Phylogenetic analysis of enteroaggregative and diffusely adherent Escherichia coli.

The phylogenetics of the various pathotypes of diarrheagenic Escherichia coli are not completely understood. In this study, we identified several plasmid and chromosomal genes in the pathogenic enteroaggregative E. coli (EAEC) prototype strain 042 and determined the prevalence of these loci among EAEC and diffusely adherent E. coli strains. The distribution of these genes is analyzed within an evolutionary framework provided by the characterization of allelic variation in housekeeping genes via multilocus enzyme electrophoresis. Our data reveal that EAEC strains are heterogeneous with respect to chromosomal and plasmid-borne genes but that the majority harbor a member of a conserved family of virulence plasmids. Comparison of plasmid and chromosomal relatedness of strains suggests clonality of chromosomal markers and a limited transfer model of plasmid distribution.

Organization of biogenesis genes for aggregative adherence fimbria II defines a virulence gene cluster in enteroaggregative Escherichia coli.

Several virulence-related genes have been described for prototype enteroaggregative Escherichia coli (EAEC) strain 042, which has been shown to cause diarrhea in human volunteers. Among these factors are the enterotoxins Pet and EAST and the fimbrial antigen aggregative adherence fimbria II (AAF/II), all of which are encoded on the 65-MDa virulence plasmid pAA2. Using nucleotide sequence analysis and insertional mutagenesis, we have found that the genes required for the expression of each of these factors, as well as the transcriptional activator of fimbrial expression AggR, map to a distinct cluster on the pAA2 plasmid map. The cluster is 23 kb in length and includes two regions required for expression of the AAF/II fimbria. These fimbrial biogenesis genes feature a unique organization in which the chaperone, subunit, and transcriptional activator lie in one cluster, whereas the second, unlinked cluster comprises a silent chaperone gene, usher, and invasin reminiscent of Dr family fimbrial clusters. This plasmid-borne virulence locus may represent an important set of virulence determinants in EAEC strains.

In vitro effects of a high-molecular-weight heat-labile enterotoxin from enteroaggregative Escherichia coli.

The pathogenic mechanisms of enteroaggregative Escherichia coli (EAggEC) infection are not fully elucidated. In this work we show that an ammonium sulfate precipitate of culture supernatant of EAggEC strain 049766 increased the potential difference (PD) and the short-circuit current (Isc) in rat jejunal preparations mounted in Ussing chambers. The precipitate contained two major proteins of 108 and 116 kDa, which were partially copurified by chromatography in DEAE-cellulose. This chromatographic fraction (peak I) increased jejunal PD and Isc in a dose-dependent manner, accompanied by a decrease in tissue electrical resistance. These effects were inhibited by incubation of peak I at 75 degreesC for 15 min or for 1 h with proteinase K at 37 degreesC. Rabbit polyclonal antibodies against peak I containing both the 108- and 116-kDa proteins inhibited the enterotoxic effect. Specific polyclonal antibodies raised against the 108-kDa but not against the 116-kDa protein inhibited the enterotoxic effect, suggesting that the 108-kDa protein is the active toxic species. Moreover, another EAggEC strain (065126) producing the 116-kDa protein but not the 108-kDa protein had no effect on rat jejunal mucosa in the Ussing chamber. The >100-kDa fraction derived from prototype EAggEC strain 042, which also expressed both 108- and 116-kDa proteins, also produced an enterotoxic effect on rat jejunal preparations in Ussing chambers; however, the same strain cured of its 65-MDa adherence plasmid did not. A subclone derived from the 65-MDa plasmid expressing the 108-kDa toxin (and not the 116-kDa protein) elicited rises in Isc. Tissue exposed to any preparation containing the 108-kDa toxin exhibited similar histopathologic changes, characterized by increased mucus release, exfoliation of cells, and development of crypt abscesses. Our data suggest that some EAggEC strains produce a ca. 108-kDa enterotoxin/cytotoxin which is encoded on the large virulence plasmid.

Pet, an autotransporter enterotoxin from enteroaggregative Escherichia coli.

Enteroaggregative Escherichia coli (EAEC) is an emerging cause of diarrheal illness. Clinical data suggest that diarrhea caused by EAEC is predominantly secretory in nature, but the responsible enterotoxin has not been described. Work from our laboratories has implicated a ca. 108-kDa protein as a heat-labile enterotoxin and cytotoxin, as evidenced by rises in short-circuit current and falls in tissue resistance in rat jejunal tissue mounted in an Ussing chamber. Here we report the genetic cloning, sequencing, and characterization of this high-molecular-weight heat-labile toxin. The toxin (designated the plasmid-encoded toxin [Pet]) is encoded on the 65-MDa adherence-related plasmid of EAEC strain 042. Nucleotide sequence analysis suggests that the toxin is a member of the autotransporter class of proteins, characterized by the presence of a conserved C-terminal domain which forms a beta-barrel pore in the bacterial outer membrane and through which the mature protein is transported. The Pet toxin is highly homologous to the EspP protease of enterohemorrhagic E. coli and to EspC of enteropathogenic E. coli, an as yet cryptic protein. In addition to its potential role in EAEC infection, Pet represents the first enterotoxin within the autotransporter class of secreted proteins. We hypothesize that other closely related members of this class may also produce enterotoxic effects.

Aggregative adherence fimbria II, a second fimbrial antigen mediating aggregative adherence in enteroaggregative Escherichia coli.

