Tension-activated channels in the mechanism of osmotic fitness in Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa (PA) is an opportunistic pathogen with an exceptional ability to adapt to a range of environments. Part of its adaptive potential is the ability to survive drastic osmolarity changes. Upon a sudden dilution of external medium, such as during exposure to rain, bacteria evade mechanical rupture by engaging tension-activated channels that act as osmolyte release valves. In this study, we compare fast osmotic permeability responses in suspensions of wild-type PA and Escherichia coli (EC) strains in stopped-flow experiments and provide electrophysiological descriptions of osmotic-release channels in PA. Using osmotic dilution experiments, we first show that PA tolerates a broader range of shocks than EC. We record the kinetics of cell equilibration reported by light scattering responses to osmotic up- and down-shocks. PA exhibits a lower water permeability and faster osmolyte release rates during large osmotic dilutions than EC, which correlates with better survival. To directly characterize the PA tension-activated channels, we generate giant spheroplasts from this microorganism and record current responses in excised patches. Unlike EC, which relies primarily on two types of channels, EcMscS and EcMscL, to generate a distinctive two-wave pressure ramp response, PA exhibits a more gradual response that is dominated by MscL-type channels. Genome analysis, cloning, and expression reveal that PA possesses one MscL-type (PaMscL) and two MscS-type (PaMscS-1 and 2) proteins. In EC spheroplasts, both PaMscS channels exhibit a slightly earlier activation by pressure compared with EcMscS. Unitary currents reveal that PaMscS-2 has a smaller conductance, higher anionic preference, stronger inactivation, and slower recovery compared with PaMscS-1. We conclude that PA relies on MscL as the major valve defining a high rate of osmolyte release sufficient to curb osmotic swelling under extreme shocks, but it still requires MscS-type channels with a strong propensity to inactivation to properly terminate massive permeability response.

Figure 1.

Osmotic survival correlates with osmolyte exchange rates. (A) Fractions of WT PA (PA14) and EC (Frag1) surviving osmotic down-shock as assayed by plate counts. The symbols and bars represent mean and standard deviation (n = 6). The x axis represents the end osmolarity upon a downshift from the initial 1,200 mOsm. (B and C) Stopped-flow recordings of small-angle light scattering changes upon mixing of suspensions of PA (B) and EC (C) with a 10-fold excess of low-osmolarity media (means of five sequential trials). The numbers by curves denote the osmolarity at the end of downshift from 1,200 mOsm (downshift endpoint). The 1,200 mOsm medium (HiLB) was standard LB supplemented with 413 mM NaCl. The scattering traces reflect the kinetics of dissipation of osmolytes contributing to the refractive index of the cytoplasm. The fitting of the scattering traces was done with nonexponential Eq. 1 (see Materials and methods) starting from the point of steepest downfall (see Fig. S3 and S4). a.u., arbitrary units. (D) The osmolyte release rates (1/τ) extracted from fits. The shock magnitude axis is aligned with A. Error bars represent standard deviation (n = 4). The osmolyte release rates are nearly equal at moderate shocks; however, at high shocks, specifically in PA, the rate sharply increases, which correlates with higher survival. For all experiments, the cultures were taken in early logarithmic phase (OD₆₀₀ of 0.25).

Figure 2.

The up-schock experiments record the rates of cell shrinkage and permit estimations of water permeability. (A and B) Stopped-flow recordings of small-angle light scattering upon mixing of suspensions of PA (A) or EC (B) grown in MLB (250 mOsm) with 10-fold excess of higher-osmolarity media (indicated by the traces). Experimental traces (blue) are overlaid with monoexponential fits (red). The insets show shrinkge rates as functions of shock magnitude. The error bars reperesent standard deviations (n = 4). Increased concentration of intracellular solutes and the accompanying increase of refractive index produce an increase in scattering. (C and D) Both cultures were grown to OD₆₀₀ of 0.25 and imaged under DIC. (E and F) Histograms of length (E) and width (F) distributions were generated from microscopic measurements on 300–400 cells of each type. The mean sizes of PA and EC cells were 1.4 × 0.8 µm and 3.0 × 1.3 µm (length × width). The assumption that cells are cylinders with spherical ends produces surface areas of 3.5 µm² and 11.3 µm² and cell volumes of 0.57 µm³ and 2.94 µm³, respectively. The SA:V ratios are 6.18 µm⁻¹ and 3.85 µm⁻¹ for PA and EC, respectively.

