A Cytotoxic Type III Secretion Effector of Vibrio parahaemolyticus Targets Vacuolar H+-ATPase Subunit c and Ruptures Host Cell Lysosomes

Abstract

Vibrio parahaemolyticus is one of the human pathogenic vibrios. During the infection of mammalian cells, this pathogen exhibits cytotoxicity that is dependent on its type III secretion system (T3SS1). VepA, an effector protein secreted via the T3SS1, plays a major role in the T3SS1-dependent cytotoxicity of V. parahaemolyticus . However, the mechanism by which VepA is involved in T3SS1-dependent cytotoxicity is unknown. Here, we found that protein transfection of VepA into HeLa cells resulted in cell death, indicating that VepA alone is cytotoxic. The ectopic expression of VepA in yeast Saccharomyces cerevisiae interferes with yeast growth, indicating that VepA is also toxic in yeast. A yeast genome-wide screen identified the yeast gene VMA3 as essential for the growth inhibition of yeast by VepA. Although VMA3 encodes subunit c of the vacuolar H + -ATPase (V-ATPase), the toxicity of VepA was independent of the function of V-ATPases. In HeLa cells, knockdown of V-ATPase subunit c decreased VepA-mediated cytotoxicity. We also demonstrated that VepA interacted with V-ATPase subunit c, whereas a carboxyl-terminally truncated mutant of VepA (VepAΔC), which does not show toxicity, did not. During infection, lysosomal contents leaked into the cytosol, revealing that lysosomal membrane permeabilization occurred prior to cell lysis. In a cell-free system, VepA was sufficient to induce the release of cathepsin D from isolated lysosomes. Therefore, our data suggest that the bacterial effector VepA targets subunit c of V-ATPase and induces the rupture of host cell lysosomes and subsequent cell death.

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

“…It is made up of two heterologous subunits: The cytoplasmic V 1 domain hydrolyzes ATP, which energizes the translocation of protons through the membrane-bound V o proton channel, and into the lumen of the acidic organelle. A recent study indicated that the Vibrio effector VopQ (vp1680, VepA) interacts with Vma3p (subunit c) of the highly conserved V-ATPase V o proton translocation domain (6). Our analyses revealed that HisVopQ interacts with Vma6 (subunit d) of the V o domain when incubated with highly enriched preparations of yeast vacuoles (Fig.…”

“…A previous report suggested that VopQ causes rupture of lysosomes in vivo and in vitro, resulting in the release of luminal enzymes (6). Because our findings are in direct contrast to this previously published work, we revisited these studies.…”

“…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 VopQ interacts with a V o subunit of the highly conserved eukaryotic vacuolar-type H + -ATPase (V-ATPase) and induces lysosomal rupture in vitro and in vivo (6). These authors hypothesized that lysosomes were "suicide bags" that rupture and lead to the release of luminal contents that are responsible for T3SS1-mediated cell death.…”

Lysosomal cell death at a glance

“…Similarly, the cytotoxicity of enniatin mycotoxins (Ivanova et al, 2012), and venom toxins from cobra (Feofanov et al, 2005) and South American rattlesnake (Hayashi et al, 2008), have been connected with LMP. Additionally, Vibrio parahaemolyticus VepA was recently identified as a new type of LMP-inducing protein (Matsuda et al, 2012). After inoculation, VepA binds to the cytoplasmic tail of the channel-forming subunit c of vacuolar H + -ATPase and triggers leakage of lysosomal hydrolases into the cytosol in a manner that depends on the subunit c. It will be of great interest to investigate whether VepA causes the widening of the ATPase channel and whether other LMP-inducing stimuli utilize a similar mechanism.…”

How the Study of Listeria Monocytogenes has Led to New Concepts in Biology

“…These findings unveil a novel activity of bacterial cholesterol-dependent cytolysins that may interfere with important host cellular pathways linked to lysosomal functions. So far, only few pathogenic bacteria were reported to target lysosomes by mechanisms differing from the one observed with L. monocytogenes [91][92][93][94][95].…”