The ability of enteropathogenic Escherichia coli (EPEC) to form attaching and effacing intestinal lesions is a major characteristic of EPEC pathogenesis. Using TnphoA mutagenesis we have identified a chromosomal gene (eae, for E. coli attaching and effacing) that is necessary for this activity. A DNA probe derived from this gene hybridizes to 100% ofE. coli of EPEC serogroups that demonstrate attaching and effacing activity on tissue culture cells as well as other pathogenic E. coli that produce attaching and effacing intestinal lesions, such as RDEC-1 (an EPEC of weanling rabbits) and enterohemorrhagic E. coli. The predicted amino acid sequence derived from the nucleotide sequence of eae shows significant homology to that of the invasin of Yersinia pseudotuberculosis.Enteropathogenic Escherichia coli (EPEC) are an important cause of infant diarrhea in the developing world (1-5). Diarrhea caused by EPEC can be severe, as evidenced by a 30% fatality rate in a recent nursery outbreak (5). Once a serious cause of "summer diarrhea" and nursery outbreaks in industrialized countries, diarrhea due to EPEC now occurs less frequently in these areas, although outbreaks in nurseries and day-care centers are reported occasionally (6, 7).Although EPEC were the first E. coli to be recognized as a diarrheal pathogen, the elucidation of EPEC virulence factors has lagged behind that of enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), and enterohemorrhagic E. coli (EHEC). Unlike that of ETEC and EHEC, the pathogenesis of EPEC does not appear to involve a toxin and no fimbrial colonization factors have been described. A major advance in the understanding of EPEC pathogenesis was the demonstration that EPEC possess a high molecular weight plasmid which is required for full virulence in volunteers (8) and is associated with the ability to adhere to HEp-2 epidermoid carcinoma cells in a pattern described as localized adherence (9, 10). This adherence phenotype is a characteristic of E. coli of the major EPEC serotypes (11) and is detectable with a DNA probe derived from one such plasmid called the EAF probe (EPEC adherence factor).Perhaps the most important feature of EPEC pathogenesis is the ability of EPEC to produce characteristic histopathological intestinal lesions in humans or experimental animal models. This lesion has been described by Moon et al. (12) as an "attaching and effacing" (A/E) lesion and is characterized by the intimate adherence of bacteria to the enterocyte, dissolution of the brush border at the site of bacterial attachment, and disruption of the cellular cytoskeleton. Within the enterocyte, high concentrations of filamentous actin are present at the site of bacterial attachment and the enterocyte membrane is frequently seen cupping the bacteria, often forming a pedestal-like structure. The production of this lesion can occur in the absence of the EAF plasmid, as evidenced by the observation that A/E lesions are produced by EAF plasmid-cured derivatives of EPEC isolates in experimental an...

Enteropathogenic Escherichia coli (EPEC) causes a characteristic histopathology in intestinal epithelial cells called the attaching and effacing lesion. Although the histopathological lesion is well described the bacterial factors responsible for it are poorly characterized. We have identified four EPEC chromosomal genes whose predicted protein sequences are similar to components of a recently described secretory pathway (type III) responsible for exporting proteins lacking a typical signal sequence. We have designated the genes sepA, sepB, sepC, and sepD (sep, for secretion of E. coli proteins). The predicted Sep polypeptides are similar to the Lcr (low calcium response) and Ysc (yersinia secretion) proteins of Yersinia species and the Mxi (membrane expression of invasion plasmid antigens) and Spa (surface presentation of antigens) regions of Shigella flexneri. Culture supernatants of EPEC strain E2348/69 contain several polypeptides ranging in size from 110 kDa to 19 kDa. Proteins of comparable size were recognized by human convalescent serum from a volunteer experimentally infected with strain E2348/69. A sepB mutant of EPEC secreted only the 110-kDa polypeptide and was defective in the formation of attaching and effacing lesions and protein-tyrosine phosphorylation in tissue culture cells. These phenotypes were restored upon complementation with a plasmid carrying an intact sepB gene. These data suggest that the EPEC Sep proteins are components of a type III secretory apparatus necessary for the export of virulence determinants.Enteropathogenic Escherichia coli (EPEC) causes infantile diarrhea throughout the world. EPEC infections result in the formation of attaching and effacing (AE) lesions which are characterized by effacement of intestinal microvilli, intimate adherence of bacteria to enterocytes, and accumulation of polymerized actin and other cytoskeletal components in the eukaryotic cell. Filamentous actin accumulates below the bacteria, resulting in the formation of cup-like pedestals (1, 2). Several signal transduction mechanisms have been associated with AE lesion formation, including tyrosine phosphorylation of a 90-kDa host cell protein (Hp9O) (3), fluxes in inositol phosphate levels (4), increased intracellular Ca2+ levels (5), and phosphorylation of myosin light chain (6). We recently described a large (35-kb) region in the EPEC chromosome, termed LEE (locus of enterocyte effacement), that encodes all of the virulence determinants for AE lesion formation so far identified (7). Two chromosomal loci within the LEE, eaeA and eaeB (eae, for E. coli attaching and effacing

