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James B Kaper

Publications and source records attributed to James B Kaper.

At least 19 recordsLinked to original sources
PubMed2006

Shiga toxin binding in normal and inflamed human intestinal mucosa.

Shiga toxins are associated with haemolytic uraemic syndrome but human intestinal epithelium does not express the Gb3 receptor. We describe Gb3 expression and Shiga toxin binding in histologically normal intestine and demonstrate that the pattern is unaltered in inflammatory disease states. Gb3 expression and Shiga toxin binding were identified in Paneth cells in both normal and inflamed mucosae.

Antigens, Tumor-Associated, Carbohydrate↗
PubMed2006

Identification and characterization of "pathoadaptive mutations" of the cadBA operon in several intestinal Escherichia coli.

The dysenteric Shigella spp. and enteroinvasive Escherichia coli (EIEC) have evolved from commensal E. coli by the acquisition of a virulence plasmid and inactivation of genes of the cad locus encoding lysine decarboxylase (LDC) by so-called pathoadaptive mutation. As horizontal gene transfer and recombination occurs frequently in E. coli we were interested to see if similar pathoadaptive mutations are commonly present in other intestinal pathotypes. Therefore, we examined 140 intestinal E. coli strains of various pathotypes and the ECOR collection for their ability to decarboxylate lysine, and identified 25 strains that were unable to do so. Complementation of a Shiga toxin-producing E. coli and two enteropathogenic E. coli strains, both LDC-negative, with the intact cad locus restored LDC activity and resulted in a reduction in adherence to tissue culture cells. We investigated the cad locus for possible alterations by using hybridization and PCR techniques and compared the results with the alterations reported for Shigella spp. and EIEC strains. Interestingly, the alterations of the cad genes were similar to those previously reported, pointing towards a parallel evolution of LDC silencing in different intestinal E. coli pathotypes.

Amino Acid Transport Systems↗
PubMed2006

Long polar fimbriae and tissue tropism in Escherichia coli O157:H7.

In vitro organ culture has demonstrated the human intestinal tropism of enterohaemorrhagic Escherichia coli O157:H7 for follicle associated epithelium overlying Peyer's patches of the terminal ileum. Long polar (LP) fimbriae are considered to mediate the attachment of Salmonella enterica serovar Typhimurium to Peyer's patch epithelium and, as homologous genes have been identified in O157:H7, we hypothesised that LP fimbriae in O157:H7 may perform the same function. However, mutation of LP fimbriae in O157:H7 strain 85/170 resulted in the novel phenotype of proximal and distal small intestinal colonisation with attaching/effacing lesion formation, while retaining adhesion to follicle associated epithelium. Application of whole genome DNA array technology did not identify changes in known fimbrial genes that could explain the change in tropism, but highlighted several genes that require further investigation. LP fimbrial genes are the first genes to be identified outside the locus of enterocyte effacement pathogenicity island that influence O157:H7 human intestinal tissue tropism.

Adhesins, Escherichia coli↗
PubMed2006

Role of Shiga toxin versus H7 flagellin in enterohaemorrhagic Escherichia coli signalling of human colon epithelium in vivo.

Enterohaemorrhagic Escherichia coli O157:H7 (EHEC) is a clinically important foodborne pathogen that colonizes human colon epithelium and induces acute colonic inflammation, but does not invade the epithelial cells. Whereas Shiga toxin (Stx) and bacterial flagellin have been studied for their ability to upregulate the production of proinflammatory chemokines by cultured human colon cancer cell lines, the relevance of studies in colon cancer cell lines to the production of proinflammatory signals by normal epithelial cells in EHEC-infected human colon is not known. We show herein that Stx does not bind to human colon epithelium in vivo. Moreover, globotriaosylceramide (Gb3/CD77) synthase, the enzyme required for synthesis of the Gb3/CD77 receptor for Stx, was not expressed by normal or inflamed human colon epithelium in vivo. In contrast, Toll-like receptor (TLR) 5, the receptor for bacterial flagellin, was expressed by normal human colon epithelium and by colon epithelium in human intestinal xenografts. EHEC H7 flagellin instilled in the lumen of human colon xenografts that contain an intact human epithelium upregulated the expression of epithelial cell proinflammatory chemokines, which was accompanied by a subepithelial influx of neutrophils. Isogenic mutants of EHEC that lacked flagellin did not significantly upregulate prototypic neutrophil and dendritic cell chemoattractants by model human colon epithelia, irrespective of Stx production. We conclude that EHEC H7 flagellin and not Stx is the major EHEC factor that directly upregulates proinflammatory chemokine production by human colon epithelium in vivo.

