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

Abstract

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.

FIG. 1

Map of the virulence region of pAA2. Black arrows represent AAF-related reading frames discussed in this paper. The pet and astA genes are enterotoxins previously published. IS-like elements are indicated by boxes; the boxes with horizontal lines represent directly repeated IS-like elements that are homologous to IS1353. Boxes with vertical stripes represent complete IS1 elements. Line, 1 kb. The inset at the top shows the upstream region of the aafD gene. Indicated are the predicted ATG start (italics), ribosomal binding site, and −10 and −35 promoter regions. Upstream from the predicted aafD promoter lies a region with high homology to the araI₁ half-site, shown to be the binding site of the AraC protein (35). This region is thus a candidate binding site of the AraC homolog AggR, under which the aafD gene is positively regulated (see text).

FIG. 2

RNA dot blots of aafD, aafA, and aafC transcripts in the presence and absence of a functional aggR gene product. Bacterial RNA prepared from L-broth cultures of 042 or 042 aggR was blotted to nitrocellulose and hybridized with probes generated by PCR. Details are presented in Materials and Methods. aafA and aafD transcripts were clearly increased in the presence of AggR; the aafC transcript was barely detectable but did not exhibit AggR dependence.

FIG. 3

Predicted secondary structure of the aggA downstream region. Nucleotide 1 is the first base of the aafA stop codon. The sequence was analyzed by using the Zuker-Stiegler algorithm processed with DNASIS version 2.10 software.