Campylobacter spp. are among the most common causes of human bacterial diarrhea worldwide. While campylobacter infections are quite common and often severe, relatively little is known about mechanisms of pathogenesis. Campylobacters are generally considered invasive, and invasiveness appears to be associated with disease in the ferret diarrheal disease model (3, 42). In addition, numerous cytotoxins in campylobacters have been described (39), but only cytolethal distending toxin (CDT) has been well characterized (29,30,40). CDT has been found in a variety of other bacteria including Escherichia coli (17, 33), Shigella spp. (26), Haemophilus ducreyi (12), Actinobacillus actinomycetemcomitans (25,35,36), and Helicobacter hepaticus (43). Although CDT has been shown to block eukaryotic cells in the G 2 phase of the cell cycle (6,28,40,43), its role in disease caused by such a diverse group of pathogens remains unclear. However, there is some evidence suggesting a role for CDT in diarrheal disease. An epidemiological study in Bangladesh showed a trend towards increased numbers of CDTpositive E. coli cells in diarrheal cases compared to asymptomatic controls, but the difference did not reach statistical significance (1). When fed to suckling mice, partially purified CDT from Shigella dysenteriae produced watery diarrhea and tissue damage in the descending colon (26).Campylobacter entercolitis is typically associated with a local acute inflammatory response and involves intestinal tissue damage. It is thought that the host inflammatory response may mediate many of the clinical symptoms (20), and inflammatory cytokine responses are recognized components of enteric infections. Interleukin-8 (IL-8) is a proinflammatory cytokine, a potent chemotactic factor for many immune effector cells, and a mediator of localized inflammatory responses. Helicobacter pylori, a primary cause of gastritis in humans, is known to induce IL-8 release from epithelial cells (15,34). Salmonella enterica serovar Typhimurium, Listeria monocytogenes, and Shigella spp. have also been shown to elicit IL-8 secretion from intestinal epithelial cells in vitro during invasion (9, 19). We have previously shown that Campylobacter jejuni also induces IL-8 secretion from intestinal epithelial cells by a process which correlated with adherence and/or bacterial invasion (14). In this report we demonstrate that C. jejuni mediates IL-8 secretion from intestinal epithelial cells by multiple mechanisms. One of these mechanisms, as previously described, involves adherence and/or invasion (14), while a second mechanism is mediated directly by CDT. MATERIALS AND METHODSBacterial strains and growth conditions. Campylobacters were routinely grown on Mueller-Hinton (MH) agar (Difco, Detroit, Mich.) under microaerobic conditions or in biphasic MH cultures. MH medium was supplemented with kanamycin to a final concentration of 50 g/ml or chloramphenicol at a final concentration of 20 g/ml in some cases. For counterselection of E. coli DH5␣ (RK212.1) donors (11, 21) in comp...

Enteroaggregative Escherichia coli (EAggEC) have been implicated as diarrheal pathogens in several settings. Some EAggEC produce a distinct heat-stable enterotoxin named EAST1. The distribution and prevalence of the EAST1 gene in selected groups of bacterial enteropathogens were determined by colony hybridization. One hundred percent of 75 O157:H7 enterohemorrhagic E. coli (EHEC), 41% of 227 EAggEC, 41% of 149 enterotoxigenic E. coli, 22% of 65 enteropathogenic E. coli (EPEC), and 38% of 47 E. coli stool isolates from asymptomatic children hybridized with an EAST1 DNA probe. None of 55 enteroinvasive E. coli, 12 Yersinia enterocolitica, or 20 Vibrio cholerae non-O1 strains were EAST1 probe-positive. Concordance between EAST1 genotype and enterotoxicity was shown in examined strains of EAggEC, EHEC, and EPEC. The gene encoding EAST1 is more broadly distributed among diarrheogenic E. coli than previously known and may represent an additional determinant in the pathogenesis of E. coli diarrhea.

Colonization factor antigen I (CFA/I) is the archetype of eight genetically related fimbriae of enterotoxigenic Escherichia coli (ETEC) designated class 5 fimbriae. Assembled by the alternate chaperone pathway, these organelles comprise a rigid stalk of polymerized major subunits and an apparently tip-localized minor adhesive subunit. We examined the evolutionary relationships of class 5-specific structural proteins and correlated these with functional properties. We sequenced the gene clusters encoding coli surface antigen 4 (CS4), CS14, CS17, CS19, and putative colonization factor antigen O71 (PCFO71) and analyzed the deduced proteins and the published homologs of CFA/I, CS1, and CS2. Multiple alignment and phylogenetic analysis of the proteins encoded by each operon define three subclasses, 5a (CFA/I, CS4, and CS14), 5b (CS1, CS17, CS19, and PCFO71), and 5c (CS2). These share distant evolutionary relatedness to fimbrial systems of three other genera. Subclass divisions generally correlate with distinguishing in vitro adherence phenotypes of strains bearing the ETEC fimbriae. Phylogenetic comparisons of the individual structural proteins demonstrated greater intrasubclass conservation among the minor subunits than the major subunits. To correlate this with functional attributes, we made antibodies against CFA/I and CS17 whole fimbriae and maltose-binding protein fusions with the amino-terminal half of the corresponding minor subunits. Anti-minor subunit Fab preparations showed hemagglutination inhibition (HAI) of ETEC expressing homologous and intrasubclass heterologous colonization factors while anti-fimbrial Fab fractions showed HAI activity limited to colonization factor-homologous ETEC. These results were corroborated with similar results from the Caco-2 cell adherence assay. Our findings suggest that the minor subunits of class 5 fimbriae may be superior to whole fimbriae in inducing antiadhesive immunity.

