Cyclic AMP Receptor Protein-Dependent Repression of Heat-Labile Enterotoxin ▿

Enterotoxigenic Escherichia coli is a major cause of acute diarrheal illness worldwide and is responsible for high infant and child mortality rates in developing nations. Two types of enterotoxins, one heat labile and the other heat stable, are known to cause diarrhea. The expression of soluble heat-labile toxin is subject to catabolite (glucose) activation, and three binding sites for cAMP receptor protein (CRP or CAP) were identified upstream and within the toxin promoter by DNase I footprinting. One CRP operator is centered at −31.5, thus encompassing the promoter's −35 hexamer. Potassium permanganate footprinting revealed that the occupancy of this operator prevents RNA polymerase from forming an open complex in vitro. However, the operator centered at −31.5 is not sufficient for full repression in vivo because the deletion of the other two CRP binding sites partially relieved the CRP-dependent repression of the heat-labile toxin promoter. In contrast to heat-labile toxin, CRP positively regulates the expression of heat-stable toxin. Thus, the conditions for the optimal expression of one enterotoxin limit the expression of the other. Since glucose inhibits the activity of CRP by suppressing the pathogen's synthesis of cyclic AMP (cAMP), the concentration of glucose in the lumen of the small intestine may determine which enterotoxin is maximally expressed. In addition, our results suggest that the host may also modulate enterotoxin expression because cells intoxicated with heat-labile toxin overproduce and release cAMP

Repression of the Inner Membrane Lipoprotein NlpA by Rns in Enterotoxigenic Escherichia coli

The expression of the inner membrane protein NlpA is repressed by the enterotoxigenic Escherichia coli (ETEC) virulence regulator Rns, a member of the AraC/XylS family. The Rns homologs CfaD from ETEC and AggR from enteroaggregative E. coli also repress expression of nlpA. In vitro DNase I and potassium permanganate footprinting revealed that Rns binds to a site overlapping the start codon of nlpA, preventing RNA polymerase from forming an open complex at nlpAp. A second Rns binding site between positions −152 and −195 relative to the nlpA transcription start site is not required for repression. NlpA is not essential for growth of E. coli under laboratory conditions, but it does contribute to the biogenesis of outer membrane vesicles. As outer membrane vesicles have been shown to contain ETEC heat-labile toxin, the repression of nlpA may be an indirect mechanism through which the virulence regulators Rns and CfaD limit the release of toxin

CfaD-Dependent Expression of a Novel Extracytoplasmic Protein from Enterotoxigenic Escherichia coli

H10407 is a strain of enterotoxigenic Escherichia coli (ETEC) that utilizes CFA/I pili to adhere to surfaces of the small intestine, where it elaborates toxins that cause profuse watery diarrhea in humans. Expression of the CFA/I pilus is positively regulated at the level of transcription by CfaD, a member of the AraC/XylS family. DNase I footprinting revealed that the activator has two binding sites upstream of the pilus promoter cfaAp . One site extends from positions −23 to −56, and the other extends from positions −73 to −103 (numbering relative to the transcription start site of cfaAp ). Additional CfaD binding sites were predicted within the genome of H10407 by computational analysis. Two of these sites lie upstream of a previously uncharacterized gene, cexE . In vitro DNase I footprinting confirmed that both sites are genuine binding sites, and cexEp :: lacZ reporters demonstrated that CfaD is required for the expression of cexE in vivo. The amino terminus of CexE contains a secretory signal peptide that is removed during translocation across the cytoplasmic membrane through the general secretory pathway. These studies suggest that CexE may be a novel ETEC virulence factor because its expression is controlled by the virulence regulator CfaD, and its distribution is restricted to ETEC

Residues near the Amino Terminus of Rns Are Essential for Positive Autoregulation and DNA Binding▿

Most members of the AraC/XylS family contain a conserved carboxy-terminal DNA binding domain and a less conserved amino-terminal domain involved in binding small-molecule effectors and dimerization. However, there is no evidence that Rns, a regulator of enterotoxigenic Escherichia coli virulence genes, responds to an effector ligand, and in this study we found that the amino-terminal domain of Rns does not form homodimers in vivo. Exposure of Rns to the chemical cross-linker glutaraldehyde revealed that the full-length protein is also a monomer in vitro. Nevertheless, deletion analysis of Rns demonstrated that the first 60 amino acids of the protein are essential for the activation and repression of Rns-regulated promoters in vivo. Amino-terminal truncation of Rns abolished DNA binding in vitro, and two randomly generated mutations, I14T and N16D, that independently abolished Rns autoregulation were isolated. Further analysis of these mutations revealed that they have disparate effects at other Rns-regulated promoters and suggest that they may be involved in an interaction with the carboxy-terminal domain of Rns. Thus, evolution may have preserved the amino terminus of Rns because it is essential for the regulator's activity even though it apparently lacks the two functions, dimerization and ligand binding, usually associated with the amino-terminal domains of AraC/XylS family members