Animals↗
PubMed2006

Nucleotide sequence analysis of the enteropathogenic Escherichia coli adherence factor plasmid pMAR7.

The complete nucleotide sequence was determined for pMAR7, an enteropathogenic Escherichia coli (EPEC) adherence factor (EAF) plasmid that contains genes encoding a type IV attachment pilus (Bfp) and the global virulence regulator per. Prototypic EAF plasmid pMAR7 is self-transmissible, unlike the smaller EAF plasmid pB171, which has no genes encoding conjugative functions. The tra locus, a highly conserved 33-kb segment found in pMAR7, is similar to the tra (conjugation) region of the F plasmid. ISEc13 copies flanking the pMAR7 tra region could potentially mobilize or delete the tra genes. Hybridization of 134 EPEC strains showed that a complete tra region is present only in strains of the EPEC1 clonal group. This study confirms EPEC's potential for dissemination of virulence attributes by horizontal transfer of the EAF plasmid.

Bacterial Adhesion↗
PubMed2006

The lpf gene cluster for long polar fimbriae is not involved in adherence of enteropathogenic Escherichia coli or virulence of Citrobacter rodentium.

Using the enteropathogenic Escherichia coli (EPEC) genome sequence, we found that EPEC E2348/69 has an lpfABCDE gene cluster homologous (about 60% identical at the protein level) to the Salmonella long polar fimbria (LPF) operon. To determine whether this operon is essential for adherence, the lpfABCD(E2)(3) genes were deleted from EPEC strain E2348/69 by allelic exchange. Analysis of the resulting EPECDeltalpfABCD(E23) strain showed no change in adherence to HeLa cells or to human intestinal biopsy cells in the in vitro organ culture (IVOC) system compared to the wild type. Sera from volunteers experimentally infected with E2348/69 showed no antibody response to the major subunit protein, LpfA. These results suggested that the lpf(E23) gene cluster is not necessary for EPEC adherence and attaching/effacing (A/E) lesion formation on human biopsy samples and is not expressed during human infection. We also identified an lpf gene cluster in Citrobacter rodentium strain ICC168 (lpf(cr)). A DeltalpfA(cr) mutant of ICC168 retained wild-type adherence and A/E lesion-forming activity on HeLa cells. C3H/HeJ mice were infected with a wild-type C. rodentium strain and its lpfA(cr) isogenic mutant. Both strains were recovered at high levels in stools, and there were no significant differences between the groups both in terms of the number of CFU/organ (colon and cecum) and in terms of the amount of hyperplasia, as measured by weight. Similar results were observed in a second mouse strain, C57BL/6. These data suggest that in addition to playing no apparent role in EPEC pathogenesis, lpf(cr) is not required for C. rodentium virulence in either the C3H/HeJ or C57BL/6 mouse model.

Animals↗
PubMed2005

Towards a vaccine for attaching/effacing Escherichia coli: a LEE encoded regulator (ler) mutant of rabbit enteropathogenic Escherichia coli is attenuated, immunogenic, and protects rabbits from lethal challenge with the wild-type virulent strain.

