Characterization of Mitochondrially Targeted T3SS2 Effectors in Vibrio parahaemolyticus

Vibrio parahaemolyticus (V. parahaemolyticus) is a Gram-negative bacterial pathogen that is the leading cause of bacterial seafood-borne acute gastroenteritis. All strains of V. parahaemolyticus harbor the type III secretion system 1 (T3SS1), which is dispensable for virulence in humans. Pathogenic strains harbor an additional type III secretion system (T3SS2) that mediates host cell invasion to form a protective replicative niche and is responsible for the enterotoxic effects. To date, only nine T3SS2 effectors have been identified within the T3SS2 pathogenicity island that contains 30-50 additional uncharacterized proteins. In the first part of this study, we performed a proteomics screen to identify T3SS2 effectors and found VPA1328, a novel T3SS2 effector recently identified as a member of the VopG family of predicted kinases. Coupling structural modeling and remote homology prediction with biochemistry, we determined that residue D205 in VPA1328 was critical for its catalytic activity. Using cell biology, we found VPA1328 localizes to the mitochondrial matrix of eukaryotic cells. Interestingly, while expression of VPA1328 did not affect cell death or mitochondrial membrane potential in HeLa cells, it decreased mitochondrial oxygen consumption rates and inhibited yeast growth. This growth phenotype was exacerbated by forcing the cells to use mitochondrial respiration for energy production. Metabolic analysis of the yeast expressing VPA1328 showed increases in fumarate, dihydroorotate, N-acetylputrescine, N,N-dimethylarginine, and glycerophosphocholine, suggesting changes in the available metabolic intermediates in the invaded host cell. In the second part of this study, we characterized known T3SS2 effector VPA1380 and discovered that VPA1380 localized to the host mitochondria by its N-terminal domain. By expressing untagged VPA1380 in yeast, we found that VPA1380 causes mitochondrial fragmentation in an activity-dependent manner. Tools were developed for use in future studies to elucidate host targets for VPA1380. Collectively, we report the first demonstration of kinase activity for any VopG-like effector. We provide evidence for the mitochondrial localization of VPA1328 and VPA1380. Our findings support a role for VPA1328 in altering eukaryotic mitochondrial respiration and metabolism and a possible role for VPA1380 in altering mitochondrial morphology. The results of this study will aid future work to define how Vibrio parahaemolyticus exploits the host mitochondria in invaded human cells.

Author dc:creator

• Chen, Luming

Contributors dc:contributor

• Alto, Neal
• Orth, Kim
• Friedman, Jonathan R.
• Henne, W. Mike
• Kohler, Jennifer J.

• Host-Pathogen Interactions
• Seafood
• Type III Secretion Systems
• Vibrio Infections
• Vibrio parahaemolyticus
• Bacterial Toxins

Language dc:language

en

Identifier

1482732342

OAI identifier oai:identifier

oai:utswmed-ir.tdl.org:2152.5/10441