Enteroaggregative Escherichia coli (EAEC) has been implicated as an agent of pediatric diarrhea in the developing world. We have shown previously that EAEC adheres to HEp-2 cells by virtue of a plasmid-encoded fimbrial adhesin designated aggregative adherence fimbria I (AAF/I), the genes for which have been cloned and sequenced. However, not all EAEC strains express AAF/I. Using TnphoA mutagenesis, we have characterized a novel fimbria (designated AAF/II) which mediates HEp-2 adherence of the human-pathogenic strain 042. AAF/II is 5 nm in diameter and does not bind AAF/I antiserum, as determined by immunogold transmission electron microscopy. TnphoA identified a gene (designated aafA) which bears significant homology to aggA, the fimbrial subunit of AAF/I (25% identity and 47% similarity at the amino acid level). When hyperexpressed and purified by polyhistidine tagging, the AafA protein assembled into 5-nm-diameter filaments which bound anti-AAF/II antiserum. The cloned aafA gene complemented a mutation in the aggA gene to confer fimbrial expression from the AAF/I gene cluster, manifesting phenotypes characteristic of AAF/II but not AAF/I. The aafA mutant did not adhere to human intestinal tissue in culture, suggesting a role for AAF/II in intestinal colonization. By using DNA probes for AAF/I and AAF/II derived from fimbrial biosynthesis genes, we show that AAF/I and AAF/II are each found in only a minority of EAEC strains, suggesting that still more EAEC adhesins exist. Our data suggest that AAF adhesins represent a new family of fimbrial adhesins which mediate aggregative adherence in EAEC.

Regulated expression of Clostridium perfringens enterotoxin in naturally cpe-negative type A, B, and C isolates of C. perfringens.

Clostridium perfringens enterotoxin (CPE), the virulence factor responsible for symptoms associated with C. perfringens type A food poisoning, is produced by enterotoxigenic C. perfringens type A isolates when these bacteria sporulate in the gastrointestinal tract. Less than 5% of the global C. perfringens population apparently carries the cpe gene. To assess the distribution of cpe-regulatory factors, we investigated whether the cpe gene of a C. perfringens food poisoning isolate can be expressed and properly regulated (i.e., expressed in a sporulation-associated manner) when transformed into naturally cpe-negative C. perfringens isolates. Sporulation-associated CPE expression was observed when low-copy-number plasmids carrying either a 5.7-kb DNA insert, containing the cpe open reading frame plus >1 kb each of upstream and downstream flanking sequences from C. perfringens food poisoning isolate NCTC 8239, or a 1.6-kb insert, containing only the cpe open reading frame of NCTC 8239, were electroporated into cpe-negative C. perfringens type A, B, and C isolates. Northern (RNA) blot analysis demonstrated that the sizes of the cpe message in the transformants and the naturally enterotoxigenic C. perfringens NCTC 8239 were similar and that this message was detectable only in sporulating cultures of the transformants or NCTC 8239. These studies strongly suggest that many, if not all, cpe-negative C. perfringens isolates (including type B isolates, which are not known to naturally express CPE) produce a factor(s) involved in normal (i.e., sporulation-associated) transcriptional regulation of CPE expression by C. perfringens food poisoning isolates. These findings are consistent with this CPE-regulatory factor(s) also regulating the expression of other genes in C. perfringens.

Comparison of Western immunoblots and gene detection assays for identification of potentially enterotoxigenic isolates of Clostridium perfringens.

Clostridium perfringens enterotoxin (CPE) is an important sporulation-associated virulence factor in several illnesses of humans and domestic animals, including C. perfringens type A food poisoning. Therefore, the ability to determine the enterotoxigenicity of food or fecal C. perfringens isolates with simple, rapid assays should be helpful for epidemiologic investigations. In this study, Western immunoblotting (to detect CPE production in vitro) was compared with PCR assays and digoxigenin-labeled probe assays (to detect all or part of the cpe gene) as a method for determining the enterotoxigenicity of C. perfringens isolates. The cpe detection assays yielded reliable results with DNA purified from vegetative C. perfringens cultures, while Western immunoblots required in vitro sporulation of C. perfringens isolates to detect CPE production. Several cpe-positive C. perfringens isolates from diarrheic animals did not sporulate in vitro under commonly used sporulation-inducing conditions and consequently tested CPE negative. This result indicates that cpe gene detection and serologic CPE assays do not necessarily yield similar conclusions about the enterotoxigenicity of a C. perfringens isolate. Until further studies resolve whether these cpe-positive isolates which do not sporulate in vitro can or cannot sporulate and produce CPE in vivo, it may be preferable to use cpe detection assays for evaluating C. perfringens isolate enterotoxigenicity and thereby avoid potential false-negative conclusions which may occur with serologic assays.

Cloning, nucleotide sequencing, and expression of the Clostridium perfringens enterotoxin gene in Escherichia coli.

A complete copy of the gene (cpe) encoding Clostridium perfringens enterotoxin (CPE), an important virulence factor involved in C. perfringens food poisoning and other gastrointestinal illnesses, has been cloned, sequenced, and expressed in Escherichia coli. The cpe gene was shown to encode a 319-amino-acid polypeptide with a deduced molecular weight of 35,317. There was no consensus sequence for a typical signal peptide present in the 5' region of cpe. Cell lysates from recombinant cpe-positive E. coli were shown by quantitative immunoblot analysis to contain moderate amounts of CPE, and this recombinant CPE was equal to native CPE in cytotoxicity for mammalian Vero cells. CPE expression in recombinant E. coli appeared to be largely driven from a clostridial promoter. Immunoblot analysis also demonstrated very low levels of CPE in vegetative cell lysates of enterotoxin-positive C. perfringens. However, when the same C. perfringens strain was induced to sporulate, much stronger CPE expression was detected in these sporulating cells than in either vegetative C. perfringens cells or recombinant E. coli. Collectively, these results strongly suggest that sporulation is not essential for cpe expression, but sporulation does facilitate high-level cpe expression.