The ler (LEE encoded regulator) gene product is a central regulator for the genes encoded on the locus of enterocyte effacement (LEE) pathogenicity island of attaching/effacing (A/E) pathogens, including human enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC) as well as animal isolates. Although an in vivo role for Ler in bacterial virulence has not been documented, we hypothesized that a Ler deletion mutant should be attenuated for virulence but might retain immunogenicity. The goals of this study were to genetically characterize ler of a rabbit EPEC (rEPEC) strain (O103:H2), to examine the effect of ler on in vivo virulence, and to determine if intragastric inoculation of an attenuated rEPEC ler mutant was immunogenic and could protect rabbits against subsequent challenge with the wild-type virulent parent strain. The predicted ler gene product of rEPEC strain O103:H2 shares high homology (over 95% amino acid identity) with the Lers of another rEPEC strain RDEC-1 (O15:H-) and human EPEC and EHEC. A defined internal ler deletion mutant of rEPEC O103:H2 showed reduced production of secreted proteins. Although orogastric inoculation of rabbits with the virulent parent O103:H2 strain induced severe diarrhea, significant weight loss and early mortality with adherent mucosal bacteria found at sacrifice, the isogeneic ler mutant strain was well tolerated. Animals gained weight and showed no clinical signs of disease. Examination of histological sections of intestinal segments revealed the absence of mucosal bacterial adherence. This result demonstrates an essential role for Ler in in vivo pathogenicity of A/E E. coli. Single dose orogastric immunization with the rEPEC ler mutant induced serum IgG antibody to whole bacteria (but not to intimin). Immunized animals were protected against enteric infection with the WT virulent parent strain exhibiting normal weight gain, absence of diarrhea and absence of mucosally adherent bacteria at sacrifice. Such attenuated ler mutant strains may have potential for use as oral vaccines, or as vaccine vectors for delivery of foreign antigens. It remains to be determined whether such regulatory mutants can protect against infection with A/E bacteria of differing serotypes affecting different hosts.

Adhesins, Bacterial↗
PubMed2005

Enteropathogenic Escherichia coli EspG disrupts microtubules and in conjunction with Orf3 enhances perturbation of the tight junction barrier.

EspG, a secreted effector of enteropathogenic Escherichia coli (EPEC), as well as its homologue Orf3, has been shown to disrupt microtubules (MTs) in fibroblasts and non-polarized epithelial cells. The roles of MTs and the effects of MT disruption in these cell types differ significantly. The aim of this study was to investigate the effects of EspG on polarized, host target intestinal epithelial cells. Immunofluorescent labelling of tubulin showed that EPEC caused progressive fragmentation and loss of the MT network in cells harbouring attached organisms. Immunoblots of proteins extracted from EPEC-infected cells showed a corresponding loss of alpha-tubulin. Type III secretion system (TTSS)-deficient strains had no effect on MT suggesting TTSS dependence. Mutation of espG, but not espF or map, ablated EPEC's effects on MTs for up to 6 h. Ectopic expression of EspG in HeLa cells caused MT disruption. While deletion of espG alone had no effect on the EPEC-induced decrease in transepithelial electrical resistance (TER), mutation of both espG and orf3 significantly delayed the kinetics of this response. Complementation of the double mutant with espG alone restored the kinetics of TER drop to that of wild type. Herein, we describe a previously unrecognized phenotype for the EPEC effectors EspG and Orf3.

Bacterial Adhesion↗
PubMed2005

Temporal expression of enteropathogenic Escherichia coli virulence genes in an in vitro model of infection.

The hallmark of enteropathogenic Escherichia coli (EPEC) infection is the ability of EPEC to cause attaching and effacing (A/E) lesions on intestinal epithelium. This event is reproducible in in vitro tissue culture models of infection. We used real-time PCR to measure transcription from several locus of enterocyte effacement (LEE) operons (LEE1 to LEE5) and from bfp during a 5-h infection of HEp-2 cells with EPEC. We found that after the initial formation of A/E lesions, which occurs as early as 5 min postinfection, EPEC continues to increase transcription from LEE3 to LEE5 as well as from bfp. These levels are maximized by 3 h postinfection and remain constant throughout the course of infection. This increase in transcription from LEE3 to LEE5 occurs when LEE1 (ler) transcription is decreasing. EspA, EspB, intimin, Tir, and bundle-forming pilus expression is detectable during the entire 5-h infection. These results indicate that the EPEC genes involved in localized and intimate adherence are continually expressed after the initial stages of A/E lesion formation on host cells.

