Vibrio effector protein, VopQ, forms a lysosomal gated channel that disrupts host ion homeostasis and autophagic flux

Abstract

Defects in normal autophagic pathways are implicated in numerous human diseases--such as neurodegenerative diseases, cancer, and cardiomyopathy--highlighting the importance of autophagy and its proper regulation. Herein we show that Vibrio parahaemolyticus uses the type III effector VopQ (Vibrio outer protein Q) to alter autophagic flux by manipulating the partitioning of small molecules and ions in the lysosome. This effector binds to the conserved Vo domain of the vacuolar-type H(+)-ATPase and causes deacidification of the lysosomes within minutes of entering the host cell. VopQ forms a gated channel ∼18 Å in diameter that facilitates outward flux of ions across lipid bilayers. The electrostatic interactions of this type 3 secretion system effector with target membranes dictate its preference for host vacuolar-type H(+)-ATPase-containing membranes, indicating that its pore-forming activity is specific and not promiscuous. As seen with other effectors, VopQ is exploiting a eukaryotic mechanism, in this case manipulating lysosomal homeostasis and autophagic flux through transmembrane permeation.

Keywords: microbial pathogenesis; virulence; yeast vacuole.

Fig. 1.

VopQ interacts with the V-ATPase V_o domain. (A) Two hundred micrograms of vacuoles purified from BJ3505 was incubated with or without 5 µg His₆-VopQ (23 °C, 30 min). VopQ-associated proteins were isolated using nickel beads, and eluates were separated via SDS/PAGE and visualized by Oriole stain. The Vma6p band was excised and digested with trypsin. Tryptic peptides were run through reverse-phase HPLCy/ion trap. Tandem MS/MS files were searched against a National Center for Biotechnology Information nonredundant database. Asterisks denote nonspecific bands. (B) Immunoblot analysis of VopQ-associated proteins from A: Vph1p (V_o), Vma6p (V_o), and Vma2p (V₁). (C) Immunoblot analysis of VopQ-associated proteins from HeLa cell lysates.

Fig. 2.

VopQ disrupts acidification in yeast vacuoles and HeLa lysosomes. (A) Proton translocation activity of vacuoles was measured as described (SI Materials and Methods) in the presence of 100 nM bafilomycin or increasing concentrations of His₆-VopQ. (B) HeLa cells expressing GFP-LC3 were microinjected with 10 µM GST or His₆-VopQ, incubated 10 min (37 °C, 5% CO₂), and then incubated with 75 nM LysoTracker Red for 10 min and visualized. (Scale bar, 20 µm.) (C) HeLa cells expressing GFP-LC3 were incubated with or without 25 μM chloroquine for 30 min and then either treated with 100 μM rapamycin or infected with the noted V. parahaemolyticus strains at a multiplicity of infection of 10 for 3 h. Cell lysates were separated via SDS/PAGE and immunoblotted for GFP and tubulin.

Fig. 3.

VopQ does not rupture lysosomes, but allows for release of molecules <350 Da (A) Carboxyfluorescein dye release was measured upon addition of increasing concentrations of MBP-VopQ, 100 nM heat-inactivated MBP-VopQ, or 100 nM MBP to carboxyfluorescein-containing liposomes. (B–D) Carboxyfluorescein or FITC dextran release was measured upon addition of 25 nM MBP-VopQ, 25 nM heat-inactivated MBP-VopQ, or 25 nM MBP to quenched liposomes.

Fig. 4.

VopQ does not induce lysosomal rupture. HeLa cells were loaded with 1 mg/mL TRD for 6 h and chased overnight. The following day, cells were microinjected with 10 µM GST or His₆-VopQ, incubated 10 min (37 °C, 5% CO₂), and then incubated with 75 nM LysoTracker Green for 10 min and visualized. (Scale bar, 20 µm.)

Fig. 5.

VopQ forms a gated channel in membranes. (A) Current recorded after 25 nM MBP-VopQ was incubated with a POPC/DOPS (85:15 molar ratio) bilayer. (Upper) The voltage pulse stimulation is shown. (B) The averaged ramp current after the capacitance components were subtracted. The red trace was a linear fit from 0 mV to +30 mV with a chord conductance of 448.11 ± 1.67 pS. The inactivation of the channel becomes obvious when the voltage is higher than 40 mV or lower than −20 mV. (C) VopQ single-channel activity at different transmembrane voltages. C, close state; O, open state. (D) Histogram of single-channel events at +80 mV. We estimated the pore diameters by applying the Nernst–Planck equation, , for specific ion k. The measured current in our recordings was expressed as the sum of sodium and chloride flows, and the estimated pore diameter is ∼18 Å. (E) The measured single-channel current–voltage relation. The linear fitting (red line) yields a single-channel conductance of 128.9 ± 3.4 pS. (F) VopQ (25 nM) incubated with 100% POPC membranes did not produce any channel activity (80 mV, n = 4). MBP (25 nM) incubated with POPC/DOPS membranes did not produce any channel activity (80 mV, n = 6).

Fig. 6.

VopQ is a targeted pore-forming bacterial effector. (A) Carboxyfluorescein (CF) release was measured upon addition of 25 nM MBP-VopQ, 25 nM heat-inactivated MBP-VopQ, or 25 nM MBP to liposomes in buffer with varying pH. (B) Dequenched carboxyfluorescein fluorescence was measured upon addition of 25 nM MBP-VopQ, 25 nM heat-inactivated MBP-VopQ, or 25 nM MBP to liposomes made of increasing concentrations of DOPS (0–20%). (C) Immunoblot analysis of VopQ association with vacuoles isolated from wild-type or V-ATPase mutant yeast strains at pH 5.5 or 7.5. P, vacuole pellet; S, supernatant; T, total reaction. (D) Model for VopQ localization and pore-forming activity. VopQ docks onto the V_o domain of the V-ATPase. VopQ then forms an 18-Å pore in the membrane, allowing the flow of ions and small molecules but not proteins 3 kDa or larger.