Functional Identification of Type III Secretion System 1 of Vibrio parahaemolyticus

Abstract

[한글]

[영문]Vibrio parahaemolyticus, a Gram-negative marine bacterium, is a worldwide cause of food-borne gastroenteritis. Genome sequencing of the clinical V. parahaemolyticus strain RIMD2210633 identified two sets of genes for the type III secretion system (TTSS), TTSS1 and TTSS2. Here, a TTSS1 mutant strain was constructed by removing the vcrD1 gene, a main component of TTSS1 apparatus. 2D-gel electrophoresis was performed, and the identified proteins to be secreted via TTSS1 were categorized into four groups; 1) the proteins related with flagella, 2) enzymes associated with bacterial virulence, 3) outer membrane proteins, and 4) the proteins with unidentified functions. Mice infected with wildtype V. parahaemolyticus showed 70% mortality between 5-8 hr, whereas mice infected with vcrD1 deletion mutant presented significant attenuation in mouse mortality. HEp-2 cells treated with wildtype V. parahaemolyticus caused 80% cell death, compared to vcrD1 deletion mutant strain killed 22% of HEp-2 cells. These results suggest TTSS1 of V. parahaemolyticus was involved in cytotoxic activity and mouse mortality. V. parahaemolyticus-induced cytotoxicity was not inhibited by the pan-caspase inhibitor, indicating that caspase is not involved in this process. V. parahaemolyticus also triggered phosphorylation of mitogen-activated protein kinases (MAPKs) including p38 and ERK1/2 but not JNK in HEp-2 cells. Experiments using inhibitors for each MAPK showed that p38 and ERK1/2 among MAPKs are involved in V. parahaemolyticus-induced death of HEp-2 cells. HEp-2 cells infected with ?vcrD1 mutant V. parahaemolyticus showed a similar level of LDH release in the presence of any of the three MAPK inhibitors. These results demonstrate that V. parahaemolyticus triggers activation of p38 and ERK1/2 of HEp-2 cells, eventually leading to death of HEp-2 cells, and that TTSS1 of V. parahaemolyticus is important in this process.