Adhesins, Bacterial↗
PubMed2005

Colonization of gnotobiotic piglets by a luxS mutant strain of Escherichia coli O157:H7.

Gnotobiotic piglets inoculated with Escherichia coli O157:H7, its luxS mutant derivative, or nonpathogenic E. coli were evaluated for attaching and effacing lesions. Although no differences in clinical symptoms were seen between pigs inoculated with the parent and those inoculated with the luxS mutant, the luxS mutant-inoculated pigs had a lower frequency of attaching and effacing lesions in the spiral colon than parent strain-inoculated pigs.

Animals↗
PubMed2005

SepZ/EspZ is secreted and translocated into HeLa cells by the enteropathogenic Escherichia coli type III secretion system.

Enteropathogenic Escherichia coli (EPEC) is a major bacterial cause of infantile diarrhea in developing countries and is the prototype for a group of gastrointestinal pathogens causing characteristic attaching and effacing (A/E) histopathology on intestinal epithelia. A/E pathogens utilize a type III secretion system (TTSS), encoded by the locus of enterocyte effacement (LEE) pathogenicity island, to deliver effector proteins into host cells. Here, we investigate sequence divergence of the LEE-encoded SepZ protein and identify it as a TTSS-secreted and -translocated molecule. SepZ is hypervariable among A/E pathogens, with sequences sharing between 60 to 81% amino acid identity with SepZ of EPEC. A SepZ-CyaA fusion was secreted and translocated into HeLa cells in a TTSS-dependent manner. Additionally, we determined that the first 20 amino acids of SepZ were sufficient to direct its translocation. In contrast to previous studies suggesting a role in invasion and the structure and/or regulation of the TTSS, we found that SepZ does not mediate uptake of EPEC into host cells or affect translocation and tyrosine phosphorylation of the translocated intimin receptor. Immunohistochemistry reveals that, after an extended HeLa cell infection, accumulated SepZ can be detected beneath the site of bacterial attachment in a subset of pedestal regions. To indicate its newly identified status as a translocated effector protein, we propose to rename SepZ as EspZ.

Adenylate Cyclase Toxin↗
PubMed2005

Identification and characterization of the locus for diffuse adherence, which encodes a novel afimbrial adhesin found in atypical enteropathogenic Escherichia coli.

The O26 serogroup of enteropathogenic Escherichia coli (EPEC) is one of the serogroups most frequently implicated in infant diarrhea and is also common among enterohemorrhagic E. coli (EHEC) strains. The most common O26 strains belong to EPEC/EHEC serotype O26:H11 and are generally Shiga toxin (Stx) positive. Stx-negative E. coli strains that are negative for the EPEC EAF plasmid and bundle-forming pilus (Bfp) are classified as atypical EPEC. Here, we report a novel adhesin present in an stx-negative bfpA-negative atypical EPEC O26:H11 strain isolated from an infant with diarrhea. A cloned 15-kb genomic region from this strain, designated the locus for diffuse adherence (lda), confers diffuse adherence on HEp-2 cells when expressed in E. coli K-12. Sequence analysis of lda revealed a G+C content of 46.8% and 15 open reading frames sharing homology with the E. coli K88 fae and CS31A clp fimbrial operons. The lda region is part of a putative 26-kb genomic island inserted into the proP gene of the E. coli chromosome. Hybridization studies have demonstrated the prevalence of the minor structural subunit gene, ldaH, across E. coli serogroups O5, O26, O111, and O145. A second plasmid-encoded factor that contributed to the Hep-2 adherence of this strain was also identified but was not characterized. Null mutations that abolish adherence to HEp-2 cells can be restored by plasmid complementation. Antiserum raised against the major structural subunit, LdaG, recognizes a 25-kDa protein from crude heat-extracted protein preparations and inhibits the adherence of the E. coli DH5alpha lda(+) clone to HEp-2 cells. Electron microscopy revealed a nonfimbrial structure surrounding the bacterial cell.

