Alkaline pH Is a Signal for Optimal Production and Secretion of the Heat Labile Toxin, LT in Enterotoxigenic Escherichia Coli (ETEC)

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.

IscR Regulates Synthesis of Colonization Factor Antigen I Fimbriae in Response to Iron Starvation in Enterotoxigenic Escherichia coli

“…Finally, it is important to note that although LT and ST have previously been shown to undergo differential expression (11), neither was induced under iron starvation here. Consistent with recently published data (12), our data support the hypothesis that LT and ST secretion may occur in tandem, responding in a similar manner to key environmental cues.…”

“…Furthermore, the amount of secreted LT detected in the culture supernatant dropped to undetectable levels under iron starvation. As LT production occurs early during infection (12), periplasmic LT levels were also quantified in exponentially growing cells to determine if the in vitro growth phase influenced the toxin response. Although the total amount of LT was higher during exponential growth phase, a similar decrease in LT was seen in cultures treated with deferoxamine (data not shown).…”

“…The cfaABCE (cfa) and eltAB operons, encoding the CFA/I fimbriae and the LT, are regulated in response to various environmental signals, including temperature and glucose, via the H-NS and cyclic AMP receptor protein (CRP) transcriptional regulators, respectively (9)(10)(11)(12)(13). cfa expression is further activated by the specific virulence regulator CfaD, although it remains unknown whether CfaD activation is dependent on any external cues (9).…”

Allele Variants of Enterotoxigenic Escherichia coli Heat-Labile Toxin Are Globally Transmitted and Associated with Colonization Factors

“…Under classical laboratory culture conditions, individual ETEC isolates differ in their abilities to secrete LT into the medium. Some strains retain LT predominantly in the periplasm or associated with lipopolysaccharide (LPS) in the outer membrane, while other strains secrete as much as 50% of the LT produced into the medium (3,7,11,12). When ETEC attaches to surface intestinal epithelial cells, the mature holotoxin is transferred to the host cell, where it can undergo posttranslational modifications leading to full activation.…”

“…In the periplasm, monomers assemble spontaneously or by DsbA disulfide oxidoreductase activity and are then secreted by the general (type II) secretion pathway (GSP) in a pH-dependent manner (9)(10)(11). Under classical laboratory culture conditions, individual ETEC isolates differ in their abilities to secrete LT into the medium.…”