Characterization of the plasmid from Escherichia coli RDEC-1 that mediates expression of adhesin AF/R1 and evidence that AF/R1 pili promote but are not essential for enteropathogenic disease

Abstract

RDEC-1, an Escherichia coli strain that adheres to rabbit mucosa and causes an attaching, effacing lesion, expresses the pilus adhesin AF/Rl which determines in vitro attachment to rabbit intestinal brush borders. In order to determine the role of AF/Rl pili in the pathogenesis of enteropathogenic diarrhea in rabbits, we localized the genes for AF/Rl expression, constructed an AF/R1strain, and compared the virulence of the AF/ Rl and AF/R1strains with particular attention to the development of attaching, effacing lesions. We introduced TnS into the 86-megadalton (MDa) conjugative plasmid known to mediate expression of AF/Rl pili and transferred the derivative plasmids into laboratory strain HB101. Transconjugant M5 was found to contain the 86-MDa plasmid from RDEC-1 and to express AF/Rl pili. Pilus expression on M5 was confirmed by reaction with antiserum raised against purified AF/Rl pili and allowed the bacteria to adhere to the rabbit ileum in an in vitro assay. Three Tn5 insertions in the 86-MDa plasmid were obtained which resulted in loss of AF/Rl expression. Part of the plasmid was mapped, including a region necessary for AF/Rl pilus expression. AF/R1mutant strain M34 was constructed, and its pathogenesis was investigated. M34 produced disease in rabbits but was less virulent than the parent. The characteristic effacing lesions of RDEC-1 and enteropathogenic E. coli developed in the intestine of rabbits infected with either M34 or RDEC-1, although with M34 they were much less frequent and did not involve the small bowel. We conclude that AF/Rl pilus expression is not essential for the attaching, effacing lesion but serves as an accessory virulence factor which promotes an initial interaction of RDEC-1 with normal epithelial cells. * Corresponding author. .thelial cells (M cells) over the Peyer's patch in the small intestine (13, 14). In their experiments, they used a Shigella flexneri derivative strain, D15, which contained an 85megadalton (MDa) plasmid from RDEC-1 and expressed AF/ Rl pili (10). D15 bacteria were found to adhere specifically to M cells hours after inoculation; however, they did not result in effacing lesions with elongation of microvilli characteristic of RDEC-1 (14). Hence, AF/Ri appears to mediate the attachment of bacteria to M cells early in infection but is not sufficient for attaching, effacing lesions. In the present study, we characterized the 86-MDa plasmid of RDEC-1, constructed mutants in AF/Rl pilus expression, and tested the virulence of an AF/Rl mutant with particular emphasis on its ability to promote attaching, effacing lesions of the absorptive epithelial cells of the ileum, cecum, and colon. MATERIALS AND METHODS Bacterial strains and media. E. coli RDEC-1 is 015:H-:K? and is resistant to nalidixic acid. Strains 640 and HS are commensal strains that were isolated from rabbits and humans, respectively. These were from the collection in the Department of Gastroenterology, Walter Reed Army Institute of Research. HB101 (5) and HU735, the strain containing the Tn5 donor plasmi...

A sialoglycoprotein complex linked to the microvillus cytoskeleton acts as a receptor for pilus (AF/R1) mediated adhesion of enteropathogenic Escherichia coli (RDEC-1) in rabbit small intestine.

“…Next we examined the role of AF/RI pili in the binding measured by this assay. An RDEC-1 variant (M34) with the AF/R1 gene inactivated and therefore lacking AF/Rl (Wolf et al, 1988) pili, gave no binding above background . When the E. coli RDEC-1 were cultured under the standard conditions (aerated PAB), promoting expression ofAF/R1 pili, the binding, as expected, was not inhibited by mannose.…”

“…The metabolically labeled bacteria were washed with PBS and resuspended in PBS containing 1% BSA and 0.15 M mannose before use. M34 is a variant of RDEC-1 in which the plasmid-encoded gene for AF/RI pili has been inactivated by transposon mutagenesis (Wolf et al ., 1988) .…”

Attaching-effacing Bacteria in Animals

“…2a) appears to be necessary for initial signalling leading to the development of the AE lesion, but the mediators of this attachment are poorly understood. Studies have shown a potentiating effect of adhesion factors (such as the EPEC Adherence Factor [EAF] plasmid and the REPEC-associated AF/R1 fimbrial adhesin) upon AE lesion formation in vitro (Knutton et al, 1987;Francis et al, 1991) and on AEEC virulence in vivo (Levine et al, 1985;Wolf et al, 1988). The EAF plasmid contains a gene cluster (bfp) encoding bundle-forming pili (BFP) (Stone et al, 1996), which mediate microcolony formation in culture and localized adhesion to HEp-2 cells (Giron et al, 1991).…”

“…For example, weanlingassociated REPEC O15 is reported commonly from Belgium, the Netherlands and North America, but less commonly from France and Spain (Blanco et al, 1996). REPEC O15:H-includes the prototypical strain RDEC-1 (Cantey and Blake, 1977; that encodes a plasmidborne rabbit-specific fimbrial adhesin, termed AF/ R1, which mediates non-intimate adhesion to ileal M cells and is an established virulence factor (Berendson et al, 1983;Wolf et al, 1988). RDEC-1 also elaborates a soluble factor which affects the electrical properties of rabbit ileal mucosa in vivo and may therefore have diarrhoeagenic properties (Raimondi et al, 2001).…”

Characterization of the eaeA gene from rabbit enteropathogenic Escherichia coli strain RDEC-1 and comparison to other eaeA genes from bacteria that cause attaching-effacing lesions

“…Both RDEC-1 and EPEC express pili necessary for initial attachment of the bacteria to mucosal surface. RDEC-1 express AF/Rl pili [22] and EPEC strains produce bundle-forming pili [23]. The AE lesion produced by RDEC-1 is indistinguishable from that produced by human EPEC [ 1, lo].…”