that fliC insertional mutants constructed in two wild‐type EPEC strains were markedly impaired in adherence and microcolony formation on cultured cells. An E. coli K‐12 strain harbouring the EPEC H6 fliC gene on a plasmid showed discrete adhering clusters on HeLa cells, albeit to less extent than the wild‐type EPEC strain. Flagella purified from EPEC bound to cultured epithelial cells and antiflagella antibodies blocked adherence of several EPEC serotypes. We determined that eukaryotic cells in culture stimulate expression of flagella by motile and non‐motile EPEC. Isogenic strains mutated in perA (a transcriptional activator), bfpA (a type IV pilin), luxS (a quorum‐sensing autoinducer gene) and in the type III secretion genes were reduced for motility in Dulbecco’s modified Eagle medium (DMEM) motility agar and produced none or few flagella when associated with epithelial cells. Growth of these mutants in preconditioned tissue culture medium restored motility and their ability to produce flagella, suggesting the influence of a signal provided by mammalian cells that triggers flagella production. This study shows for the first time that the flagella of EPEC are directly involved in the adherence of these bacteria and supports the existence of a molecular relationship between the two existing type III secretion pathways of EPEC, the EPEC adherence factor (EAF) plasmid‐encoded regulator, quorum sensing and epithelial cells.

A plasmid‐encoded type IV fimbrial gene of enteropathogenic Escherichia coli associated with localized adherence

Enteropathogenic Escherichia coli (EPEC) form adherent microcolonies on the surface of tissue culture cells in a pattern termed localized adherence. Localized adherence requires the presence of a large EPEC adherence factor (EAF) plasmid. Recently a bundle‐forming pilus has been described in EPEC possessing the EAF plasmid. An analysis of 22 non‐invasive EPEC TnphoA mutants revealed that seven have insertions in the EAF plasmid and are incapable of localized adherence. We report here the mapping of the TnphoA insertions in these mutants. The nucleotide sequence of the gene interrupted in these TnphoA mutants (bfpA) was determined and found to correspond to the N‐terminal amino acid sequence of the major structural protein of the bundle‐forming pilus. The bfpA gene bears sequence similarities to members of the type IV fimbrial gene family and encodes a potential site for processing by a prepilin peptidase. A plasmid containing bfpA as the only open reading frame directs the synthesis of a protein recognized by antiserum raised against the bundle‐forming pilus. TnphoA mutants at this locus are unable to synthesize BfpA, but synthesis is restored by introduction of a plasmid containing the cloned gene. The minimum fragment of DNA required to restore localized adherence is considerably greater than that required to restore BfpA synthesis. BfpA expression, as assessed by alkaline phophatase activity in bfpA::TnphoA mutants, is affected by temperature and growth medium. These studies describe an EPEC plasmid‐encoded fimbrial gene, a candidate for the elusive EPEC adherence factor responsible for localized adherence.

Identification and Characterization of lpfABCC′DE, a Fimbrial Operon of Enterohemorrhagic Escherichia coli O157:H7

ABSTRACT The mechanisms underlying the adherence of Escherichia coli O157:H7 and other enterohemorrhagic E. coli (EHEC) strains to intestinal epithelial cells are poorly understood. We have identified a chromosomal region (designated lpfABCC′DE) in EHEC O157:H7 containing six putative open reading frames that was found to be closely related to the long polar (LP) fimbria operon (lpf) of Salmonella enterica serovar Typhimurium, both in gene order and in conservation of the deduced amino acid sequences. We show that lpfABCC′DE is organized as an operon and that its expression is induced during the exponential growth phase. The lpf genes from EHEC strain EDL933 were introduced into a nonfimbriated (Fim−) E. coli K-12 strain, and the transformed strain produced fimbriae as visualized by electron microscopy and adhered to tissue culture cells. Anti-LpfA antiserum recognized a ca. 16-kDa LpfA protein when expressed under regulation of the T7 promoter system. The antiserum also cross-reacted with the LP fimbriae in immunogold electron microscopy and Western blot experiments. Isogenic E. coli O157:H7 lpf mutants derived from strains 86-24 and AGT300 showed slight reductions in adherence to tissue culture cells and formed fewer microcolonies compared with their wild-type parent strains. The adherence and microcolony formation phenotypes were restored when the lpf operon was introduced on a plasmid. We propose that LP fimbriae participate in the interaction of E. coli O157:H7 with eukaryotic cells by assisting in microcolony formation.