Figure 3.

Steps in the giant spheroplast preparation as viewed by DIC microscopy. (A) Intact PA (PA14) cells. (B and C) Filamentous forms induced by carbenicillin (B) and giant spheroplasts after lysozyme digestion (C).

Figure 4.

The composition of MS channel populations in EC and PA as revealed by pressure ramp experiments. (A and B) Pressure ramps applied to excised patches of native MS channel populations in EC (A) and PA (B) produce comparable conductance responses. The characteristic double-wave pattern is observed in EC patches. The channel population in EC patch contains ∼60 MscS and 50 MscL channels. PA always shows a smaller proportion of low-threshold MscS-like channels compared with a more dominant MscL-like channel population (∼80 per patch; see Table 2 and Fig. S5). The symmetric recording buffer contained 200 KCl, 90 mM MgCl₂, 10 mM CaCl₂, and 10 mM HEPES, pH 7.2.

Figure 5.

The puse-step-pulse protocol reveals adaptable fractions of channel populations in PA and EC. (A–D) The low- and high-threshold subpopulations of native channels in PA (A and B) and EC (C and D) exhibit distinctive adaptive behaviors. Exposure of excised patches to prolonged moderate tension produces massive inactivation of the native low-threshold channel population. (A) The first 0.1-s pressure pulse invokes ∼50% of total patch conductance, engaging the low-threshold population. The following 10-s step of variable amplitude conditions the low-threshold population, and the last pulse, equal in amplitude to the first, reveals the much smaller population that remains active after the conditioning step. When the same protocol utilizes saturating pulses (engaging the entire channel population) and a broader range of conditioning steps, a larger fraction of the population remains active. The experiment shows that the low-threshold population in PA is especially prone to inactivation. Red arrows indicate current levels produced by the remaining channel population at the end compared with the amplitude of response to the initial pulse (black arrows).

Figure 6.

Current to voltage relationships for unitary currents measured for PaMscL, PaMscS-1, and PsMscS-2. The examples of single-channel currents are shown as insets in each panel. Based on the Goldman equation, the E_rev shift for PaMscS-1 in response to 1:5 (pipette/bath) gradient of KCl (relative to symmetric conditions) of −6.1 mV predicts the permeability ratio P_CI/P_K of 1.5. For PaMscS-2, the E_rev shift of 23 mV (5:1 pipette/bath gradient) predicts P_CI/P_K of 4.4. Reversal potentials are indicated by blue arrows. The three MS channels were expressed in EC MJF 641 cells and recorded under symmetric and asymmetric ionic conditions. In the experiment with PaMscS-2, the gradient was inverted (5:1) because, for yet unknown reasons, no channel activity was observed in the opposite configuration.

Figure 7.

Midpoint determination of PA MscS-like channels using MscL as an intrinsic tension gauge. (A and B) The homologues PaMscS-1 (A) and PaMscS-2 (B) were expressed in PB113 EC cells carrying native MscL. Each of these channels generates a “wave” of current with its own midpoint. The p_MscS/p_MscL midpoint ratios for both MscS homologues (∼0.5) are slightly lower than that of EC MscS (∼0.6), indicating that these channels open at lower tension. PaMscS-2 is expressed at a much lower level despite full induction.

Figure 8.

The inactivation and recovery of PaMscS-1 and PaMscS-2. (A and C) Pulse-step-pulse protocols show that both MscS-like homologues from PA inactivate. The degree of inactivation is determined as the ratio of current at the end (red arrows) to the initial test pulse response of full population (black arrows). PaMscS-2 displays ∼60% inactivation, which is more than PaMscS-1 and about twice what is seen in its EC counterpart. (B and D) The recovery from inactivation shows that PaMscS-2 recovers much more slowly than PaMscS-1, indicating a more stable inactivated state.

Figure 9.

Relative ability of PA MS channels to rescue EC MJF641 from osmotic down-shock. At moderate osmotic down-shocks, PaMscL and PaMscS-1 were good at rescuing the MJF641. Surprisingly, PaMscS-2 did not serve as a good emergency valve, and the cells displayed poor survival statistically indistinguishable from channel-free MJF641. The plate counts are shown as means with standard deviations (n = 8).