The Haemolysin Co-regulated Protein And Its Related Pathogenicity In Salmonella Enteritidis

Salmonella enteritidis Infection is one of the leading zoonoses, seriously harmful to the health of human beings and livestock and poultry breeding. Salmonella enterica contains over2,500diverse serotypes with different host ranges, and cause diseases with severity ranging from subclinical colonization to serious systemic disease. Among these serotypes, Salmonella Enteritidis is a kind of aggressive, pantropic host zoonoses, which infects a broad range of hosts including humans and all kinds animals. Salmonella Enteritidis causes a subclinical infection in poultry, and infected hens can become chronic carriers, leading to a severe systemic disease and major economic losses. Human consumption of contaminated poultry products results in an acute self-limiting gastroenteritis, being responsible for60%～80%of the estimated human food poisoning events.Interaction between bacteria and their hosts is guided by a communication/signaling interplay which aims to influence the host response. As contact-dependently pathogenic bacteria, Salmonella enteritidis use specialized protein secretion systems to deliver proteins and toxins into the target cells are crucial for bacterial virulence and survival.Type VI secretion systems represent an emerging paradigm for protein secretion in Gram-negative bacteria from plant and animal, and it is widely considered to be potential virulence of gram-negative bacteria. The T6SSs are commonly encoded in pathogenicity islands, for example, enteropathogenic Escherichia coil (EPEC) pheU, Pseudomonas aeruginosa HSI (Hcp-secretion island) and Salmonella enterica centrosome7island. Bioinformatics and comparative genomics analysis show that the T6SS gene clusters are located in5different genomic islands(SPI-6, SPI-19, SPI-20, SPI-21and SPI-22), which are highly conserved and with different evolution process. T6SSs encoded in SPI-19are widely present in five toxic serotypes. Among these, Dublin, Weltevreden, Agona and Gallinarum carry the highly conserved T6SS encoded in SPI-19, while serotype Enteritidis presents a SPI-19with sixteen ORFs, including three T6SS core component: SEN1002(Hcp-like proteins), SEN1003(ImpA homolog) and SEN1004(a truncated IcmF). Haemolysin co-regulated protein encoded in Salmonella enteritidis is just as haemolysin co-regulated proteinl encoded in Salmonella Gallinarum that includes most of the T6SS functions. According to the recent studies, Salmonella Gallinarum is a direct descendant of Salmonella enteritidis and the transfer of a complete SPI-19form Salmonella Gallinarum into Salmonella enteritidis increased the level for the bacterial colonizing the ileum, liver and spleen of infected chickens. The SPI-19T6SS structural differences from others was thought to be associated with the interaction of pathogenic bacteria and the host macrophages, therefore, hcp gene may play an important role in the formation process of salmonella enteritis virulence. Further study of the virulence related functions of SPI-19coding hcp from Salmonella enteritidis will lay a solid foundation for the research of pathogenesis and control the widespread of Salmonella enteritidis.To investigate the virulence-related function of hemolysin-coregulated protein Hcp encoded by SPI-19in Salmonella enteritidis, we knocked out hcp gene in SPI-19from Salmonella enteritidis domestic standard strain50336and SD-2, constructed two hcp deletion mutants by γRed recombination system and two complemented strains50336△hcp/phcp and SD-2Ahcp/phcp, respectively. Invasion, adhesion and survival assays in human epithelial cell line Caco-2and chicken macrophage cell line HD11were carried out to evaluate the virulence between wild-type and the hcp deletion isogenic mutants. The results showed that the ability of adherence and invasion in Caco-2for mutants50336Ahcp decreased about39.1%and47.9%, respectively, the SD-2AHcp were46.5%and58.9%. Phagocytosis of50336△hcp and SD-2Ahcp compared with the wild strains after2hours incubation revealed the significant difference, respectively in decline35.7%and37.2%inHD11. The Real Time-PCR tests showed significant reduction about the expression level among the genes encoded by fliC, sefA, ompA and ompC between the wild type strain and Ahcp mutants. Compared to the parent strain, the expression level of fliC was decrease at28.7%(50336strain) and37.1%(SD-2strain), the expression level of sefA from SEF14was reduced at14.7%(50336strain) and18.8%(SD-2 strain), the expression level of ompA was declined at37%(50336strain) and47.5%(SD-2strain), the expression level of ompC was descend38%(50336strain) and29.7%(SD-2strain). Experiments show that hcp gene encoded by SPI-19can strengthen the virulence of Salmonella enteritidis50336and SD-2. And the expression level among the genes encoded by fliC, sefA, ompA and ompC is