The Estuary Effect: Variations in Temperature and Salinity Alter msh Promoter Activity in Vibrio cholerae

Vibrio cholerae , the facultative pathogen underlying cholera, naturally inhabits warm aquatic estuaries. Environmental persistence is enhanced by the ability of V. cholerae to colonize host reservoirs and form multicellular biofilms, causing seasonally endemic outbreaks in many tropical regions. Most toxigenic strains utilize the type IVa mannose-sensitive hemagglutinin (MSHA) pilus for host reservoir colonization and biofilm formation. Temperature and salinity can alter V. cholerae biofilm formation, yet their impact on MSHA production specifically remains largely unknown. Here, we utilized transcriptional reporters of predicted msh promoters ( msh -P1/ msh -P2/ msh -P3) and functional assays, to determine temperature and salinity impacts on msh expression and pilus biogenesis. Under standard laboratory conditions (30°C, 1% NaCl) only msh -P1/P2 are active and inversely-coordinated with one another. Both msh -P1/P2 activity were elevated by high temperature (37°C) and low salinity (0.25%/0.5% NaCl), and reduced by low temperature (20°C/25°C) and high salinity (2%/3% NaCl). Temperature-mediated alterations in promoter activity were not immediately reflected in changes to cell-surface MSHA levels, whereas high salinity led to decreased MSHA production. Combining high temperature (37°C) and high salinity (2%/3% NaCl), attenuated the salinity-mediated reduction of msh -P1/P2 activity. Biofilm biomass levels were only substantially heightened at 25°C and 20°C, likely a result of no temperature-dependent changes in cell-surface MSHA, and additional temperature-controlled biofilm regulation previously described. We also found msh -P1/P2 promoter activity and MSHA production varies widely across toxigenic O1 and O139 serogroups despite complete sequence homology. These results shed new light on how key signals regulate MSHA pilus production to support V. cholerae persistence in aquatic environments.

Toxigenic Vibrio cholerae bacteria, are the causative agent of the life-threatening pandemic diarrheal disease cholera. Along with sporadic outbreaks often following catastrophic natural disasters or large-scale breakdowns of civil sanitation infrastructure, cholera is endemic to ∼50 countries across primarily Asia and Africa, driven by V. cholerae 's survival in brackish estuary waters. This survival is enhanced by the ability of V. cholerae to interact with estuarial surfaces, via the type IVa mannose-sensitive hemagglutinin (MSHA) pilus. This study identifies the impacts of key estuarial signals, temperature and salinity, on msh gene promoter activity and pilus production. Our results indicate that promoter activity and pilus production is reduced at lower temperatures and higher salinities, which perhaps helps to explain V. cholerae 's preferential habitation of warm brackish aquatic environments.