Model of total LT production and secretion.

<p>A) The total production defined at LT both in the pellet fraction and in the supernatant fraction was higher in E2863ΔCRP than in the wild-type E2863 confirming that transcription of the <i>eltAB</i> operon is induced in the absence of cAMP-CRP. Secretion was decreased in E2863ΔCRP demonstrating that CRP is a positive regulator of LT secretion. B) At pH 5 secretion of LT is inhibited and the outer environment and the periplasmic space is acidic and the proton gradient favors an influx of protons into the cytosol. In order to keep the cytosol close to neutral there is a net outflux of protons from the cytosol. At neutral pH the cytosol is kept slightly more alkaline than the periplasm favouring a normal proton gradient and some LT is secreted At pH 9 the proton concentration is lower in the periplasm than the cytosol creating an inverted proton gradient and LT production and secretion is induced in response to the alkaline conditions in the periplasm. C) The inverted proton gradient poses a stress for the bacterial cell resulting in lower growth rate at pH 9 while growth at pH 5 supported growth to higher OD values of bacterial cells than pH 9.</p

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

<div><p>Enterotoxigenic <i>Escherichia coli</i> (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 <i>in vitro</i> and <i>in vivo</i>. 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.</p></div

Effect of the pH on the total production (A) and secretion (B) of LT toxin.

<p>The wild-type (E2863 wt) and cAMP receptor protein deficient strain (E2863ΔCRP) were grown in LBK media adjusted to different pH values as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074069#s2" target="_blank">Material and Methods</a>. The concentration of LT toxin was determined by GM1-ELISA in the pellet and supernatant. Total production was defined as the total LT present in the pellet and supernatant and percentage of secretion as the ratio of LT in the supernatant to total LT. Bars represent the mean and error bars represent the standard deviations of 9 independent cultures. A Student t test was used to calculate P values, *P<0.05, **P<0.01, ***p<0.001.</p

Transcription of the <i>eltAB</i> operon is repressed by CRP independently of pH.

<p>The gene expression levels were determined for E2863wt and E2863ΔCRP by q-PCR as transcripts per bacteria calculated from OD values of the cultures in LBK media adjusted at different pH. A) pH 5, B) pH 7 and C) pH 9. The bars represent the mean value of 4 analyses. A Student t test was used to calculate the P values, *P<0.05, **P<0.01, ***p<0.001.</p

Regulation by CRP of the production and secretion of LT toxin.

<p>H10407 wild-type strain (H10407wt), cAMP receptor protein deficient strain (H10407ΔCRP), the crp complemented strain (ΔCRP-Rec), E2863 wild-type strain (E2863 wt) and cAMP receptor protein deficient strain (E2863ΔCRP) were cultured in LBK medium aerobically at 37°C. The concentrations of LT toxin were determined by GM1-ELISA in the pellet and supernatant fractions after 3 and 5 hours of growth and from overnight culture and results were pooled. Bars represent the mean and error bars represent the standard deviations of 9 independent experiment. Total production was defined as the total LT present in the pellet and supernatant and secretion as the ratio of LT supernatant to total LT. The number of transcripts of <i>eltAB</i> gene that encode LT toxin was quantified by q-PCR. A Student t test was used to calculate P values. *P<0.05, **P<0.01, ***p<0.001 A) Total production of H10407. B) Percentage of secretion of H10407. C) Total production of E2863 D) Percentage of secretion of E2863 E) Transcription of <i>eltAB</i> gene of E2863.</p

Production and transcription of the LT toxin depended on CRP is higher at early hours of the bacteria growth.

<p>E2863 wt and E2863ΔCRP were growth in LBK media and samples were taken at 3, 5 hours and from overnight cultures. GM1 ELISA was used to measure the total production and secretion of LT toxin. Expression of the toxin was quantified in <i>eltAB</i> gene for LT toxin by q-PCR. Bars represent the mean and error bars represent the standard deviations of n = 9 (ELISA) and n = 3 (q-PCR). A Student t test was used to calculate P values, *P<0.05, **P<0.01. A) Total production. B) Percentage of secretion C) Number of transcripts of <i>eltAB</i> gene.</p

CRP is a positive regulator of LT secretion <i>in vivo</i>.

<p>The wild-type (E2863 wt) and cAMP receptor protein deficient strain E2863ΔCRP) were appropriately diluted in PBS and orogastrically inoculated into infant mice. Three hours post-infection 8 small intestines per bacterial strain were collected and used for estimation of LT secretion by GM1-ELISA as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074069#s2" target="_blank">Material and Methods</a>. Bars represent the geometric mean and error bars represent the standard deviations of 3 independent experiments. A Student t test was used to calculate P values, *P<0.05.</p

Determinations of the relative amounts of Ogawa antigen in LPS preparations of the generated Hikojima strains MS1568 and MS1580 based on amounts of methylated and un–methylated perosamine analyzed with mass spectrometry in relation to similar analyses of LPS preparations from reference Inaba Phil6973 and Ogawa Cairo 50 strains.

<p>The area under the curve for the methylated part (m/z ratio 756.6) divided by the area under the curve for the non–methylated part (m/z ratio 742.6) subtracted with the background (this ratio from the Inaba strain Phil6973) was used for the two Hikojima strain (MS1568 and MS1580) LPS preparations to calculate their percentage of Ogawa LPS (compared to the 100% Ogawa in the LPS from the Cairo50 reference strain). Diagrams are from one of 3 such experiments showing closely similar patterns and with the calculated mean ± SEM percentages Ogawa antigen indicated for MS1568 and MS1580.</p

Colony blot results.

<p>O1 Inaba <i>V. cholerae</i> strain JS1569 was transformed with expression plasmids carrying different mutant <i>wbeT</i> genes. Different mutations in the <i>wbeT</i> gene give different levels of expression of the Ogawa antigen as seen by different levels of staining following labelling with Ogawa–specific antibodies. The plasmids were isolated and the <i>wbeT</i> genes sequenced. The mutations present in the different clones are indicated.</p

A schematic figure over the construction of the suicide plasmid and its incorporation into and it’s the removal from the genome.

<p>(A) Amplification of the mutated <i>wbeT</i> gene the upstream DNA was generated to be fused together in a primerless PCR. (B) The fused sequenced were used in another primerless PCR together with the kanamycin resistant gene flaked by FRT–sites and the downstream sequenced of <i>wbeT</i> gene. (C) The result of the second primerless PCR used to be incorporated into the pMT–suicide–sacB plasmid for integration into the genome of O1 <i>V. cholerae</i>. (D) Primer orientation of the wbe region before and (E) after gene manipulation.</p