Analysis of Vibrio parahaemolyticus Virulence Systems
Calder, Thomas James
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Vibrio parahaemolyticus is a Gram-negative halophilic bacterium and one of the leading causes of food-borne gastroenteritis from the consumption of raw or undercooked seafood. The pathogenicity of V. parahaemolyticus is attributed to several virulence factors, including two hemolysins and two type III secretion systems (T3SS1 and T3SS2). Herein, we compare the virulence of V. parahaemolyticus POR strains, which harbor a mutation in the T3SS needle apparatus, to the V. parahaemolyticus CAB strains, which contain mutations in transcriptional regulators for the T3SSs. Additionally, we characterize a novel T3SS2 effector termed VPA1380. From this study, we demonstrate that each structural or regulatory mutant of T3SS1 or T3SS2 alters the pathogenicity of the bacterium in a different manner. POR and CAB strains exhibited differences in biofilm growth, but shared similar levels of swarming motility and effector production/secretion. Additionally, while the cytotoxicity of these strains was similar, the CAB2 (T3SS1 regulatory mutant) strain was strikingly more invasive than the comparable POR2 (T3SS1 structural mutant) strain. In summary, by creating structural or regulatory mutations in either T3SS1 or T3SS2, differential downstream effects on other virulence systems were observed. Effector proteins secreted from T3SS2 have been previously shown to promote colonization of the intestinal epithelium, invasion of host cells, and destruction of the epithelial monolayer. In this study, we identify VPA1380, a T3SS2 effector protein that is toxic when expressed in yeast. Bioinformatic analyses revealed that VPA1380 is highly similar to the inositol hexakisphosphate (IP6)-inducible cysteine protease domain of several large bacterial toxins. Mutations of conserved catalytic residues and of residues in the putative IP6-binding pocket abolished toxicity in yeast. Furthermore, VPA1380 was not toxic in yeast cells deficient for the production of IP6. Therefore, our findings suggest that VPA1380 is a cysteine protease that requires IP6 as an activator. Additionally, VPA1380 appeared to disrupt trafficking of dextran and transferrin, which may be due to VPA1380’s potential interaction with important retrograde factors. Elucidating the host targets and cellular effects of VPA1380 is important for understanding the pathogenic nature of V. parahaemolyticus for diagnostic and treatment and purposes.