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/R1 which determines in vitro attachment to rabbit intestinal brush borders. In order to determine the role of AF/R1 pili in the pathogenesis of enteropathogenic diarrhea in rabbits, we localized the genes for AF/R1 expression, constructed an AF/R1- strain, and compared the virulence of the AF/R1+ and AF/R1- strains with particular attention to the development of attaching, effacing lesions. We introduced Tn5 into the 86-megadalton (MDa) conjugative plasmid known to mediate expression of AF/R1 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/R1 pili. Pilus expression on M5 was confirmed by reaction with antiserum raised against purified AF/R1 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/R1 expression. Part of the plasmid was mapped, including a region necessary for AF/R1 pilus expression. AF/R1- mutant 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/R1 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.