SummaryThe MtrC-MtrD-MtrE efflux pump system confers resistance to macrolide antibiotics and antimicrobial substances of the host innate defense. Clinical isolates with increased resistance to erythromycin and azithromycin frequently harbor mutations in the mtrR structural gene, which encodes a repressor of the mtrCDE operon, or the mtrR promoter region. The MtrC-MtrD-MtrE system is important for gonococcal survival in the murine genital tract, and derepression of the mtrCDE operon via deletion of mtrR confers increased fitness in vivo. Here we compared isogenic strains with naturally occurring mtrR locus mutations for differences in mtrCDE expression and pump-related phenotypes. Mutations upstream of mtrC, including those within the MtrR binding region and a novel mutation that increases mtrC RNA stability conferred the highest levels of derepression as measured by mtrCDE transcription and resistance to antibiotics, progesterone, and antimicrobial peptides. In contrast, mutations within the mtrR coding sequence conferred low to intermediate levels of derepression. In vivo, the mtr mutants were more fit than the wild type strain, the degree to which paralleled in vitro resistance gradients. These studies establish a hierarchy of mtrR locus mutations with regard to regulation of pump efflux, and suggest selection for more derepressed mutants may occur during mixed infections.

Active efflux of antimicrobial substances is likely to be an important bacterial defense against inhibitory host factors inherent to different body sites. Two well-characterized multidrug resistance efflux systems (MtrCDE and FarAB-MtrE) exist in Neisseria gonorrhoeae, a bacterial pathogen of the human genital mucosae. In vitro studies suggest that the MtrCDE and FarAB-MtrE efflux systems protect the gonococcus from hydrophobic antimicrobial substances that are likely to be present on mucosal surfaces. Here we report that a functional MtrCDE efflux system, but not a functional FarAB-MtrE system, enhances experimental gonococcal genital tract infection in female mice. Specifically, the recovery of mtrD and mtrE mutants, but not a farB mutant, from mice inoculated with mutant or wild-type gonococci was reduced compared with that of the wild-type strain. Competitive-infection experiments confirmed the survival disadvantage of MtrCDE-deficient gonococci. This report is the first direct evidence that a multidrug resistance efflux system enhances survival of a bacterial pathogen in the genital tract. Additionally, experiments using ovariectomized mice showed that MtrCDEdeficient gonococci were more rapidly cleared from mice that were capable of secreting gonadal hormones. MtrCDE-deficient gonococci were more sensitive to nonphysiological concentrations of progesterone in vitro than were wild-type or FarAB-MtrE-deficient gonococci. These results suggest that progesterone may play an inhibitory role in vivo. However, hormonally regulated factors rather than progesterone itself may be responsible for the more rapid clearance of mtr-deficient gonococci from intact mice.Bacterial colonization of mucosal surfaces is challenged by components of the host innate immune response, including hydrophobic, membrane-damaging compounds such as bile salts, fatty acids, and antibacterial peptides. Gram-negative bacteria have evolved elaborate active efflux systems, which together with the low permeability of the outer membrane confer inherent resistance to these antimicrobial substances. One class of active efflux systems, the multidrug resistance pumps, is remarkable in the capacity to recognize structurally dissimilar substrates, including diverse antibiotics (34,35). Two such systems, namely, the mtrCDE-encoded (6, 12, 13, 36) and farAB-encoded (22) efflux systems, have been well characterized in Neisseria gonorrhoeae. Like other multidrug resistance pumps, the mtrCDE-and farAB-encoded efflux systems are composed of three components that function together to capture the substrate in the inner membrane and transport it through the periplasm and out to the external milieu via an outer membrane pore (34,49). A third efflux system that utilizes a transporter homologous to NorM of Vibrio parahaemolyticus was recently identified in N. gonorrhoeae and Neisseria meningitidis (37).The gonococcal MtrCDE ("mtr" stands for multiple transferable resistance) system is encoded by an operon consisting of three genes, mtrC, mtrD, and mtrE (11), and ...

