Vibrio cholerae

Classification and Genomics

V. cholerae organisms are oxidase-positive facultative anaerobes that can be identified using a variety of biochemical tests and selective media. The organisms are characteristically highly motile and bear a unipolar sheathed flagellum. V. cholerae is classified into more than 200 serogroups; differences in the composition of the O-specific polysaccharide (OSP) chains of lipopolysaccharide (LPS) molecules establish the chemical basis for the distinct antigenicity of the serogroups. The O1 serogroup of V. cholerae was the only serogroup associated with epidemic cholera until 1992, when V. cholerae O139 emerged. However, some non-O1, non-O139 V. cholerae serogroups have given rise to gastroenteritis outbreaks, but not to large epidemics. In general, the majority of V. cholerae isolates from the environment including those of V. cholerae O1 do not produce the virulence factors, such as cholera toxin (CT), that are required to cause cholera. The O1 serogroup is divided into two principal serotypes, Inaba and Ogawa, which differ by the presence of a 2- O -methyl group in the nonreducing terminal sugar of the Ogawa OSP, which is absent from Inaba OSP. Switching of serotypes during epidemics is well described; the driving forces underlying switching are not known, but they do not appear to be random. CT-producing (toxigenic) O1 serogroup strains are also divided into two biotypes based on a number of microbiologic and biochemical differences. The classic biotype of V . cholerae O1 caused the second and sixth cholera pandemics and probably the earlier and intervening pandemics as well, whereas the El Tor biotype of V. cholerae O1 is the cause of the ongoing seventh cholera pandemic. The classic biotype of V . cholerae is now likely extinct.

V. cholerae, similar to many other bacterial enteric pathogens, is classified as a Gammaproteobacteria. In contrast to most Gammaproteobacteria such as Escherichia coli, whose genomes consist of single circular chromosomes, the genome of V. cholerae is multipartite. The V. cholerae genome (and that of all Vibrio spp.) is unequally divided between two circular chromosomes. Its large chromosome (≈3 kb) contains the vast majority of the essential genes as well as genes implicated in pathogenicity, whereas the small chromosome (≈1 kb) is enriched for genes of unknown function. A plausible explanation for the evolution of the bipartite V . cholerae genome is that a remote ancestor of V. cholerae (and all vibrios) acquired a large plasmid that subsequently obtained essential genes and other chromosome-like features. Acquisition of virulence-linked genes from other (unknown) donor organisms via lateral gene transfer has played a central role in the evolution of pathogenic V. cholerae . For example, the genes encoding CT are encoded within a bacteriophage that infected and integrated its DNA into the genome of a nontoxigenic precursor of toxigenic V. cholerae O1.

Pathogenicity

V. cholerae has no known natural vertebrate host besides humans. However, the pathogen is not dependent on humans for its propagation; V. cholerae grows in brackish estuaries and coastal seawaters, often in close association with copepods or other zooplankton. V. cholerae can also grow in water of lower salinity when it is warm and adequate organic material is available. Because patients with severe cholera can excrete up to 20 L of “rice water” stool laden with ≈10 V. cholerae cells/mL per day, humans greatly facilitate the propagation and dissemination of the pathogen. Humans usually become infected with V. cholerae after ingestion of contaminated water or food, although there may be direct fecal-oral spread between people that does not involve water or food. The infectious dose in volunteer studies carried out in the United States is relatively high. Low stomach pH is known to lower the infectious dose in volunteers; additional factors likely impact the infectious dose as well. Because the frequency of hypochlorhydria (caused by chronic Helicobacter pylori infection) in cholera endemic regions is often high, the V. cholerae infectious dose may be considerably lower in these regions.