Adhesion pili (fimbriae) play a critical role in initiating the events that lead to intestinal colonization and diarrheal disease by enterotoxigenic Escherichia coli (ETEC), an E. coli pathotype that inflicts an enormous global disease burden. We elucidate atomic structures of an ETEC major pilin subunit, CfaB, from colonization factor antigen I (CFA/I) fimbriae. These data are used to construct models for 2 morphological forms of CFA/I fimbriae that are both observed in vivo: the helical filament into which it is typically assembled, and an extended, unwound conformation. Modeling and corroborative mutational data indicate that proline isomerization is involved in the conversion between these helical and extended forms. Our findings affirm the strong structural similarities seen between class 5 fimbriae (from bacteria primarily causing gastrointestinal disease) and class 1 pili (from bacteria that cause urinary, respiratory, and other infections) in the absence of significant primary sequence similarity. They also suggest that morphological and biochemical differences between fimbrial types, regardless of class, provide structural specialization that facilitates survival of each bacterial pathotype in its preferred host microenvironment. Last, we present structural evidence for bacterial use of antigenic variation to evade host immune responses, in that residues occupying the predicted surface-exposed face of CfaB and related class 5 pilins show much higher genetic sequence variability than the remainder of the pilin protein.crystal structure ͉ pili ͉ diarrheal disease ͉ adhesion ͉ proline isomerization

SummaryFimbrial filaments assembled by distinct chaperone pathways share a common mechanism of intersubunit interaction, as elucidated for colonization factor antigen I (CFA/I), archetype of enterotoxigenic Escherichia coli (ETEC) Class 5 fimbriae. We postulated that a highly conserved b-strand at the major subunit N-terminus represents the donor strand, analogous to interactions within Class I pili. We show here that CFA/I fimbriae utilize donor strand complementation to promote proper folding of and interactions between CFA/I subunits. We constructed a series of genetic variants of CfaE, the CFA/I adhesin, incorporating a C-terminal extension comprising a flexible linker and 10-19 of the N-terminal residues of CfaB, the major subunit. Variants with a donor strand complement (dsc) of Ն12 residues were recoverable from periplasmic fractions. Genetic disruption of the donor b-strand reduced CfaE recovery. A hexahistidine-tagged variant of dsc 19CfaE formed soluble monomers, folded into b-sheet conformation, displayed adhesion characteristic of CFA/I, and elicited antibodies that inhibited mannose-resistant haemagglutination by ETEC expressing CFA/I, CS4 and CS14 fimbriae. Immunoelectron microscopy indicated that CfaE was confined to the distal fimbrial tip. Our findings provide the basis to elucidate structure and function of this class of fimbrial adhesins and assess the feasibility of an adhesin-based vaccine.

CfaE is the minor, tip-localized adhesive subunit of colonization factor antigen I fimbriae (CFA/I) of enterotoxigenic Escherichia coli and is thought to be essential for the attachment of enterotoxigenic E. coli to the human small intestine early in diarrhea pathogenesis. The crystal structure of an in cis donor strand complemented CfaE was determined, providing the first atomic view of a fimbrial subunit assembled by the alternate chaperone pathway. The in cis donor strand complemented variant of CfaE structure consists of an N-terminal adhesin domain and a C-terminal pilin domain of similar size, each featuring a variable immunoglobulin-like fold. Extensive interactions exist between the two domains and appear to rigidify the molecule. The upper surface of the adhesin domain distal to the pilin domain reveals a depression consisting of conserved residues including Arg 181 , previously shown to be necessary for erythrocyte adhesion. Mutational analysis revealed a cluster of conserved, positively charged residues that are required for CFA/I-mediated hemagglutination, implicating this as the receptor-binding pocket. Mutations in a few subclass-specific residues that surround the cluster displayed differential effects on the two red cell species used in hemagglutination, suggesting that these residues play a role in host or cell specificity. The C-terminal donor strand derived from the major subunit CfaB is folded as a ␤-strand and fits into a hydrophobic groove in the pilin domain to complete the immunoglobulin fold. The location of this well ordered donor strand suggests the positioning and orientation of the subjacent major fimbrial subunit CfaB in the native assembly of CFA/I fimbriae.Microbial adherence to host surfaces represents a critical, early step in pathogenesis. Pili or fimbriae are one of several types of macromolecules that serve this function, projecting from the bacterial surface to dock with specific receptors on the host cell surface within preferred niches (1, 2). Enterotoxigenic Escherichia coli (ETEC) 3 is a common bacterial cause of diarrhea of both humans and domesticated animals (3, 4). As a human pathogen, it is second only to rotavirus as a cause of infant mortality from diarrhea in resource-limited countries and the leading cause of travelers' diarrhea (5, 6). ETEC is a noninvasive pathogen that expresses fimbrial colonization factors to mediate small intestinal adherence and elaborates enterotoxins that induce fluid and electrolyte secretion (7-10). Colonization factor antigen I (CFA/I) is one of the most prevalent adhesive fimbriae associated with human disease and is representative of the largest class (Class 5) of human-specific ETEC fimbriae (11)(12)(13)(14). Sequence analysis of the bioassembly components further divides this class of fimbriae into three subclasses, 5a, 5b, and 5c (15, 16).Studies of the biogenesis of CFA/I and related Class 5 fimbriae, referred to as the alternate chaperone (AC) pathway, have revealed four essential proteins (17-19). A periplasmic chaperone (C...

