The pore-forming bacterial effector, VopQ, halts autophagic turnover

Abstract

Vibrio parahemolyticus Type III effector VopQ is both necessary and sufficient to induce autophagy within one hour of infection. We demonstrated that VopQ interacts with the V_o domain of the conserved vacuolar H⁺-ATPase. Membrane-associated VopQ subsequently forms pores in the membranes of acidic compartments, resulting in immediate release of protons without concomitant release of lumenal protein contents. These studies show how a bacterial pathogen can compromise host ion potentials using a gated pore-forming effector to equilibrate levels of small molecules found in endolysosomal compartments and disrupt cellular processes such as autophagy.

Defects in autophagic pathways are implicated in numerous human diseases such as neurodegenerative diseases and cancer, as well as defense against bacterial invasion. For example, the induction of autophagy is important in the degradation of intracellular pathogens such as Mycobacterium tuberculosis. However, other intracellular pathogens, such as Legionella pneumophila and Coxiella burnetii, have evolved strategies to subvert autophagy and use it to their own advantage by interrupting autophagosome maturation. Because of the extent to which pathogenic microorganisms must manipulate their host cells during infection, multiple studies have used bacterial virulence proteins as a powerful tool to uncover novel host signaling mechanisms and to further study host-pathogen interactions.

We recently characterized the biochemical activity of the virulence factor VopQ, a novel Vibrio parahemolyticus (V. para) protein with no homology to any proteins outside of the Vibrios. V. para is a Gram-negative marine bacterium, that is a major cause of gastroenteritis due to the consumption of contaminated raw or undercooked seafood, which may be life threatening in patients that are immune compromised or have underlying conditions such as liver diseases. Each of the two chromosomes of V. para. harbor a type 3 secretion system (T3SS), designated T3SS1 and T3SS2, enabling the translocation of bacterial proteins, or effectors, into the eukaryotic host. T3SS1 orchestrates a temporally regulated cell death mediated by the induction of autophagy, followed by cell rounding, and eventual lysis of the host cell. VopQ induces autophagy during infection, and elucidating its molecular mechanism not only provides a better understanding of V.para. pathology, but also offers new insights into the mechanism of eukaryotic autophagy.

Previously, recombinant VopQ was shown to be necessary and sufficient to induce rapid autophagosomal accumulation in HeLa cells. In order to understand the activity of VopQ, we sought to find interacting proteins in eukaryotic model systems. Using pulldown assays, we show that VopQ interacts with at least one V_o-specific subunit of the conserved V-ATPase proton pump, the main electrogenic proton pump responsible for the acidification of intracellular organelles such as the lysosome. The V-ATPase consists of two distinct and separable domains: the V₁ ATP-hydrolyzing domain and the V_o proton translocation domain. In order to understand how VopQ affects V-ATPase activity, we isolated yeast vacuoles and tested V-ATPase activity in the presence of VopQ in vitro. Surprisingly, VopQ immediately deacidifies the vacuole at low nanomolar concentrations, without hindering ATP hydrolysis activity. This phenotype was consistent during V. para infections where VopQ is necessary to induce deacidification of lysosomes. Using the pH-dependent dye LysoTracker, we also show that VopQ is sufficient to immediately deacidify lysosomes upon microinjection of recombinant VopQ.

Due to the rapid deacification observed with VopQ, we hypothesized that VopQ either disrupts the V_o proton channel or forms a lysosomal pore. With synthetic liposomes encapsulating fluorescent dyes of differing molecular weights, we showed that VopQ forms a pore independent of the V-ATPase structure and does not translocate molecules larger than 3 kDa. Consistent with this observation, we confirmed that VopQ does not allow large molecules, such as a 10-kDa dextran or proteases, to escape the lysosome during an infection. Furthermore, lipid bilayer electrophysiology experiments showed that VopQ forms a gated channel with an approximate pore size of 18Å, in agreement with our liposome dye release assays.

The ability of VopQ to form pores in a V-ATPase-independent manner suggests that the activity of VopQ is not specific to the lysosomal membrane. The ability of VopQ to bind liposomes, however, is based purely on electrostatics: in the liposome assay at pH 5.5, VopQ is positively charged due to its pI of 6, and binds to negatively charged liposomes made of POPC/DOPS. Raising the reaction pH or eliminating negatively charged lipids from the liposome completely abrogates VopQ pore-forming activity. When using purified yeast vacuolar membranes, we find that the association of VopQ with the vacuolar membrane is completely dependent upon the V-ATPase at physiological pH (7.5). Therefore, VopQ requires the V-ATPase to identify, bind and form pores on eukaryotic endolysosomal membranes. This is the first example of a targeted bacterial effector that forms pores on lysosomal membranes to alter ion homeostasis and autophagic flux. We show that VopQ, like lysosomotropic agents such as chloroquine, equilibrates the lysosome and causes autophagosomal accumulation through inhibiting the turnover of autophagosomes.

Bacterial effectors often mimic eukaryotic activities and in this case, VopQ may function similarly to the lysosomal MTOR-dependent ATP-sensitive sodium channel that senses the metabolic state of the cell, alters lysosomal membrane potential accordingly, and appears to regulate autophagic recycling of nutrients. Similarly, VopQ forms a gated ion channel in a strategic location to collapse membrane potentials, thereby directly altering autophagic flux. Furthermore, VopQ binds directly to the V-ATPase V_o domain, which appears to play a key role in the regulation of autophagy through amino acid sensing, and even more directly, autophagosome-lysosome membrane fusion (Fig. 1). Future studies involving membrane fusion model systems may help to further elucidate the role of VopQ and the V-ATPase in autophagosome fusion with the lysosome.

Figure 1. VopQ (red cylinder) binds to the V-ATPase V_o domain and forms a channel, resulting in deacidification of lysosomes and inhibition of autophagosome-lysosome fusion.

Sreelatha A, Bennett TL, Zheng H, Jiang QX, Orth K, Starai VJ. Vibrio effector protein, VopQ, forms a lysosomal gated channel that disrupts host ion homeostasis and autophagic flux. Proc Natl Acad Sci U S A. 2013;110:11559–64. doi: 10.1073/pnas.1307032110.

10.4161/auto.26449