Adhesins, Escherichia coli↗
PubMed2004

Characterization of the second long polar (LP) fimbriae of Escherichia coli O157:H7 and distribution of LP fimbriae in other pathogenic E. coli strains.

A second region containing five genes homologous to the long polar fimbrial operon of Salmonella enterica serovar Typhimurium is located in the chromosome of enterohemorrhagic Escherichia coli (EHEC) O157:H7. A non-fimbriated E. coli K-12 strain carrying the cloned EHEC lpf (lpf2) genes expressed thin fibrillae-like structures on its surface and displayed reduced adherence to tissue culture cells. Neither mutation in the lpfA2 gene in either the parent or lpfA1 mutant strains showed an effect in adherence or in the formation of A/E lesions on HeLa cells. lpfA2 isogenic mutant strains adhere to Caco-2 cells almost as well as the wild-type at 5 h, but they were deficient in adherence at early time points. A collection of diarrheagenic E. coli strains were investigated for the presence of lpfA1 and lpfA2 and results showed that these genes are present in specific serogroups which are phylogenetically related. Our results suggest that LP fimbriae 2 may contribute to the early stages of EHEC adhesion and that genes encoding the major LP fimbrial subunits are present in a small group of EHEC and EPEC serotypes.

Bacterial Adhesion↗
PubMed2004

Pathogenic Escherichia coli.

Few microorganisms are as versatile as Escherichia coli. An important member of the normal intestinal microflora of humans and other mammals, E. coli has also been widely exploited as a cloning host in recombinant DNA technology. But E. coli is more than just a laboratory workhorse or harmless intestinal inhabitant; it can also be a highly versatile, and frequently deadly, pathogen. Several different E. coli strains cause diverse intestinal and extraintestinal diseases by means of virulence factors that affect a wide range of cellular processes.

Animals↗
PubMed2004

SepL, a protein required for enteropathogenic Escherichia coli type III translocation, interacts with secretion component SepD.

Enteropathogenic Escherichia coli (EPEC), an important cause of infantile diarrhoea in the developing world, disrupts host cell microvilli, causes actin rearrangements and attaches intimately to the host cell surface. This characteristic phenotype, referred to as the attaching and effacing (A/E) effect, is encoded on a 36 kb pathogenicity island called the locus of enterocyte effacement (LEE). The LEE includes genes involved in type III secretion and translocation, the eae gene encoding an outer membrane adhesin known as intimin, the tir gene for the translocated intimin receptor, a regulator and various genes of unknown function. Among this last group is sepL. To determine the role of SepL in EPEC pathogenesis, we constructed and tested a non-polar sepL mutant. We found that this sepL mutant is deficient for A/E and that it secretes markedly reduced quantities of those proteins involved in translocation (EspA, EspB and EspD), but normal levels of those proteins presumed to be effectors (Tir, EspF and EspG). Despite normal levels of secretion, the mutant strain was unable to translocate EspF and Tir into host cells and formed no EspA filaments. Fractionation studies revealed that SepL is a soluble cytoplasmic protein. Yeast two-hybrid and affinity purification studies indicated that SepL interacts with the LEE-encoded protein SepD. In contrast to SepL, we found that SepD is required for type III secretion of both translocation and effector proteins. Together, these results demonstrate that SepL has a unique role in type III secretion as a functional component of the translocation system that interacts with an essential element of the secretion machinery.

Bacterial Adhesion↗