Structure and secretion of CofJ, a putative colonization factor of enterotoxigenic Escherichia coli

Abstract

SummaryEnterotoxigenic Escherichia coli (ETEC) colonize the human gut, causing severe cholera-like diarrhoea. ETEC utilize a diverse array of pili and fimbriae for host colonization, including the Type IVb pilus CFA/III. The CFA/III pilus machinery is encoded on the cof operon, which is similar in gene sequence and synteny to the tcp operon that encodes another Type IVb pilus, the Vibrio cholerae toxin co-regulated pilus (TCP). Both pilus operons possess a syntenic gene encoding a protein of unknown function. In V. cholerae, this protein, TcpF, is a critical colonization factor secreted by the TCP apparatus. Here we show that the corresponding ETEC protein, CofJ, is a soluble protein secreted via the CFA/III apparatus. We present a 2.6 Å resolution crystal structure of CofJ, revealing a large β-sandwich protein that bears no sequence or structural homology to TcpF. CofJ has a cluster of exposed hydrophobic side-chains at one end and structural homology to the pore-forming proteins perfringolysin O and α-haemolysin. CofJ binds to lipid vesicles and epithelial cells, suggesting a role in membrane attachment during ETEC colonization.

Crystal Structure of the Minor Pilin CofB, the Initiator of CFA/III Pilus Assembly in Enterotoxigenic Escherichia coli

“…In ETEC strain 31-10, the cof operon is located on a 55-kb virulence plasmid (81) that is unstable and not amenable to genetic manipulation (41). The cof operon was cloned into plasmid pACYC184, producing pcof, which was transformed into several E. coli expression strains (DH5␣, HB101, and MC4100 (41)).…”

“…Expression of pcof in E. coli MC4100 -The vector pcof contains the entire ETEC cof operon inserted into the cloning vector pACYC184 (ATCC) between restriction enzyme sites EcoNI and EagI-HF (41). pcof was transformed into electrocompetent E. coli MC4100 cells, and cells containing this plasmid were selected with Cm.…”

“…Deletion of Minor Pilin Genes-The cofB gene was disrupted in pcof using the -RED recombinase system (62) used previously to generate the pcof⌬cofA construct (41). The central portion of the cofB gene (residues 45-462) was targeted for deletion using the CofB-RED-For/-Rev primers to PCR-amplify the kan cassette in plasmid pKD4 using Q5 DNA polymerase.…”

“…The T4P and T2S systems are functionally related as several T4P systems have secretory functions. The V. cholerae toxin coregulated pilus (TCP) apparatus secretes a protein, TcpF, which is required for colonization of the infant mouse (39,40), and CFA/ III secretes CofJ (41).…”

Vibrio cholerae and Escherichia coli heat-labile enterotoxins and beyond

“…Among them are two members of the type IV pilus family: longus pilus and the colonization factor antigen III (CFA/III) [255,256]. The pili machineries are encoded in genes clustered in the lng and cof operons, which are highly similar in sequence to each other (57-97% sequence identity), and, to a lesser extent, to the V. cholerae tcp operon (14-46% sequence identity), with which they share synteny [257]. Taken together with the observation referred to here that ETEC requires host cell contact for cAMP production, these bacteria appear to utilize a similar system of coordinated attack as V. cholerae.…”