| Vibrio anguillarum is ubiquitous in marine envrioment. It is an etiological agent that cause septicemia in marifish. For survive in harsh marine environment, marine bacteria have developed many strategies to response to various ecological stress. Very little is known about the mechanism of ecological adaption of V. anguillarum. Type Ⅵ secretion system (T6SS) is a newly found bacterial protein secretion system that function in bacterial pathogenesis and stress response. To get a better understand the mechanism of ecological adaption of V. anguillarum, the present study was carried out to investigate the functional expression of T6SS of V. anguillarum and its role in antibacterial activites. The results are listed as following:1. The characteristics of T6SS expression and secretion of V. anguillarum MHK3 in different culture conditions. Hcp (Hemolysin Coregulated Protein) is an secreted protein and its secretion is dependent of T6SS. Hcp secrection is a hallmark of functional T6SS. In this study, by using qRT-PCR and Western blot analysis, expression and secretion of Hcp were assayed at various culture conditions, including temperature at 10℃,16℃,28℃ and 37 ℃, NaCl concetration at 0.2%,0.5%,1.0%,3.0% and 5.0%, and pH at 6,7,8,9 and 10. The results showed that Hcp expressed and secreted at 10-37 ℃. Hcp mRNA levels of MHK3 were found to be decresed with the increase of temperature, and the highest Hcp mRNA expression level was found at 10 ℃. The Hcp level in intracellular proteins (ICP) was similar at 10-37 ℃; the Hcp level in extracellular proteins (ECP) was high at 10-28℃, but low at 37℃. Hcp expressed and secreted at 0.5-5% NaCl. Hcp mRNA levels was high at 3% NaCl but low at other NaCI concentrations. Hep level in ICP and ECP were high in 0.5-5% NaCl but low in 0.2% NaCI. Hcp expressed at pH 6-10. Hcp mRNA levels was high in pH 8 but low in other pH values. Hcp levels in ICP and ECP were decreased with the increase of pH. Hcp did not detected in ECP of MHK3 grew at pH 10. Test of protein stability showed that Hcp remained stability after its secretion.These results demonstrated that MHK3 exerts its T6SS functional in respose to various environmental changes by expression and secretion of Hcp.2. The influence of quraum sensing regulator luxO on the expression and secretion of T6SS. An in-frame deletion mutant of luxO in V. anguillarum MHK3 was constructed. The growth of AluxO mutant was significantly lower than the wild-type MHK3. Hcp expression and secretion of ΔluxO and ΔvasC (vasC is one component of MHK3 T6SS genes cluster) were determined at various growth stages (OD540nm=0.1,0.6,1.0,2.0). The results showed that Hcp mRNA expression levels were significantly lower than those of the wild-type MHK3 during the whole growth phase. Hcp protein was not detected in ICP and ECP fractions of ΔluxO, while Hcp protein was detected only in ICP of AvasC These results indicated that luxO have an influence on the Hcp expression by positively regulating the Hcp expression of MHK3 at the transcriptional level; deletion of vasC might cause deficiency on the intact of T6SS and thereafter affects the secretion of Hcp of MHK3.3. Antibacterial activity mediated by MHK3 functional T6SS. This study is to determine the antibacterial activity of V. anguillarum MHK3, which has an functinal T6SS (Hcp+), and ΔluxO and AvasC, both having deficiency in T6SS function (Hcp). When MHK3 (Hcp+) co-incubated with Edwardsiella tarda or Escherichia coli respectively for 4 hours, the bacterial counts of E. tarda and E. coli decreased by 2-3 logs, while the bacterial counts of MHK3 did not changed. When ΔluxO (Hcp-) or ΔvasC (Hcp-) co-incubated with E. tarda or E. coli, counts of MHK3 as well as counts of E. tarda and E. coli did not change differently. These results indicated that a functional T6SS of MHK3 mediated anti-bacterial activity of V. anguillarum by inhibition of the growth of E. tarda and E. coli. |
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