Abstract
Enterotoxigenic Escherichia coli (ETEC) cause secretory diarrhea in children and travelers to endemic areas. ETEC spreads through the fecal-oral route. After ingestion, ETEC passes through the stomach and duodenum before it colonizes the lower part of the small intestine, exposing bacteria to a wide range of pH and environmental conditions. This study aimed to determine the impact of external pH and activity of the Cyclic AMP receptor protein (CRP) on the regulation of production and secretion of heat labile (LT) enterotoxin. ETEC strain E2863wt and its isogenic mutant E2863ΔCRP were grown in LBK media buffered to pH 5, 7 and 9. GM1 ELISA, cDNA and cAMP analyses were carried out on bacterial pellet and supernatant samples derived from 3 and 5 hours growth and from overnight cultures. We confirm that CRP is a repressor of LT transcription and production as has been shown before but we show for the first time that CRP is a positive regulator of LT secretion both in vitro and in vivo. LT secretion increased at neutral to alkaline pH compared to acidic pH 5 where secretion was completely inhibited. At pH 9 secretion of LT was optimal resulting in 600 percent increase of secreted LT compared to unbuffered LBK media. This effect was not due to membrane leakage since the bacteria were viable at pH 9. The results indicate that the transition to the alkaline duodenum and/or exposure to high pH close to the epithelium as well as activation of the global transcription factor CRP are signals that induce secretion of the LT toxin in ETEC.
Highlights
Diarrhea caused by Enterotoxigenic Escherichia coli (ETEC) remains a major cause of mortality and morbidity worldwide
The expression of heat labile enterotoxin LT has convincingly been shown to be repressed at the level of transcription by the cyclic AMP receptor protein Cyclic AMP receptor protein (CRP) in the main laboratory ETEC strain H10407 [16]
To investigate both LT production and secretion regulated by CRP, LT toxin was measured by GM1 ELISA in the LT/STh/STp-expressing strain H10407wt, the isogenic H10407DCRP and the corresponding CRP complemented recombinant strain DCRP Rec grown in unbuffered LBK media
Summary
Diarrhea caused by Enterotoxigenic Escherichia coli (ETEC) remains a major cause of mortality and morbidity worldwide. ETEC is a leading cause of infectious diarrhea in young children in developing countries and represent the principal etiological agent of travelers diarrhea [1,2,3]. LT is a multimeric A-B5-type toxin, consisting of a single A subunit, LTA, and a ring of five B subunits, LTB. LTB mediates the toxins binding properties and LTA ADP ribosylates host G proteins [4,5]. LTA acts on Gsa, a protein that governs the activity of adenylate cyclase in eukaryotic cells. The ADP ribosylation of Gsa results in the overproduction of cyclic AMP (cAMP), which causes increased chloride secretion and disrupts sodium absorption [4]. Each subunit of LT is translated separately from a bicistronic message and transported to the periplasm, where holotoxin assembly either might occur spontaneously [7,8] or with the aid of the disulfide bridge forming protein DsbA [9,10,11]
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