closely related to hcp gene.Bacterial biofilm formation is mainly affected by bacteria AI-2quorum sensing system and regulated by adhesins existed on the surface of bacteria. When we deleted the hcp gene encoded by SPI-19in salmonella enteritidis, the expression level of SEF14fimbriae and flagella were decreased. To investigate the role of the hcp gene of Salmonella enteritidis reference strains50336and SD-2in their biofilm formation. Crystal violet assays were used to compare the biofilm forming ability of the protype strains and hcp gene in-frame deletion mutants. We also measured the biofilm forming ability of the series of mutants, i.e.50336∞sefA,50336△fliC, SD-2△sefA and SD-2△fliC. The AI-2bioassay was performed to measure QS activity. The result show that compared to the parent strain, the biofilm forming ability of50336△hcp and SD-2△hcp were decreased67%and69%; the biofilm growth level of50336△fliC and SD-2AfliC were decreased49%and54%, respectively. There was no significant influence on the biofilm formation for the sefA gene deletion mutant. AI-2activity in the mutant strains was not significantly different from the protype. Our date demonstrated that the hcp gene of SPI-19appreciably impact the formation of biofilm in salmonella enteritidis.The hcp gene encoded by SPI-19in Salmonella enteritidis reference strains CMCC(B)50336and SD-2were amplified by PCR and sequenced, then inserted into the multiple clone sites of the pET-28a(+) prokaryotic expression vector. The positive recombinant plasmid was screened and transformed into BL21(DE3) E.coli. After being inducted with the optimal concentration of IPTQ the expected20kDa engineered protein rHcp from the recombinant E.coli carrying pET28-hcp was detected by SDS-PAGE and affinity-purified. ICR mice were immunized for three times with50μg affinity-purified recombinant antigen and the specific mouse antibody against the protein was titrated using indirect ELISA, the specificity of the immune serum was confirmed by Western blot. Results showed that the nucleotide sequence of the hcp gene of50336and SD-2share100%identity with other hcp genes in GenBank database. SDS-PAGE analysis showed that the express protein was20kDa in molecular mass. ELISA title of the immunized mouse serum reached at1:8000. The specificity of the immunized serum to Hcp protein was determined in SPI-19hcp-positive Salmonella enteritidis and Salmonella Gallinarum. These results suggested that the recombinant hcp(rHcp) had good antigenicity, and prove that Hcp protein is just structural proteins in Salmonella enteritidis and Salmonella Gallinarum, which are not secreted in vitro, and provide important bio-material for further study of rHcp function in Salmonella enteritidis.To investigate the pathogenicity of the hcp gene encoded by SPI-19in Salmonella enteritidis, the pathogenicity of hcp gene in-frame deletion mutants50336△hcp and SD-2△hcp was tested when compared with the two parent strains CMCC(B)50336and SD-2and complemented strains50336Ahcp/phcp and SD-2△hcp/phcp in6-week-old BALB/c mice and1day-old Qingyuan chickens infected via subcutaneous inoculation, respectively. The animal infection experiment results showed, that the LD50for the two mutants were79.43cfu and125.89cfu in the mice, and89.13×107cfu and158.49×107cfu in the chickens, that the LD50for the two complemented were50.12cfu and19.95cfu in the mice, and56.23×107cfu and39.81×107cfu in the chickens, that the LD50for the two parents were19.95cfu and7.94cfu in the mice, and25.12×107cfu and14.13×107cfu in the chickens. Furthermore, compared with the wild group, the107cfu mutant infected chickens with the107cfu mutants for average weight-gain at10-day-old were about5.04g and7.83g more. These suggested the pathogenicity of Salmonella enteritidis decreased sharply when knocked out the hcp gene.In a word, the results presented in this study suggest hcp gene is a vital virulence factor of Salmonella enteritidis, which plays an important role involved in the expression level among the genes encoded by fliC, sefA, ompA and ompC, and Salmonella enteritidis adhering to Caco-2cells in vitro, anti-phagocytosis to HD11and forming biofilm. And animal infection experiments test its function in the molecular pathogenesis mechanism. Exploring the relationship between Chinese indigenous chicken lines and the susceptibility of Salmonella Enteritidis will do some work in finding new clues about the eradication of this disease as well as to tackle the food safety and hygiene problem. At the same time, these bring us new vision that whether hcp involves in the process of Salmonella enteritidis and Salmonella Gallinarum to colonize the host cells as adhesion, so that we can enrich the concept of adhesion of Salmonella. It also plays an vital part in providing necessary basis materials in further research in the virulence of Salmonella enteritidis and Salmonella Gallinarum.