Synergistic role of curli and cellulose in cell adherence and biofilm formation of attaching and effacing Escherichia coli and identification of Fis as a negative regulator of curli.

Curli are adhesive fimbriae of Escherichia coli and Salmonella enterica. Expression of curli (csgA) and cellulose (bcsA) is co-activated by the transcriptional activator CsgD. In this study, we investigated the contribution of curli and cellulose to the adhesive properties of enterohaemorragic (EHEC) O157:H7 and enteropathogenic E. coli (EPEC) O127:H6. While single mutations in csgA, csgD or bcsA in EPEC and EHEC had no dramatic effect on cell adherence, double csgAbcsA mutants were significantly less adherent than the single mutants or wild-type strains to human colonic HT-29 epithelial cells or to cow colon tissue in vitro. Overexpression of csgD (carried on plasmid pCP994) in a csgD mutant, but not in the single csgA or bscA mutants, led to significant increase in adherence and biofilm formation in EPEC and EHEC, suggesting that synchronized over-production of curli and cellulose enhances bacterial adherence. In line with this finding, csgD transcription was activated significantly in the presence of cultured epithelial cells as compared with growth in tissue culture medium. Analysis of the influence of virulence and global regulators in the production of curli in EPEC identified Fis (factor for inversion stimulation) as a, heretofore unrecognized, negative transcriptional regulator of csgA expression. An EPEC E2348/69Deltafis produced abundant amounts of curli whereas a double fis/csgD mutant yielded no detectable curli production. Our data suggest that curli and cellulose act in concert to favour host colonization, biofilm formation and survival in different environments.

Flagellin of Enteropathogenic Escherichia coli Stimulates Interleukin-8 Production in T84 Cells

ABSTRACT The type III secretion system (TTSS) of enteropathogenic Escherichia coli (EPEC) has been associated with the ability of these bacteria to induce secretion of proinflammatory cytokines, including interleukin-8 (IL-8), in cultured epithelial cells. However, the identity of the effector molecule directly involved in this event is unknown. In this study, we determined that the native flagellar filament and its flagellin monomer are activators of IL-8 release in T84 epithelial cells. Supernatants of wild-type EPEC strain E2348/69 and its isogenic mutants deficient in TTSS (escN) and in production of intimin (eae), grown in Luria-Bertani broth, elicited similar amounts of IL-8 secretion by T84 cells. In contrast, supernatants of EPEC fliC mutants and of B171, a nonflagellated EPEC strain, were defective in inducing IL-8 release, a phenotype that was largely restored by complementation of the fliC gene in the mutant lacking flagella. Purified flagella from E. coli K-12, EPEC serotypes H6 and H34, and enterohemorrhagic E. coli serotype H7 all induced IL-8 release in T84 cells. Induction of IL-8 by purified flagella or His-tagged FliC from EPEC strain E2348/69 was dose dependent and was blocked by a polyclonal anti-H6 antibody. Finally, the mitogen-activated protein kinases (Erk1 and -2 and Jnk) were phosphorylated in flagellin-treated T84 cells, and inhibition of the p38 and Erk pathways significantly decreased the IL-8 response induced by EPEC flagellin. Our data clearly indicate that FliC of EPEC is sufficient to induce IL-8 release in T84 cells and that activation of the Erk and p38 pathways is required for IL-8 induction.