Host immune responses, including the characteristic influx of neutrophils, against Neisseria gonorrhoeae are poorly understood; adaptive immunity is minimal and nonprotective. We hypothesize that N. gonorrhoeae selectively elicits Th17-dependent responses which recruit innate defense mechanisms including neutrophils and antimicrobial proteins that it can resist. We found that N. gonorrhoeae induced production of IL-17 in mouse T cells and of Th17-inducing cytokines in mouse and human antigen-presenting cells in vitro. IL-17 was induced in the iliac lymph nodes in vivo in a female mouse model of genital tract gonococcal infection. Antibody blockade of IL-17 or deletion of the major IL-17 receptor in IL-17RA-knockoutmice led to prolonged infection and diminished neutrophil influx. Genital tract tissue from IL-17RA-knockout mice showed reduced production of neutrophil-attractant chemokines in response to culture with N. gonorrhoeae. These results imply a crucial role for IL-17 and Th17 cells in the immune response to N. gonorrhoeae.

Expanding efforts to develop preventive gonorrhea vaccines is critical because of the dire possibility of untreatable gonococcal infections. Reverse vaccinology, which includes genome and proteome mining, has proven very successful in the discovery of vaccine candidates against many pathogenic bacteria. However, progress with this approach for a gonorrhea vaccine remains in its infancy. Accordingly, we applied a comprehensive proteomic platform-isobaric tagging for absolute quantification coupled with two-dimensional liquid chromatography and mass spectrometry-to identify potential gonococcal vaccine antigens. Our previous analyses focused on cell envelopes and naturally released membrane vesicles derived from four different Neisseria gonorrhoeae strains. Here, we extended these studies to identify cell envelope proteins of N. gonorrhoeae that are ubiquitously expressed and specifically induced by physiologically relevant environmental stimuli: oxygen availability, iron deprivation, and the presence of human serum. Together, these studies enabled the identification of numerous potential gonorrhea vaccine targets. Initial characterization of five novel vaccine candidate antigens that were ubiquitously expressed under these different growth conditions demonstrated that homologs of BamA (NGO1801), LptD (NGO1715), and TamA (NGO1956), and two uncharacterized proteins, NGO2054 and NGO2139, were surface exposed, secreted via naturally released membrane vesicles, and elicited bactericidal antibodies that cross-reacted with a panel of temporally and geographically diverse isolates. In addition, analysis of polymorphisms at the nucleotide and amino acid levels showed that these vaccine candidates are highly conserved among N. gonorrhoeae strains. Finally, depletion of BamA caused a loss of N. gonorrhoeae viability, suggesting it may be an essential target. Together, our data strongly support the use of proteomics-driven discovery of potential vaccine targets as a sound approach for identifying promising gonococcal antigens. Molecular & Cellular

