The dynamic hypoosmotic response of Vibrio cholerae relies on the mechanosensitive channel MscS.

Vibrio cholerae adapts to osmotic down-shifts by releasing metabolites through two mechanosensitive (MS) channels, low-threshold MscS and high-threshold MscL. To investigate each channel's contribution to the osmotic response, we generated ΔmscS, ΔmscL, and double ΔmscL ΔmscS mutants in V. cholerae O395. We characterized their tension-dependent activation in patch-clamp, and the millisecond-scale osmolyte release kinetics using a stopped-flow light scattering technique. We additionally generated numerical models describing osmolyte and water fluxes. We illustrate the sequence of events and define the parameters that characterize discrete phases of the osmotic response. Survival is correlated to the extent of cell swelling, the rate of osmolyte release, and the completeness of post-shock membrane resealing. Not only do the two channels interact functionally, but there is also an up-regulation of MscS in the ΔmscL strain, suggesting transcriptional crosstalk. The data reveal the role of MscS in the termination of the osmotic permeability response in V. cholerae.

MscS inactivation and recovery are slow voltage-dependent processes sensitive to interactions with lipids.

Mechanosensitive channel MscS, the major bacterial osmolyte release valve, shows a characteristic adaptive behavior. With a sharp onset of activating tension the channel population readily opens, but under prolonged action of moderate tension it inactivates. The inactivated state is non-conductive and tension insensitive, which suggests that the gate becomes uncoupled from the lipid-facing domains. Because the distinct opening and inactivation transitions are both driven from the closed state by tension transmitted through the lipid bilayer, here we explore how mutations of two conserved positively charged lipid anchors, R46 and R74, affect 1) the rates of opening and inactivation and 2) the voltage dependences of these transitions. Previously estimated kinetic rates for opening-closing transitions in wild-type MscS at low voltages were 3-6 orders of magnitude higher than the rates for inactivation and recovery. Here we show that MscS activation exhibits a shallow nearly symmetric dependence on voltage, whereas inactivation is substantially augmented and recovery is slowed down by depolarization. Conversely, hyperpolarization impedes inactivation and speeds up recovery. Mutations of R46 and R74 anchoring the lipid-facing helices to the inner interface to an aromatic residue (W) do not substantially change the activation energy and closing rates, but instead change the kinetics of both inactivation and recovery and essentially eliminate their voltage dependence. Uncharged polar substitutions (S or Q) for these anchors produce functional channels but increase the inactivation and reduce the recovery rates. The data clearly delineate the activation-closing and the inactivation-recovery pathways and strongly suggest that only the latter involves extensive rearrangements of the protein-lipid boundary associated with the uncoupling of the lipid-facing helices from the gate. The discovery that hyperpolarization robustly assists MscS recovery suggests that membrane potential is one of the factors that regulates osmolyte release valves by putting them either on "ready" or "standby" based on the cell's metabolic state.

The dynamic hypoosmotic response of Vibrio cholerae relies on the mechanosensitive channel MscS.

Like other intestinal bacteria, the facultative pathogen Vibrio cholerae adapts to a wide range of osmotic environments. Under drastic osmotic down-shifts, Vibrio avoids 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 individual contribution to V. cholerae osmotic permeability response, we generated individual ΔmscS, ∆mscL, and double ΔmscL ΔmscS mutants in V. cholerae O395 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. The ΔmscL mutant showed comparable release kinetics to WT, but a higher osmotic survival, while ΔmscS had low survival, comparable to the double ΔmscL ΔmscS mutant. 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 the ΔmscL strain 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 in Vibrio.

MscS inactivation and recovery are slow voltage-dependent processes sensitive to interactions with lipids.

