Type Ⅵ Secretion Systems Of Vibrio Parahaemolyticus:Function And Regulation

Vibrio parahaemolyticus, a halophilic bacterium in marine environments, is a most important foodbrone pathogen in the world. It causes wound infectious, septicemia and most commonly acute gastroenteritis in humans, it also causes disease in invertebrates, including abalone, Haliotis diversicolor supertexta and shrimp.The major virulence factors of V. parahaemolyticus include thermostable direct hemolysin (TDH), TDH related hemolysin (TRH) and two sets of type III secretion system (T3SS). PCR identification of tdh, trh, vcrDl (structure gene of T3SS1) and vcrD2(structure gene of T3SS2) in33clinical isolates of V. parahaemolyticus and511environmental isolates stored in our lab found that the fraction of these genes were100%,9.1%,100%and87.9%respectively in clinical strains and11.2%,19.4%,100%,1.2%in environmental strains. T3SS1was present in all V. parahaemolyticus. which is consisted with previous reports.tdh and T3SS2exist higher in clinical strains than in environmental strains, which was one feather of pathogenic factors.Type VI secretion system, a new secretion only found in proteobacteria, devotes environment adapting and pathogenicity of bacteria. Bioinformatic analysis of whole genome of V. parahaemolyticus RIMD2210633reveals two clusters of T6SS. One is on Chromosone I, which is called T6SS1; the other is on Chromosome II, which is named T6SS2. T6SS1is identical to T6SS in Vibrio cholerae, which has90.9%possession in clinical isolates and25%in environment isolates. T6SS2, however, is identical to T6SS in those marine bacteria, which exists in all the V. parahaemolyticus isolates.To confirm if both T6SS1and T6SS2can translocate and secrete proteins in vitro, a lot of mutant strains target on structure proteins icmF1and icmF2, as well as translocons hcpl and hcp2was made. Hcpl and Hcp2polyclonal antibody were immunized using V. parahaemolyticus clinical strain HZ as background strain. Western blot was performed to analysis the expression and translocation of Hcpl and Hcp2in pellets and supernatants of HZ and its mutants. We found that Hcp2can only be detected in pellet, but not in supernatant after deleting icmF2, the structure gene of T6SS2. However, Hcpl can not be detected neither in pellet nor in supernatant in HZ and all its mutants. When expressed using plasmid, Hcpl can be detected in pellet but not in supernatant even in the wild-type strain. The result of real-time RT PCR revealed that the transcription of hcpl was10times lower than hcp2, and icmF1was about17times lower than icmF2. These results highly show that T6SS2is an active secretion system in vitro, which can translocate and secrete proteins. However, T6SS1is an inactive secretion system in vitro, and hcp1, even the whole genes of T6SS1cluster were in a low transcriptional and expressional level.To investigate the function of T6SS, icmF1, icmF2, hcp1or hcp2mutants were made based on DTTT background, a triple mutant strain void of tdh, vcrDl and vcrD2. Functional analysis targets two major parts. One is about bacteria infection, including cell adherence, cytotoxicity, autophagy, apoptosis and hemolysis. The other is about environment adapt, including growth further, acid tolerance and biofilm.Both T6SS1and T6SS2devote cell adherence. After deleting icmF1and hcp1, the adherence to Caco-2and HeLa cell were both decreased. However, deleting icmF2and hcp2only lead cell adherence decreasing to HeLa cell. T6SS1do play functions in cell assay, and Hcp1antibody in cell culture can decrease adherence of wild-type strain to Caco-2.T6SS2induces autophagy. When autophagy happened, cytosolic LC3-Ⅰ converted to membrane associated LC3-Ⅱ. In our experiment, LC3-Ⅱ was decreased when macrophage RAW264.7was infected with△icmF2,△hcp2and△vgrG2mutant strains for4h. No significant change of LC3-Ⅱ was seen when infected with△icmF1. In a cell line stably expressing a GFP-tagged LC3, LC3-Ⅱ accumulates slowly when infected with△icmF2, compared to DTTT and adding2μmol/mL Rapamycin. These results indicated that T6SS2induces autophagy of RAW264.7.T6SS2induces autophagy of RAW264.7through VgrG2protein, a translocon of T6SS2. In a cell line expressing GFP-tagged VgrG2, LC3-Ⅱ increased obviously in western blot. Further in a cell cultured with prokaryotic VgrG2protein, LC3-Ⅱ also accumulated quickly. No matter intracellular or extrinsic VgrG2induce autophagy, indicating that the autophagy pass-way might change in the process of the phage tail-associated protein VgrG2across into host membrane.Neither T6SS1nor T6SS2induce cytotoxicity, hemolysin and apoptosis. And both T6SS1and T6SS2were not involved in acid tolerance and biofilm of V. parahaemolyticus.In T6SS2cluster, VPA1045and VPA1049were found to contain the Che-Y domain, belonging to response regulate family of two-component regulators. Deletion of either Vpa1045or Vpa1049did not affect expression and transcription of Hcp2, but decreased translocation of Hcp2in the supernatant of V. parahaemotycus. Bacterial adherence to the HeLa monolayer was also significantly reduced infected with△Vpa1045or△Vpa1049mutant strain comparing to their parent strain. So we concluded that the two-component regulators VPA1045and VPA1049might regulate T6SS2of V. parahaemolyticus.