MSCS is a Critical Component for Osmotic Survival of Vibrio Cholerae

Abstract

Vibrio cholerae (Vc), an opportunistic pathogen, adapts to a wide range of osmotic environments. In events of drastic osmotic down-shifts, Vc avoids mechanical rupture by releasing ions and small osmolytes through MscS and MscL, the two major bacterial mechanosensitive (MS) channel species. To investigate contributions of these channels to Vibrio’s osmotic permeability response, we generated DmscL, DmscS and double DmscL DmscS mutants in Vc O395. Patch-clamp recordings confirmed large and generally homogenous populations of MscS in DmscL and MscL in DmscS, whereas in the double mutant we observed low densities of minor MS channels resembling MscS or MscM. Stopped-flow experiments and analysis of light-scattering kinetics detected no change in water permeability. The DmscL strain, however, showed a moderate slowdown of the osmolyte release kinetics and a drastically increased osmotic survival, exceeding WT. The DmscS mutant exhibited markedly slower osmolyte release, increased fraction of permeable osmolytes, and lowest survival. Deletion of both channels changed the release characteristics suggesting the presence of permeant structures which do not reseal (membrane cracks), also producing low survival. qPCR experiments indicated that mscS transcription is about 1.4 fold upregulated in DmscL compared to WT. This is consistent with the higher density of MscS channels observed by electrophysiology in this strain and with the highest rescuing efficiency. The up-regulation of MscS in the absence of MscL also suggests a special crosstalk mechanism in mechanosensitive channel expression. Modeling of the scattering kinetics accurately predicts separate stages of cell swelling, tension generation, osmolyte release, and termination of permeation resulting in a stable level of cell population scattering. In the absence of inactivating MscS, the final level is unstable, signifying incomplete resealing of the membrane. We conclude that MscS is the most critical component in the Vibrio permeability response.