James B. Kaper

Longus: a long pilus ultrastructure produced by human enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) causes an acute cholera‐like diarrhoea in both humans and animals. We describe a new pilus termed longus produced by ETEC, which can extend for over 20 microns from the cell surface. Longus is composed of a repeating subunit of 22 kDa and its NH2‐terminal amino acid sequence revealed homology with the toxin‐coregulated pilus of Vibrio cholerae, the bundle‐forming pilus of enteropathogenic E. coli and type IV pilins of some Gram‐negative bacterial pathogens. The longus structural gene (IgA) is encoded in a large plasmid and was cloned in a 5 kb fragment, which proved to be sufficient for pilus production and assembly in E. coli K‐12. The presence of IngA was restricted to human ETEC strains. In contrast to other ETEC pili, IngA was widely distributed among ETEC strains independent of their geographical origin, serotype, toxin production, or other pili antigens expressed. Longus is a new member of the type IV pili family, which may represent a highly conserved intestinal colonization factor of ETEC. Common antigenic determinants exist among longus and their pilin subunits, produced by heterologous ETEC. Longus could be significant in the immuno‐prophylaxis of diarrhoeal disease caused by ETEC, especially against those strains in which no colonization factors have been identified and that produce heat‐stable toxin only.

Temporal Expression of Enteropathogenic Escherichia coli Virulence Genes in an In Vitro Model of Infection

ABSTRACT 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.

Simultaneous expression of CFA/I and CS3 colonization factor antigens of enterotoxigenic Escherichia coli by ΔaroC, ΔaroD Salmonella typhi vaccine strain CVD 908

Among the known colonization factors of enterotoxigenic Escherichia coli (ETEC), CFA/I and CS3 (the common antigen in the CFA/II family of fimbrial antigens) are two of the most prevalent fimbrial antigens found in clinical isolates but are never expressed by the same wild-type strain. We manipulated the genetic determinants encoding CS3 and CFA/I fimbriae so that these two important colonization factors are expressed simultaneously in attenuated Salmonella typhi live oral vaccine strain CVD 908, including after growth in liquid medium (CFA/I is poorly expressed by wild-type ETEC in broth culture). The recombinant fimbrial structures produced by CVD 908 are morphologically indistinguishable from the CS3 fibrillae and CFA/I rod-like fimbriae produced by ETEC, and are recognized by monospecific CS3 and CFA/I antibodies. This prototype construct may prove useful in investigating the live vector approach to immunoprophylaxis of ETEC diarrheal disease.

Numerical taxonomy ofKlebsiella pneumoniae strains isolated from clinical and nonclinical sources

A total of 180 clinical and nonclinical isolates ofKlebsiella pneumoniae, for which 99 characteristics were recorded, were subjected to numerical taxonomy analysis. Of these strains, 172 clustered into five major groups, with an overall similarity of 64%. Intragroup similarities ranged from 77 to 82%, with the subgroups corresponding to the speciesK. pneumoniae sensu stricto, K. oxytoca, andKlebsiella spp. 1, 2, and 3. Biochemical tests useful in distinguishing the species included production of indole, degradation of pectate, growth at 10°C, fecal coliform response, production of urease, fermentation of inulin andd-tartrate, utilization ofl-arginine and gentisate andm-hydroxybenzoate, and pigment formation ond-gluconate ferric citrate agar.

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

Enteroaggregative Escherichia Coli, A New Diarrheal Agent

The ability of Escherichia coli strains to adhere to HEp-2 cells in tissue culture correlates with their capacity to cause diarrhea. Three distinct patterns of adherence have been described: localized, diffuse and aggregative (1). Localized adherence (LA) is characteristic of enteropathogenic E. coli of classical infant diarrhea O:H serotypes (2). This property requires the presence of EPEC Adherence Factor (EAF) genes which are located on a plasmid (2, 4, 5). Several recent epidemiological studies have clearly demonstrated that E. coli isolates exhibiting LA are isolated significantly more often from infants and young children with diarrhea than from matched controls (3, 6, 7); the vast majority of such isolates fall into classical EPEC O serogroups (3). Diffuse adherence E. coli (DAEC) are probably the least studies and understood of the HEp-2 cell adherent E. coli. In the one strain extensively studied, diffuse adherence requires the expression of antigenically distinct fimbriae, the structural the expression of antigenically distinct fimbriae, the structural genes of which are chromosomal (8). There is controversy over whether of not DAEC are diarrheagenic (1, 3, 6, 7, 9, 10).