Identification of Novel Components Influencing Colonization Factor Antigen I Expression in Enterotoxigenic <i>Escherichia coli</i>

<div><p>Colonization factors (CFs) mediate early adhesion of Enterotoxigenic <i>Escherichia coli</i> (ETEC) in the small intestine. Environmental signals including bile, glucose, and contact with epithelial cells have previously been shown to modulate CF expression in a strain dependent manner. To identify novel components modulating CF surface expression, 20 components relevant to the intestinal environment were selected for evaluation. These included mucin, bicarbonate, norepinephrine, lincomycin, carbon sources, and cations. Effects of individual components on surface expression of the archetype CF, CFA/I, were screened using a fractional factorial Hadamard matrix incorporating 24 growth conditions. As most CFs agglutinate erythrocytes, surface expression was evaluated by mannose resistant hemagglutination. Seven components, including porcine gastric mucin, lincomycin, glutamine, and glucose were found to induce CFA/I surface expression <i>in vitro</i> in a minimal media while five others were inhibitory, including leucine and 1,10-phenanthroline. To further explore the effect of components positively influencing CFA/I surface expression, a response surface methodology (RSM) was designed incorporating 36 growth conditions. The optimum concentration for each component was identified, thereby generating a novel culture media, SP1, for CFA/I expression. CFs closely related to CFA/I, including CS4 and CS14 were similarly induced in SP1 media. Other epidemiologically relevant CFs were also induced when compared to the level obtained in minimal media. These results indicate that although CF surface expression is complex and highly variable among strains, the CF response can be predicted for closely related strains. A novel culture media inducing CFs in the CF5a group was successfully identified. In addition, mucin was found to positively influence CF expression in strains expressing either CFA/I or CS1 and CS3, and may function as a common environmental cue.</p></div

Predicted and experimental read-outs of bacterial culture in SP1 media based on the response surface methodology.

<p>Experimental value is an average of at least 6 replicates</p><p>Predicted and experimental read-outs of bacterial culture in SP1 media based on the response surface methodology.</p

Effect of culture components on CFA/I surface expression, LT secretion, and growth.

<p>The effect of individual components on CFA/I surface expression (A), LT secretion (B) and bacterial growth based on final density after overnight culture (C) was determined. The CFA/I expressing strain 258909–3 was cultured overnight in M9-low media supplemented with component combinations defined in the Hadamard matrix. CFA/I surface expression was evaluated by MRHA, LT toxin secretion by ELISA-GM1 of culture supernatant, and growth by OD₆₀₀. Components with a significant effect on a given response are shown in grey and those with no effect in black, with dashed lines representing the threshold of significance for each endpoint (p<5%). Coefficients are indicated for each component and indicate the relative strength of the response. PGM: porcine gastric mucin; 1,10 o-phen: 1,10-phenanthroline.</p

Evaluation of CF surface expression following growth in optimized culture media SP1.

<p>CF surface expression was evaluated by MHRA in ETEC strains following overnight growth in M9, SP1, or CFA media in the absence or presence of 0.15% bile salts (BS). CF surface expression was evaluated in strains expressing fimbriae within the CF5a family (A) or other CFs (B). Data are expressed as the minimal hemagglutination titer (MHT) required for agglutination of human or bovine erythrocytes transformed in log₂ for normal distribution. ND indicates not detected. The hemagglutination pattern of CS17-expressing strain is shown in (C). *Strains expressing CS14 or CS17 were cultivated in M9 or CFA media supplemented with BS, while all other strains were cultivated in M9 or CFA in the absence of BS. Data are shown as average of at least three independent replicates, with bars representing the SEM. Statistical analyses were performed using the Mann Whitney test (A), with surface expression in SP1 and CFA media compared to basal M9 media. *p<0.01, ** p<0.001.</p

Effect of PGM on surface expressed CFA/I or CS1/CS3.

<p>ETEC strains expressing CFA/I (H10407) or CS1/CS3 (E24377A) were cultured in M9 media alone or supplemented with all components found to have a positive effect (p<10%) on CFA/I via the Hadamard matrix including glucose, aspartic acid, glutamine, FeSO₄, bicarbonate, lincomycin and EGTA (PC) in the presence or absence of porcine gastric mucin (PGM). MRHA was performed following overnight growth, with the minimal hemagglutination titer (MHT) expressed in log₂. Means of at least two independent replicates are shown with error bars representing the standard error of the mean (SEM). Statistical analyses were performed using the Mann-Whitney test, * p<0.05, ** p<0.01. ND: not detected.</p

ETEC virulence response in M9 media as compared to the CFA reference media.

<p>CFA and M9 were compared using the Student t-test, p-val is p-value, ns is not significant. Values are ± SEM. MHT: minimal hemagglutination titer</p><p>ETEC virulence response in M9 media as compared to the CFA reference media.</p

Identification of an optimum condition for CFA/I surface expression using response surface methodology.

<p>Identification of an optimum condition for CFA/I surface expression using response surface methodology.</p

Virulence traits and origin of potato soft-rot pathogens.

*<p>The potential of each strain to induce a tuber soft-rot was evaluated in potato host plant seven days after infection by intra-medulla injection.</p>#<p>The potential of each strain to induce a hypersensitive response (HR) was evaluated in tobacco non host plant 24 hours after leaves infiltration.</p><p>CFBP, Collection Française de Bactéries Phytopathogènes, Institut National de la Recherche Agronomique, Angers, France.</p

Bacterial QS signal producers or biosensors, and plasmids.

<p>Gm^r and Tet^r indicate resistance to gentamicin and tetracycline, respectively.</p><p>NAHSL, N-acyl homoserine lactones; AI-2, Autoinducer-2; HHQ, 4-hydroxy-2-heptylquinoline; PQS, Pseudomonas quinolone signal.</p

Characterization of signaling molecules produced by potato soft-rot pathogens.

*<p>Production of different N-acyl homoserine lactones (NAHSL) was determined by thin-layer chromatography (TLC) and HPLC-MS/MS. For quantification, NAHSL were extracted from PGA minimal medium culture at late exponential phase (optimal production).</p>#<p>Autoinducer-2 (AI-2) activity was determined using biosensor <i>V. harveyi</i> BB170.</p>°<p>Production of 4-hydroxy-2-heptylquinoline (HHQ) and Pseudomonas quinolone signal (PQS) were determined by TLC.</p>†<p>Production of indole-3-acetic acid (IAA), indole-3-propionic acid (IPA), indole-3-butyric acid (IBA) and kynurenic acid (KA) were determined by HPLC-UV. For quantification, indolic compounds were extracted from M9 minimal medium supplemented with l-tryptophan culture at stationary growth phase (optimal production).</p>§<p>Production of γ-amino butyric acid (GABA) was determined by ELISA test.(+) positive detection by the biosensor; (−) not detected or below threshold.</p