Adhesin Degradation Accelerates Delivery of Heat-labile Toxin by Enterotoxigenic Escherichia coli

Koushik Roy,Rita Kansal,Scott R Bartels,David J Hamilton,Salwa Shaaban,James M Fleckenstein

doi:10.1074/jbc.m111.251546

Abstract

Many enteric pathogens, including enterotoxigenic Escherichia coli (ETEC), produce one or more serine proteases that are secreted via the autotransporter (or type V) bacterial secretion pathway. These molecules have collectively been referred to as SPATE proteins (serine protease autotransporter of the Enterobacteriaceae). EatA, an autotransporter previously identified in ETEC, possesses a functional serine protease motif within its secreted amino-terminal passenger domain. Although this protein is expressed by many ETEC strains and is highly immunogenic, its precise function is unknown. Here, we demonstrate that EatA degrades a recently characterized adhesin, EtpA, resulting in modulation of bacterial adhesion and accelerated delivery of the heat-labile toxin, a principal ETEC virulence determinant. Antibodies raised against the passenger domain of EatA impair ETEC delivery of labile toxin to epithelial cells suggesting that EatA may be an effective target for vaccine development.

Highlights

Infectious diarrheal disease caused by the enterotoxigenic Escherichia coli (ETEC)2 accounts for hundreds of thousands of deaths each year in areas where sanitation and clean water remain scarce [1]
By contrast, we observed that a mutant strain deficient in the production of another putative virulence protein, the EatA autotransporter, appeared more adherent than wild type bacteria using in vitro intestinal epithelial cell monolayer adhesion assays
Antibodies raised against the passenger domain enhanced adherence of the WT strain but had no appreciable effect on the mutant (Fig. 1d), further suggesting that EatA normally modulates the interaction of ETEC with target host cells

Summary

Deletion of eltA encoding A subunit of LT

H10407 eatA::CmR mutant FϪ ⌬lacX74 galE thi rpsL ⌬phoA (Pvull) ⌬ara-714 leu::Tn10. LMG194 with fliC::KmR F-mcrA ⌬(mrr-hsdRMS-mcrBC) ⌽80lacZ⌬M15 ⌬lacX74 recA1 araD139 ⌬ (ara leu) 7697 galU galK rpsL (StrR) ⌬eltAB (pJL001) ⌬eltAB (pBADmycHisA). Separation and Detection of Proteins in Culture Supernatants— After growth of bacteria in Luria broth for the indicated time, cultures were centrifuged at 10,000 ϫ g for 20 min to pellet bacteria, and supernatants of either H10407 or the jf904 eatA mutant were recovered, sterile-filtered through a 0.45-␮m filter (Millipore), and concentrated by ultrafiltration as above. Ϳ100 ␮g of purified mutant EatA passenger domain was transferred onto nitrocellulose, and the membrane was blocked with 1% BSA in TBS-T for 30 min at room temperature. The A subunit of LT by EatA, the purified passenger domain (ϳ9 pmol) was mixed with LT holotoxin (116 pmol), and after incubation for 1 h at 37 °C, proteins were separated by SDS-PAGE and stained with Coomassie Blue.

RESULTS

LT eltA

DISCUSSION