Cholera, a debilitating enteric disease of humans, is characterized by massive diarrhea that is frequently lethal when untreated. Typically arising from ingestion of contaminated drinking water, the disease is caused by toxigenic strains of the gram-negative bacterium Vibrio cholerae through their colonization of the small intestine and production of cholera toxin (CT), an enterotoxin (5, 15). The bacterium occurs in coastal, bay, and estuarine waters and in association with aquatic plankton as an autochthonous member of aquatic microbial communities. Along with other environmental factors, salinity is thought to govern the distribution of V. cholerae in aquatic environments and consequently might play a key role in the incidence and seasonal occurrence of the disease (2, 3, 7-11, 16, 17, 22, 25, 26, 30, 31, 34). Understanding how the bacterium adapts to changes in salinity in its transitions between aquatic habitats, sources of drinking water, and the human intestine may therefore provide critical insight into the interrelated issues of the environmental dynamics of the bacterium and its pathogenic interactions with humans. In this issue, Shikuma and Yildiz (29) report a transcript profiling approach leading to identification of V. cholerae genes differentially regulated by salinity (osmolarity). Their identification and analysis of salinity-responsive genes, and in particular the identification of a new osmolarity-responsive regulator, OscR, open up new territory on how V. cholerae copes with and responds adaptively to changes in salinity during its transitions between the environment and the human intestine.
Salinity and cholera.
Cholera is endemic in coastal, estuarine, and riverine waters of Bangladesh and India. In these locations, the disease undergoes pronounced seasonal cycles, with two peaks per year, one in the dry season and one following the monsoon rains. The cycles appear to correlate with sea surface temperature, rainfall, and other climate variables that could influence the abundance of the bacterium and its transmission from environmental reservoirs (4, 12, 13, 22, 23, 27). During the dry season, incursion of seawater due to low river flow might transport the bacterium inland from coastal habitats and salinities in sources of drinking water presumably increase to levels that would support the survival and reproduction of V. cholerae (16, 17). During monsoons, flood conditions could then lead to widespread dissemination of the bacterium into freshwater habitats (13). The distribution of V. cholerae in aquatic habitats correlates with salinity, and although it reproduces more rapidly at moderate salinities, similar to those of coastal seawater, V. cholerae survives and can reproduce at the very low salinities associated with some freshwater sources (9, 14, 16, 17, 21, 33). This ability might play a key role in the incidence of cholera in areas where the disease is endemic by allowing V. cholerae, atypically among many Vibrio species, to persist in sources of drinking water and thereby to come into contact with humans. Furthermore, colonization of external surfaces and gut tracts of copepods and other aquatic plankton, which potentially serve as vectors for the bacterium, might further enhance the survival of V. cholerae in freshwater systems and thereby increase its contact with humans (3, 4, 25, 26).
In addition to coping with a range of salinities in its aquatic habitats, V. cholerae is likely to experience substantial changes in osmolarity when it is ingested into the human gastrointestinal tract, establishes itself in the small intestine and causes the release of fluids and ions from intestinal tissue, and is then expelled back into the aquatic environment in the ensuing diarrhea. Given these transitions, it is not surprising that genes involved in virulence are subject to control by osmolarity (6, 15, 18) and that the bacterium expresses genes late in the infection that enhance its survival in the aquatic environment (28).
Salinity-regulated genes.
The transcriptional profiling reported by Shikuma and Yildiz (29), using a strain of V. cholerae O1 El Tor grown at different salinities, identified over 300 genes regulated twofold or greater by salinity. Many of the genes were annotated as carrying out metabolic and cellular processes, including genes involved in pathogenesis. Over 40% of the salinity-regulated genes, however, were identified as coding for hypothetical proteins. In the several specific cases examined using chromosomal lacZ fusions, osmolarity of growth medium, rather than salinity per se, was the factor controlling expression. The regulated genes were grouped in three broad classes by their responses to salinity, i.e., upregulated at high, mid-range, or low salinity. Notable among genes upregulated at high salinity, some of which had been identified previously as responsive to ionic or osmotic medium composition (24, 32), were those coding for Na+/H+ antiporters and for transport and biosynthesis of compatible solutes.
Genes upregulated at mid-range salinity.
The expression of the two main virulence factors of V. cholerae, CT and toxin-coregulated pilus, is known to be regulated by salinity (osmolarity) (6, 18, 19); osmotic regulation is thought to operate via ToxR, a transmembrane transcriptional regulator that controls the bacterium's complex virulence gene regulatory network (5, 15, 19). Shikuma and Yildiz (29) demonstrate through transcript profiling that the CT genes (ctxA and ctxB), tcp, the regulatory genes tcpP, tcpH, and toxT, and the ToxR-regulated genes ompU and ompT exhibit maximal expression at mid-range salinities. Furthermore, vps and rbm, key genes involved in biofilm formation, were identified as upregulated at mid-range salinities. Biofilm formation was shown to be modulated by salinity, apparently under the control of the vps transcriptional regulators VpsT and VpsR.
OscR, a new osmolarity-responsive regulator.
Among the genes whose expression levels were highest at low salinity were those for certain membrane-derived oligosaccharides and for production of polyamine. Particularly interesting in this class, however, is a gene designated oscR (osmolarity-controlled regulator). The transcript levels for oscR were two- to threefold higher at low than at moderate salinity (osmolarity). OscR is a member of the IclR family of transcriptional regulators, which are found in a variety of gram-negative and gram-positive bacteria and which function as repressors, activators, or both; these proteins contain a GAF domain, which binds cyclic nucleotides and sodium ions (1, 20). Hypothetical proteins homologous to OscR are found in other Vibrio species and other gram-negative bacteria (29).
Mutation analysis, i.e., using a ΔoscR mutant, revealed that OscR functions to negatively regulate the expression of many genes and to increase the expression of some, but apparently only at low osmolarity. Specifically, several of the vps genes are downregulated in the presence of OscR at low osmolarity. Apparently, either OscR or a regulatory factor controlled by OscR functions to repress vps gene expression under low-osmolarity conditions. This pattern of gene expression correlates with altered biofilm formation at low osmolarity. An implication of this control is that biofilm formation may be less adaptive to V. cholerae under low osmolarity conditions, although it is unclear at present why biofilm formation would not be equally beneficial to the bacterium at different salinities. Conversely, the expression of flagellin genes, e.g., flaB and flaD, and motility apparently are induced in the presence of OscR, but again, only under low-osmolarity conditions (29). These findings suggest that the bacterium senses and mounts a substantial and highly specific adaptational response to low osmolarity.
Perspective.
It is tempting to speculate that the ability of V. cholerae to persist under low-salinity conditions, thereby allowing opportunities for contact between the bacterium and humans via sources of drinking water, is a necessary preadaptation for causing disease. Regardless of that possibility, the identification of OscR is an important first step in characterizing the low-osmolarity response of V. cholerae. Given the large number of genes controlled by OscR at low osmolarity (29), defining this response and examining its possible relationship to human disease will undoubtedly involve much additional, interesting work.
FOOTNOTES
↵▿ Published ahead of print on 24 April 2009.
The views expressed in this Commentary do not necessarily reflect the views of the journal or of ASM.
- American Society for Microbiology
