问号钩端螺旋体趋化信号转导基因cheAs及cheX的研究

Leptospira is a kind of peculiar microorganism in evolution and taxology. Pathogenic leptospira will cause Leptospirosis Disease, which was legal class B infectious disease in China. Though after 1980s the incidence of Leptospirosis Disease was relative stable, the explosive infection frequently comes up in infected areas. In recent years with the increased incidence, Leptospirosis Disease has been detected in ninety one countries and regions. .Chemotaxis is one kind of bacterial movement, which is also the way to avoid the harmful environment, and toward to the beneficial side. In addition, some research showed that chemotaxis can help pathogens pass hosts' tissue barrier which may funtion in pathogenesis. .Chemotaxis and motility are critical for the virulence pathogenic Leptospires which enable bacteria to sense respond to numerous changes in their environment. Furthermore, chemotaxis can enable bacteria to penetrate host tissue barriers during infection. L. interrogans serovar lai strains 017 and KH-1 were shown to movetoward hemoglobin and this movement was shown to be related to their virulence. .Four strains of pathogenic leptospira have been sequenced since 2003.From the genome information, we found that Leptospiral chemotaxis signal transduction system is complex. Our previous study showed the function of chemotaxis genes chew, cheY and cheB.Combine with our previous data, bioinformatics analysis of cheA1, cheA2 and cheX put forward the hypothesis of leptospirosis interrogans chemotaxis signal transduction pathway：CheA1 has dual function- - kinase and phosphatease function in the chemotaxis phosphorelay pathway of Leptospira interrogans; CheA1 and/or CheA2 together with CheB and CheR to keep the methylation feedback pathway..We intend to use molecular biology and bioinformatics technology to study the function and the regulation of these chemotaxis-associated genes. It will hopeful to know more about the pathogenesis on Leptospira interrogans and supply experimental evidence to therapy in Leptospirosis Disease.

钩端螺旋体在进化和分类上较特殊，所致钩体病是我国法定报告传染病中流行范围广泛且情况复杂的一种人畜共患病。趋化特性是钩体致病机制的重要组成部分，课题组前期在完成钩体赖株测序工作的基础上，重点研究了钩体的趋化基因cheWs、cheYs和cheBs，发现了它们在趋化信号转导途径中的功能及其特殊性，并结合对趋化基因cheA1、cheA2及cheX的生物信息学分析，提出钩体胞内趋化基因信号转导通路的如下假说：钩体CheA1在钩体趋化信号转导通路磷酸基团转移途径中起到了磷酸激酶与磷酸酯酶的双重功能，将磷酸集团转移给CheYs后，与CheX同时对CheYs进行去磷酸化；CheA1和/或CheA2与CheBs及CheRs共同维持甲基化反馈通路。我们拟结合生物信息学与分子生物学技术对cheAs及cheX的功能、调控进行深入研究，验证假说，初步完善钩体胞内趋化信号转导通路，为明确钩体致病机制打下基础。

钩端螺旋体在进化和分类上较特殊，所致钩体病是我国法定报告传染病中流行范围广泛且情况复杂的一种人畜共患病，而趋化特性是钩体致病机制的重要组成部分。本研究在课题组前期工作的基础上提出钩体胞内趋化基因信号转导通路的假说，拟结合生物信息学与分子生物学技术对cheAs 及cheX 的功能、调控进行深入研究，验证假说，初步完善钩体胞内趋化信号转导通路。.在研究中我们首先针对各个待研究基因进行了系统的生物信息学分析，发现问号钩端螺旋体的CheA1蛋白可能具有双重功能。cheA2 基因编码的蛋白则中负责结合并将磷酸基团信号传递给下游反应调节蛋白的P2结构域被一段未知功能的氨基酸序列取代，该结构域的缺失可能会导致信号难以向下游传递。.同时我们还在问号钩端螺旋体cheA1、cheA2、cheX基因在E.coli cheA 及cheX基因缺陷株中进行异源互补实验，发现CheA1和CheA(E)可在一定程度恢复RP9535的趋化能力，而CheX可以互补E.coli cheZ 缺陷株的趋化能力。.通过CheAs的体外磷酸基团转移实验的结果我们发现，CheA1以及CheA1中只包含P1、P2、P3、P4、P5结构域的截短CheA1(1-700)蛋白具有自磷酸化能力，且可以将磷酸基团转移给下游的反应调节蛋白，而其它反应调节蛋白则没有接收上游传递的磷酸基团的能力。问号钩端螺旋体中的另一个组氨酸蛋白激酶CheA2则和我们预期的结果不一致：最初，从结构域分析来看，我们认为P2结构域的确实可能会导致磷酸基团不能向反应调节蛋白传递，但是实验结果表明该蛋白的自磷酸化能力已经丧失，说明在问号钩端螺旋体趋化过程中，该蛋白的功能已经完全丧失。同时，CheA1还可以将磷酸化的CheY1快速去磷酸化。.以上的研究结果揭示了问号钩体趋化信号转导过程中起作用的基因，并且证实了CheA1具有激酶和磷酸酯酶的双重功能。.通过本研究，对问号钩端螺旋体趋化系统胞内信号传导中的磷酸化系统有了深入了解，从而对钩体的趋化信号转导途径有了进一步的认识，为研究钩体真正的致病机打下良好的基础。