The dynamic hypoosmotic response ofVibrio choleraerelies on the mechanosensitive channel MscS

Abstract

AbstractLike other intestinal bacteria, the facultative pathogenVibrio choleraeadapts to a wide range of osmotic environments. Under drastic osmotic down-shifts,Vibrioavoids mechanical rupture by rapidly releasing excessive metabolites through mechanosensitive (MS) channels that belong to two major types, low-threshold MscS and high-threshold MscL. To investigate each channel’s individual contribution toV. cholerae’sosmotic permeability response, we generated individualΔmscS, ΔmscL, and doubleΔmscL ΔmscSmutants inV. choleraeO395 and characterized their tension-dependent activation in patch-clamp experiments, as well as their millisecond-scale osmolyte release kinetics using a stopped-flow light scattering technique. We additionally generated numerical models reflecting the kinetic competition of osmolyte release with water influx. Both mutants lacking MscS exhibited delayed osmolyte release kinetics and decreased osmotic survival rates compared to WT. TheΔmscLmutant showed comparable release kinetics to WT, but a higher osmotic survival, while ΔmscShad low survival, comparable to the doubleΔmscLΔmscSmutant. By analyzing release kinetics following rapid medium dilution, we illustrate the sequence of events and define the set of parameters that characterize discrete phases of the osmotic response. Osmotic survival rates are directly correlated to the extent and duration of cell swelling, the rate of osmolyte release and the onset time, and the completeness of the post-shock membrane resealing. Not only do the two channels interact functionally during the resealing phase, but there is also a compensatory up-regulation of MscS in theΔmscLstrain suggesting some transcriptional crosstalk. The data reveal the advantage of the low-threshold MscS channel in curbing tension surges, without which MscL becomes toxic, and the role of MscS in the proper termination of the osmotic permeability response inVibrio.