After passage through the stomach, V. cholerae has the unusual capacity to survive and multiply in the small intestine. Although many processes and gene products contribute to the organism's ability to colonize the small intestine, toxin coregulated pili (TCP) are the most critical identified factors primarily dedicated to mediating colonization. The major subunit of these pili is encoded by tcpA, the first gene of the tcp operon. Most of the other genes of the operon encode the machinery for the biogenesis of TCP, but tcpE encodes a protein found at the tip of the pilus, and tcpF encodes a protein secreted by the TCP assembly apparatus that is also required for colonization. TCP are thought to promote V. cholerae intestinal colonization in at least three ways: TCP (1) mediate bacterium-bacterium interactions, enabling microcolony formation within the small intestine; (2) confer protection against toxic factors produced in the intestine; and (3) promote attachment to the intestinal epithelium. The tcp operon is found within the TCP pathogenicity island, a chromosome segment ≈41 kb that also encodes additional virulence-associated factors, which appears to have been acquired via horizontal gene transfer by a nonpathogenic ancestor of contemporary V. cholerae . In addition, the type VI secretion system may be induced in the intestine, promoting colonization potentially by enabling interactions with the microbiota.

While colonizing the small intestine, V. cholerae secretes CT, the AB ₅ subunit–type protein toxin that causes the secretory diarrhea that is characteristic of cholera. The sufficiency of CT to elicit cholera-like diarrhea was demonstrated experimentally; volunteers fed as little as 5 µg of purified CT developed severe diarrhea indistinguishable from cholera. Thus cholera is a toxin-mediated disease, and V. cholerae is a noninvasive mucosal pathogen. The pentameric B subunit of CT binds to GM1, a glycosphingolipid on the surface of epithelial cells. The CT-GM1 interaction targets the toxin through a retrograde trafficking pathway from the plasma membrane to the Golgi apparatus and endoplasmic reticulum and then to the cytoplasm. In the cytoplasm, the enzymatic A subunit of CT mediates the transfer of adenosine diphosphate ribose from nicotinamide adenine dinucleotide to the G protein that regulates adenylate cyclase activity, leading to elevation in the intracellular cyclic adenosine monophosphate (cAMP) concentration. Increased cAMP levels promote chloride ion (Cl ⁻ ) secretion by intestinal crypt cells and decreased absorption by villous cells. Water moves from epithelial cells into the bowel lumen to maintain osmolality, and diarrhea results when the resorptive capacity of the remainder of the gut is exceeded. Additional actions of CT (besides dysregulation of adenylate cyclase) likely contribute to the secretory response underlying cholera. Also, other factors besides CT contribute to the diarrheal response because volunteers who ingest V. cholerae deficient for ctxA often develop mild diarrhea.

The genes encoding CT, ctxA and ctxB (ctxAB) , are not part of the ancestral V . cholerae genome; instead, these genes are embedded in the genome of CTXΦ, a filamentous bacteriophage that infected V. cholerae, rendering it toxigenic. Of note, the receptor for this phage is TCP, suggesting that a TCP+, ctxAB − strain was the precursor of contemporary pathogenic V. cholerae . The intricate interdependence of these two mobile elements—the TCP pathogenicity island and the CTX prophage—extends even further; ToxT, a key transcriptional activator of ctxAB expression, similar to the tcp operon, is encoded within the TCP island. Mobile genetic elements such as phages, plasmids, integrative conjugative elements, and pathogenicity islands have played critical roles in the evolution of most bacterial pathogens.

Many of the key virulence factors of V. cholerae such as CT and TCP are not constitutively expressed. Instead, fairly shortly after V. cholerae is ingested, as yet undefined host signals trigger the coordinated expression of the principal virulence factors of V. cholerae . A membrane-embedded transcription activator, ToxR, sits at the top of a signaling cascade that promotes expression of many of the pathogen's virulence-associated genes. There may be a specific temporal order of V. cholerae gene expression during expression. Furthermore, as the pathogen passes to more distal parts of the intestine, expression of late genes promotes V. cholerae survival outside the human host and engenders a transient hyperinfectious state, which promotes transmission of the organism to new hosts. Additional regulatory pathways such as quorum sensing systems, which modify gene expression according to bacterial density and the concentrations of autoinducer molecules, modulate expression of V. cholerae virulence. Notably, it has been suggested that crosstalk between the quorum sensing molecules of the microbiota and quorum regulation of V. cholerae impact the pathogen's capacity to colonize the intestine.