The production of a characteristic intestinal histopathology called attaching and effacing (A/E) lesions by enteropathogenic Escherichia coli (EPEC) is a major characteristic of EPEC pathogenesis. We previously identified a chromosomal gene (eae) of EPEC necessary for the production of A/E lesions on hunman tissue culture cells. Using antiserum raised to an Eae-PhoA fusion protein, we found that the eae gene encodes a 94-kDa membrane protein. This antiserum recognized a 94-kDa membrane protein in parent strain E2348/69 and a protein of similar size in E. coli HB101 carrying eae on a plasmid but did not recognize any proteins in E. coli HB101 carrying a plasmid with an internal deletion in the eae gene. Antigenically related proteins of ca. 94 kDa were detected in a collection of EPEC strains representing seven EPEC serogroups and in two EHEC strains of serotype 026:H11. Volunteer sera drawn 28 days after but not before ingestion of strain E2348/69 recognized the 94-kDa Eae protein as well as a 128-kDa Eae-PhoA fusion protein, suggesting that the Eae protein is likely to be a previously reported 94-kDa protein shown to be immunogenic in volunteers. The amount of detectable Eae protein was increased in the presence of a high-molecular-weight plasmid which is associated with the ability to produce localized adherence to tissue culture cells. These data suggest that the virulence plasmid of EPEC strain E2348/69 may have a regulatory role in the production of A/E activity.

SumlTlaryThe opacity (Opa) proteins of Neisseria gonorrhoeae are a family of outer membrane proteins demonstrating phase and antigenic variation. IV. gonorrhoeae strain FA0190 has 11 opa loci that encode at least 8 antigenically distinct Opa proteins. To determine if expression of one Opa protein or a subset of them is favored during gonococcal infection, we inoculated Opa-negative variants of strain FA1090 intraurethrally into male volunteers. The Opa phenotype of gonococci isolated from urine and urethral swab cultures from nine infected subjects was determined. Opa proteins were expressed in a large proportion of the reisolates from the infected subjects. Gonococci cultured from urine or urethral swab samples from six of the subjects were uniformly Opa positive, with the predominant Opa variants differing among subjects. Three different Opa proteins were represented as the predominant type in at least one subject each. In three subjects, there was more heterogeneity in Opa phenotype of the reisolates, including the presence of Opa-negative variants. An increase in the proportion of isolates expressing multiple Opa proteins occurred over time in most subjects. Passage of the inoculum in vitro did not result in similar changes in Opa expression. There was no detectable difference in infectivity of an Opa-negative variant and one expressing an Opa protein (OpaF) that was highly represented in reisolates from the original nine subjects. Reisolates from three infected volunteers inoculated with the OpaF variant showed continued expression of OpaF alone or in conjunction with other Opa proteins. These results demonstrate that there is strong selection for expression of one or more Opa proteins by strain FA1090 in vivo, but that no single protein is preferentially expressed during early infection in the male urethra. isseria gonorrhoeae is the causative agent of the sexually ransmitted disease gonorrhea. Humans are the exclusive host of N. gonorrhoeae; although some animal models have been described, their usefulness in studying pathogenesis has been limited by their lack of similarity to human mucosal disease (1). N. gonorrhoeae infection of men, unlike that of women, rarely leads to complications (2), and experimental infection of adult men has been used to study potential virulence factors and to evaluate candidate vaccines (3)(4)(5)(6)(7)(8).The gonococcus has a number of surface components demonstrating phase and antigenic variation. Reversible, highfrequency switches in expression result in turning on or off the synthesis of a component (phase variation) or in changing from one expressed antigenic version of a component to another (antigenic variation). These variable components include the opacity (Opa) 1 proteins of the outer membrane 1 Abbreviations used in this paper: ECL, enhanced chemiluminescence; FSM, freezer storage medium; HV, hypervariable; LOS, lip0oligosaccharide; Opa, opacity.(previously designated Protein II). Opa proteins are heat modifiable and range from 25 to 30 kD in molecular mass. Gonococ...