Mycobacterium tuberculosis produces pili during human infection

Mycobacterium tuberculosis is responsible for nearly 3 million human deaths worldwide every year. Understanding the mechanisms and bacterial factors responsible for the ability of M. tuberculosis to cause disease in humans is critical for the development of improved treatment strategies. Many bacterial pathogens use pili as adherence factors to colonize the host. We discovered that M. tuberculosis produces fine (2- to 3-nm-wide), aggregative, flexible pili that are recognized by IgG antibodies contained in sera obtained from patients with active tuberculosis, indicating that the bacilli produce pili or pili-associated antigen during human infection. Purified M. tuberculosis pili (MTP) are composed of low-molecular-weight protein subunits encoded by the predicted M. tuberculosis H37Rv ORF, designated Rv3312A. MTP bind to the extracellular matrix protein laminin in vitro, suggesting that MTP possess adhesive properties. Isogenic mtp mutants lost the ability to produce Mtp in vitro and demonstrated decreased laminin-binding capabilities. MTP shares morphological, biochemical, and functional properties attributed to bacterial pili, especially with curli amyloid fibers. Thus, we propose that MTP are previously unidentified host-colonization factors of M. tuberculosis.

Fimbriae and adherence of Stenotrophomonas maltophilia to epithelial cells and to abiotic surfaces

Stenotrophomonas maltophilia is an emerging nosocomial bacterial pathogen associated with several infectious diseases and opportunistic infections, especially in immunocompromised patients. These bacteria adhere avidly to medical implants and catheters forming a biofilm that confers natural protection against host immune defences and different antimicrobial agents. The nature of the bacterial surface factors involved in biofilm formation on inert surfaces and in adherence of S. maltophilia to epithelial cells is largely unknown. In this study, we identified and characterized fimbrial structures produced by S. maltophilia grown at 37°C. The S. maltophilia fimbriae 1 (SMF‐1) are composed of a 17 kDa fimbrin subunit which shares significant similarities with the N‐terminal amino acid sequences of several fimbrial adhesins (G, F17, K99 and 20K) found in Escherichia coli pathogenic strains and the CupA fimbriae of Pseudomonas aeruginosa. All of the clinical S. maltophilia isolates tested produced the 17 kDa fimbrin. Antibodies raised against SMF‐1 fimbriae inhibited the agglutination of animal erythrocytes, adherence to HEp‐2 cells and biofilm formation by S. maltophilia. High resolution electron microscopy provided evidence of the presence of fimbriae acting as bridges between bacteria adhering to inert surfaces or to cultured epithelial cells. This is the first characterization of fimbriae in this genus. We provide compelling data suggesting that the SMF‐1 fimbriae are involved in haemagglutination, biofilm formation and adherence to cultured mammalian cells.

Longus pilus of enterotoxigenic Escherichia coli and its relatedness to other type-4 pili--a minireview.

Longus is a long pilus produced by human enterotoxigenic Escherichia coli (ETEC) which shares significant structural and biochemical features with class-B type-4 pili. These pili include the toxin-coregulated pilus (TCP) of Vibrio cholerae, the bundle-forming pilus (BFP) of enteropathogenic E. coli and both longus and the colonization factor antigen III (CFA/III) of ETEC. These pili are produced under defined growth conditions indicating that they are under the control of different regulatory elements. While TCP is chromosomally encoded, the remaining pili are encoded on large virulence plasmids. Longus and CFA/III are closely related pili although certain DNA and protein differences also exist between them. This may account for the differences in the regulation, surface presentation, antigenicity, and prevalence of these two pilins among ETEC. Neighboring lngA, a second open reading frame termed lngB was found which encodes a protein with significant homology to proteins which are part of a type-II secretory system such as XcpV, OutC, and PulO of Pseudomonas aeruginosa, Erwinia chrysanthemi, and Klebsiella pneumoniae, respectively. This suggests that lngB may be an accessory gene involved in biogenesis of longus.