Mechanosensitive channel MscS, the major bacterial osmolyte release valve, shows a characteristic adaptive behavior. With a sharp onset of activating tension, the channel population readily opens, but under prolonged action of moderate near-threshold tension, it inactivates. The inactivated state is non-conductive and tension-insensitive, which suggests that the gate gets uncoupled from the lipid-facing domains. The kinetic rates for tension-driven opening-closing transitions are 4-6 orders of magnitude higher than the rates for inactivation and recovery. Here we show that inactivation is augmented and recovery is slowed down by depolarization. Hyperpolarization, conversely, impedes inactivation and speeds up recovery. We then address the question of whether protein-lipid interactions may set the rates and influence voltage dependence of inactivation and recovery. Mutations of conserved arginines 46 and 74 anchoring the lipid-facing helices to the inner membrane leaflet to tryptophans do not change the closing transitions, but instead change the kinetics of both inactivation and recovery and essentially eliminate their voltage-dependence. Uncharged polar substitutions (S or Q) for these anchors produce functional channels but increase the inactivation and reduce the recovery rates. The data suggest that it is not the activation and closing transitions, but rather the inactivation and recovery pathways that involve substantial rearrangements of the protein-lipid boundary associated with the separation of the lipid-facing helices from the gate. The discovery that hyperpolarization robustly assists MscS recovery indicates that membrane potential can regulate osmolyte release valves by putting them either on the 'ready' or 'standby' mode depending on the cell's metabolic state.

Differential Interactions of Piscidins with Phospholipids and Lipopolysaccharides at Membrane Interfaces.

Piscidins 1 and 3 (P1 and P3) are potent antimicrobial peptides isolated from striped bass. Their mechanism of action involves formation of amphipathic α-helices on contact with phospholipids and destabilization of the microbial cytoplasmic membrane. The peptides are active against both Gram-positive and Gram-negative bacteria, suggesting easy passage across the outer membrane. Here, we performed a comparative study of these two piscidins at the air-water interface on lipopolysaccharide (LPS) monolayers modeling the outer bacterial surface of Gram-negative organisms and on phospholipid monolayers, which mimic the inner membrane. The results show that P1 and P3 are highly surface active (log KAW ∼ 6.8) and have similar affinities to phospholipid monolayers (log Klip ≈ 7.7). P1, which is more potent against Gram negatives, exhibits a much stronger partitioning into LPS monolayers (log KLPS = 8.3). Pressure-area isotherms indicate that under increasing lateral pressures, inserted P1 repartitions from phospholipid monolayers back to the subphase or to a more shallow position with in-plane areas of ∼170 Å2 per peptide, corresponding to fully folded amphipathic α-helices. In contrast, peptide expulsion from LPS occurs with areas of ∼35 Å2, suggesting that the peptides may not form the similarly oriented, rigid secondary structures when they avidly intercalate between LPS molecules. Patch-clamp experiments on Escherichia coli spheroplasts show that when P1 and P3 reach the outer surface of the bacterial cytoplasmic membrane, they produce fluctuating conductive structures at voltages above 80 mV. The data suggests that the strong activity of these piscidins against Gram-negative bacteria begins with the preferential accumulation of peptides in the outer LPS layer followed by penetration into the periplasm, where they form stable amphipathic α-helices upon contact with phospholipids and attack the energized inner membrane.

The host-defense peptide piscidin P1 reorganizes lipid domains in membranes and decreases activation energies in mechanosensitive ion channels.

The host-defense peptide (HDP) piscidin 1 (P1), isolated from the mast cells of striped bass, has potent activities against bacteria, viruses, fungi, and cancer cells and can also modulate the activity of membrane receptors. Given its broad pharmacological potential, here we used several approaches to better understand its interactions with multicomponent bilayers representing models of bacterial (phosphatidylethanolamine (PE)/phosphatidylglycerol) and mammalian (phosphatidylcholine/cholesterol (PC/Chol)) membranes. Using solid-state NMR, we solved the structure of P1 bound to PC/Chol and compared it with that of P3, a less potent homolog. The comparison disclosed that although both peptides are interfacially bound and α-helical, they differ in bilayer orientations and depths of insertion, and these differences depend on bilayer composition. Although Chol is thought to make mammalian membranes less susceptible to HDP-mediated destabilization, we found that Chol does not affect the permeabilization effects of P1. X-ray diffraction experiments revealed that both piscidins produce a demixing effect in PC/Chol membranes by increasing the fraction of the Chol-depleted phase. Furthermore, P1 increased the temperature required for the lamellar-to-hexagonal phase transition in PE bilayers, suggesting that it imposes positive membrane curvature. Patch-clamp measurements on the inner Escherichia coli membrane showed that P1 and P3, at concentrations sufficient for antimicrobial activity, substantially decrease the activating tension for bacterial mechanosensitive channels. This indicated that piscidins can cause lipid redistribution and restructuring in the microenvironment near proteins. We conclude that the mechanism of piscidin's antimicrobial activity extends beyond simple membrane destabilization, helping to rationalize its broader spectrum of pharmacological effects.