Enteroaggregative Escherichia coli (EAEC) heat-stable enterotoxin 1 (EAST1) was originally discovered in EAEC but has also been associated with enterotoxigenic E. coli (ETEC). Multiple genomic restriction fragments from each of three ETEC strains of human origin showed homology with an EAST1 gene probe. A single hybridizing fragment was detected on the plasmid of ETEC strain 27D that also encodes heat-stable enterotoxin Ib and colonization factor antigen I. We isolated and characterized this fragment, showing that it (i) carries an allele of astA nearly identical to that originally reported from EAEC 17-2 and (ii) expressed enterotoxic activity. Sequence analysis of the toxin coding region revealed that astA is completely embedded within a 1,209-bp open reading frame (ORF1), whose coding sequence is on the same strand but in the ؊1 reading frame in reference to the toxin gene. In vitro expression of the predicted M r -ϳ46,000 protein product of ORF1 was demonstrated. ORF1 is highly similar to transposase genes of IS285 from Yersinia pestis, IS1356 from Burkholderia cepacia, and ISRm3 from Rhizobium meliloti. It is bounded by 30-bp imperfect inverted repeat sequences and flanked by 8-bp direct repeats. Based on these structural features, pathognomonic of a regular insertion sequence, this element was designated IS1414. Preliminary experiments to show IS1414 translocation were unsuccessful. Overlapping genes of the type suggested by the IS1414 core region have heretofore not been described in bacteria. It seems to offer a most efficient mechanism for intragenomic and horizontal dissemination of EAST1.Enterotoxigenic Escherichia coli (ETEC) strains are a major cause of secretory diarrhea in both humans and animals (4, 19). The basic pathogenic repertoire of ETEC includes species-specific surface adhesins that promote small intestinal colonization and enterotoxins that stimulate intestinal cell secretion (13, 24). These virulence determinants are often encoded on transmissible genetic elements (23,40). Two types of enterotoxins are elaborated by ETEC, a heat-labile (LT) and a heat-stable (ST) toxin. STI is the more predominant of two different ETEC heat-stable enterotoxins (21) and causes fluid secretion by activating membrane-bound guanylyl cyclase C (38).Enteroaggregative E. coli (EAEC) heat-stable enterotoxin 1 (EAST1) is a genetically distinct toxin that is structurally related to STI and also elevates intestinal cGMP levels (35, 36). Little is known about the pathogenic significance of EAST1 in diarrhea. In one case-control study, E. coli isolates that were genotypically positive for EAST1 were highly associated with diarrhea in Spanish children (43). It was notable that very few of the EAST1 E. coli isolates from this study hybridized with an aggregative adherence DNA probe. Three separate outbreaks of diarrhea linked to EAST1 E. coli have been reported. The index strain in a Minnesota outbreak was an O39:NM E. coli that expressed EAST1 and had the enteropathogenic E. coli gene locus for enterocyte effacement ...

Two genes involved in iron utilization in Campylobacter coli VC167 T1 have been characterized. The cfrA gene encodes a protein with a predicted M r of 77,653 which, after processing of the leader sequence, has a predicted M r of 75,635. This protein has significant sequence identity to siderophore receptors of several bacteria, and site-specific mutants defective in cfrA do not synthesize one of two major iron-repressible outer membrane proteins. An adjacent gene encodes a TonB-like protein; a mutant in this gene lost the ability to utilize hemin, ferrichrome, and enterochelin as iron sources. The cfrA and tonB genes of VC167 T1 hybridized to all strains of C. coli and most strains of C. jejuni examined but did not hybridize to several other strains of C. jejuni, suggesting that the thermophilic campylobacters can be separated into two categories based on the presence of these two iron utilization genes.