High-Throughput Screening Methods Established For Screening Type Ⅲ Inhibitor And The Research Of Metal Ions Effect On The T3SS Of Xanthomonas Campestris Pathovar Campestris

The type III secretion system (T3SS) of Xanthomonas campestris pv. campestris (Xcc) is one of the most common virulence system and the key pathogenic factor among most gram-negative bacteria, which transport effector proteins into host cells to produce disease and cause huge economic loss to agricultural production. A single device missing or not working of T3SS, would lead to significant reduction or completely lost of pathogenicity. Using the strategy of small molecule compounds inhibit the T3SS of Xcc, this research established a high-throughput screening type III inhibitor method, and explored the influence of metal ions to the T3SS of Xcc, which will help us to understand the process of pathogenic bacteria to manipulate host cell, and used to development of new antimicrobials to inhibit or disintegrate the pathogenic bacteria.In this study, we found that NiCl2(0.01μM～0.1mM), CoCl2(0.01μM～0.1mM), CdCl2(0.1μM～0.1mM) and CuCl2(0.01mM～0.1mM) under the corresponding concentrations, can inhibit both the bacteria growth and the expression of hrpBl (XC3011) at different degrees by measured the GUS report strain of8004/pLgus3011. ZnCl2(1μM～0.1mM), MnCl2(1μM～0.1mM), CaCl2(0.01mM～0.1mM) and FeSO4(0.1μM～0.1mM) under corresponding concentrations, inhibit the expression of hrpBl, rather than affect the growth of bacteria. CrCl3, AlCl3, NaCl and K2SO4in the concentrations range from0.01μM to0.1mM, neither affect on the growth of bacteria, nor influence the expression of hrpBl. MgCl2at low concentration can induce the expression of hrpBl, but inhibit the expression of hrpBl at high concentration, which declared that the growth of Xcc8004depends on the magnesium ions. In order to test the bio-control effect of metal ions, when spraying the concentration of ZnCl2, MnCl2, CaCl2and FeSO4don’t affect bacterial growth, which can affect pathogenicity of Xcc8004for the host plant in carrots at a certain extent in pathogenicity test.In order to research the inhibition of ZnCl2, MnCl2, CaCl2and FeSO4on the T3SS whether is specificity. By measuring four GUS report strains of038G09of T2SS (XC3563),8004/pLgus3011of T3SS (XC3011),117H12of T4SS (XC1638) and8004/pLgus3806(house keeping gene, XC3806), we found that ZnCl2and MnCl2could inhibit the gene expression of XC3011and XC3563, while non-significant impact on the gene expression of XC1638and XC3806by determined the GUS report strains of038G09,8004/pLgus3011,117H12and8004/pLgus3806. FeSO4could both significantly inhibit the genes expression of XC3563, XC3011and XC1638, nevertheless has no effect on XC3806, CaCl2can significantly inhibit the gene expression of XC3011, whereas no significant impact on the genes expression of XC3563, XC1638and XC3806, When the concentration of MgCl2less than0.01mM can significantly inhibit the growth of these report strains, which then lead to the gene expression of XC3563, XC3011, XC1638and XC3806with the increase of concentration of magnesium ion. The gene expression of XC3011enhanced then suppressed again first with the increase of concentration, the genes expression of XC1638and XC3563increased, however in the concentration range of determination, the influence on the gene expression of XC3806was non-significant.In this study bacterial luciferase gene(luxAB) and the effector gene promoter of XC0241were cloned to pLAFR6vector and then introduced into Xcc by triparental conjugation, yielding a high-throughput screening method (HTS) to filtrate the type III small molecular inhibitors, which added to the research method in this field by measuring the relative fluorescence intensity of small molecule compounds before and after adding.