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 V o 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.utophagy is a cellular process by which cells degrade and recycle cytoplasmic contents by encapsulating them within a distinctive double bilayer membrane vesicle for delivery to the degradative lysosome (1). Disruption of normal autophagic pathways is implicated in numerous human diseases, stressing the importance of autophagy and its proper regulation (2). Vibrio parahaemolyticus, a Gram-negative marine bacterium and a major cause of gastroenteritis due to the consumption of contaminated raw or undercooked seafood, induces autophagy during infection (3). V. parahaemolyticus harbors two type 3 secretion systems (T3SSs), molecular syringes that enable the translocation of bacterial proteins, known as effectors, into the eukaryotic host (3). The first T3SS (T3SS1) orchestrates a temporally regulated cell death mediated by the induction of autophagy, followed by cell rounding and resulting in lysis of the host cell (4). T3SS1 effector VopQ, also known as VepA (vp1680), is both necessary and sufficient for the rapid induction of autophagy, even in the presence of known chemical inhibitors of autophagy (5).VopQ is a 53-kDa protein with no apparent homology to any proteins outside of the Vibrio species. Vibrio homologs of VopQ have no known function or conserved structural domain. Previous work from our laboratory has shown that VopQ is a cytotoxic effector that accelerates host cell death and is essential in protecting V. parahaemolyticus from phagocytic uptake during infection (5). Based on microbial genetic studies, VopQ was shown to be necessary for the formation of an extensive network of autophagic vesicles in host cells within an hour of V. parahaemolyticus infection (5). Strikingly, recombinant VopQ alone is sufficient to induce this massive accumulation of autophagic vesicles, observed within minutes of microinjection of recombinant VopQ (picomolar concentrations) into eukaryotic cells (5). A recent report shows that Vo...

Vibrio effector protein VopQ inhibits fusion of V-ATPase–containing membranes

“…Accordingly, VopQ S200P still associates with yeast vacuoles in a V-ATPasedependent manner, because vacuoles lacking the V-ATPase no longer bind VopQ or VopQ S200P at pH 7.5 ( Fig. S7G), confirming previous observations of VopQ-vacuole interactions (17). Therefore, VopQ S200P still has some affinity for the V-ATPase, which is required for its channel-forming activity in vitro.…”

“…VopQ deacidifies the lumen of vacuoles through the rapid formation of an 18-Å channel in the membrane after direct interactions with V o subunits of the V-ATPase (17). A recent publication by Coonrod et al (13) shows that acidification of the yeast vacuole by the V-ATPase is absolutely required for vacuole fusion in vivo.…”

“…We used a dye-release assay that measures the release of a self-quenching concentration of carboxyfluorescein dye encapsulated in liposomes (41). Because these are protein-free liposomes, this assay tests for VopQ channel activity independent of its V-ATPase-dependent targeting activity (17). Interestingly, VopQ S200P induced carboxyfluorescein release from protein-free liposomes (17) with rates identical to wild-type VopQ (Fig.…”

“…Recent work has shown that a bacterial protein VopQ (also known as VP1680 or VepA) forms an outward rectifying, gated channel in membranes that contain the V-ATPase, resulting in the collapse of ion gradients and the disruption of autophagic flux (17). VopQ is a type III effector protein from the marine bacterium Vibrio parahaemolyticus that strongly associates with the V o domain of the eukaryotic V-ATPase (17,18).…”

“…VopQ is a type III effector protein from the marine bacterium Vibrio parahaemolyticus that strongly associates with the V o domain of the eukaryotic V-ATPase (17,18). Given the proposed role of the V o domain in fusion, we sought to examine the effect of VopQ on fusion using the well-defined biochemical model of eukaryotic membrane fusion from the budding yeast Saccharomyces cerevisiae.…”