Multiple intraintestinal signals coordinate the regulation of Vibrio cholerae virulence determinants.

Vibrio cholerae is a Gram-negative motile bacterium capable of causing fatal pandemic disease in humans via oral ingestion of contaminated water or food. Within the human intestine, the motile vibrios must evade the innate host defense mechanisms, penetrate the mucus layer covering the small intestine, adhere to and multiply on the surface of the microvilli and cause disease via the action of cholera toxin. The explosive diarrhea associated with V. cholerae intestinal colonization leads to dissemination of the vibrios back into the environment to complete this phase of the life cycle. The host phase of the vibrio life cycle is made possible via the concerted action of a signaling cascade that controls the synthesis of V. cholerae colonization determinants. These virulence proteins are coordinately synthesized in response to specific host signals that are still largely undefined. A more complete understanding of the molecular events involved in the V. cholerae recognition of intraintestinal signals and the subsequent transcriptional response will provide important information regarding how pathogenic bacteria establish infection and provide novel methods for treating and/or preventing bacterial infections such as Asiatic cholera. This review will summarize what is currently known in regard to host intraintestinal signals that inform the complex ToxR regulatory cascade in order to coordinate in a spatial and temporal fashion virulence protein synthesis within the human small intestine.

The Vibrio cholerae ToxR Regulon Encodes Host-Specific Chemotaxis Proteins that Function in Intestinal Colonization.

Virulence gene regulation in Vibrio cholerae is under the control of the ToxR-ToxT regulatory cascade. Chemotaxis and net motility have been shown to influence the infectivity of Vibrio cholerae. V. cholerae toxR mutants do not synthesize proteins required for chemotaxis towards mucus. The inability of the toxR mutant strain to recognize and swim towards mucus is due to their failure to synthesize AcfB, a methyl-accepting chemotaxis protein. AcfB has previously been shown to be involved in intestinal colonization using the infant mouse model of cholera infection. Wild type V. cholerae recognizes galactose-6-sulfate in the capillary tube assay whereas V. cholerae acfB mutants fail to migrate into the capillary tubes. Vibrio strains carrying a mutation in tcpI, a ToxR regulated gene found within the Vibrio Pathogenicity Island (VPI), which encodes a methyl accepting chemotaxis protein are fully chemotactic towards mucus and galactose-6-sulfate.

Expression of Vibrio cholerae virulence genes in response to environmental signals.

Vibrio cholerae, the causative agent of Asiatic cholera, is a gram-negative motile bacterial species acquired via oral ingestion of contaminated food or water sources. The O1 serogroup of V. cholerae is responsible for pandemic cholera and is divided into two biotypes, classical and El Tor (Butterton and Calderwood, 1995; Mekalanos, 1985). The El Tor biotype is responsible for the current cholera pandemic. In the absence of disease, the vibrio life cycle consists of a free-swimming phase in marine and estuarine environments in association with zooplankton, crustaceans, insects, and water plants. Vibrios interact with various surfaces found in the environment to generate biofilms which may promote survival (Watnick etaL, 1999). Within the host the motile vibrios must evade the innate host defense mechanisms, penetrate the mucus layer covering the intestinal villi, adhere to and colonize the epithelial surface of the small intestine, assume a non-motile phase, replicate and cause disease by secreting numerous exoproteins at the site of infection (Oliver and Kaper, 1997). The voluminous diarrhea associated with cholera infection leads to the dissemination of the vibrios back into a watery environment and thus a continuation of the environmental phase of the life cycle. The host phase of the vibrio life cycle is only possible through the action of a group of virulence genes (ToxR-regulon) controlled by a complex and incompletely understood regulatory cascade. The ToxR regulon colonization and toxin genes are coordinately expressed in response to specific host signals that have yet to be completely defined (Skorupsky and Taylor 1997). Although little is known regarding the host signals that impact the ToxR regulatory cascade, it is clear that these intraintestinal signals play an important role in maximizing the ability of the vibrios to survive and multiply within the host. Key to understanding the complex events involved in the pathogenesis of V. cholerae will be elucidating the intraintestinal signaling molecules that trigger the expression of vibrio virulence genes. Understanding the molecular basis of this host-parasite interaction will provide important information with respect to how pathogenic bacteria establish infection and provide insights leading to novel methods for treating and/or preventing bacterial infections. This review will summarize what is known regarding host signaling and the complex ToxR regulatory system employed by V. cholerae to coordinate virulence gene expression within the host.

Importance of the D and E helices of the molecular chaperone DnaK for ATP binding and substrate release.

The C-terminal domain of the molecular chaperone DnaK is a compact lid-like structure made up of five alpha-helices (alphaA-alphaE) (residues 508-608) that is followed by a 30-residue disordered, flexible region (609-638). The lid encapsulates the peptide molecule bound in the substrate-binding domain, whereas the function of the 30-residue disordered region is not known. By sequentially deleting the flexible subdomain and the individual lid helices, we deduced the importance of each structural unit to creating long-lived DnaK-peptide complexes. Here we report that (i) the alphaD helix is essential for long-lived DnaK-peptide complexes. For example, ATP triggers the dissociation of a acrylodan-labeled p5 peptide (ap5, a-CLLLSAPRR) from wtDnaK and DnaK595(A-D) with k(off) equal to 7.6 and 8.9 s(-1), respectively, whereas when the D-helix is deleted, creating DnaK578(A-C), k(off) jumps to 207 s(-1). (ii) The presence of the alphaB helix impacts the rate of the ATP-induced high-to-low affinity conformational change. For example, ATP induces this conformational change in a lidless variant, DnaK517(1/2A), with a rate constant of 442 s(-1), whereas, after adding back the B-helix (residues 518-554), ATP induces this conformational change in DnaK554(A-B) with a rate constant of 2.5 s(-1). Our interpretation is that this large decrease occurs because the B-helix of the DnaK554(A-B) is bound in the substrate-binding site. (iii) The deletion analysis also revealed that residues 596-638, which comprise the alphaE helix and the flexible subdomain, affect ATP binding. Our results are consistent with this part of the lid producing conformational heterogeneity, perhaps by binding to the ATPase domain.