LIGANDS & BINDING SPECIFICITY OF THE PASTA DOMAINS OF M TUBERCULOSIS PROTEINS

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 结核分枝杆菌明显能够在人类宿主中休眠，导致无症状的潜伏感染，这激发了人们对了解这种生物中细胞生长和细胞分裂是如何调节的极大兴趣。长期以来，人们观察到细菌培养的“废”或“条件化”培养基能刺激休眠细胞的生长，这导致了一种复苏促进因子(RPF)的鉴定，方法是从废培养基中提纯一种能够刺激放线菌黄微球菌生长的成分。RPF是一种小蛋白，在其他放线杆菌中有同源物，包括结核分枝杆菌，它有五个RPF基因。对这些基因在结核分枝杆菌中的功能研究表明，它们单独地不是复苏休眠的结核分枝杆菌细胞所必需的，并且单个RPF突变株没有其他生长或形态表型。然而，当两个或更多的RPF基因失活时，就会观察到生长或复苏缺陷。最近的证据表明，RPF是PGN水解酶，这表明休眠细胞的生长刺激可能是这些分泌蛋白的酶活性所致，可能是通过改变PGN结构或通过PGN降解产物与细菌细胞表面的相互作用而产生的。 通过生物信息学分析，确定了青霉素结合蛋白和丝氨酸/苏氨酸激酶胞外部分的一个结构域(PASA结构域)，并根据肺炎链球菌与头孢菌素类抗生素结合的PBP2X的结构，预测该结构域将与未交联的PGN前体的干肽结合。最近，枯草杆菌丝氨酸/苏氨酸蛋白激酶的意大利面域被证明既能结合完整的PGN，也能结合水解的PGN。枯草杆菌孢子与PGN孵育可促进孢子萌发，提高丝氨酸/苏氨酸的磷酸化水平。对PGN的来源和这些功能效应有一定的特异性，表明含有中位二氨基松酸(m-DAP)的PGN在刺激孢子萌发方面具有一定的特异性。 结核分枝杆菌基因组编码两种含有面团结构域的蛋白质，一种是丝氨酸/苏氨酸蛋白激酶PKnB(Rv0014c)，它的胞外区由四个意大利面结构域组成；另一种是双功能青霉素结合蛋白PBP2(PonA2，Rv3682)，它在胞外转肽酶和含有转糖酶的结构域的远端具有一个单一的意大利面结构域。在这项工作中，我们确定了PKnB定位于隔膜和细胞极点。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The apparent ability of M. tuberculosis to become dormant in the human host, leading to asymptomatic latent infection, has stimulated great interest in understanding how cell growth and cell division are regulated in this organism. A longstanding observation that "spent" or "conditioned" medium from bacterial cultures is able to stimulate growth of dormant cells, led to the identification of a resuscitation promoting factor (Rpf) by purifying from spent medium a component that was able to stimulate growth in the actinomycete Micrococus luteus. Rpf is small protein that has homologues in other actinobacteria, including M. tuberculosis, which has five rpf genes. Functional studies of these genes in M. tuberculosis has shown that individually they are not required for resuscitation of dormant Mtb cells and single rpf mutant strains do not have other growth or morphologic phenotypes. When two or more rpf genes are inactivated, however, growth or resuscitation defects are observed. The recent demonstration that the Rpf's are PGN hydrolases suggests that growth stimulation of dormant cells may result from the enzymatic activity of these secreted proteins, possibly through alterations in PGN structure or through the interaction of PGN degradation products with the bacterial cell surface. A domain found to occur in the extracellular portions of penicillin binding proteins and serine/threonine kinases (PASTA domain) was identified by bioinformatic analysis and predicted to bind to the stem peptide of un-crosslinked PGN precursors based on the structure of PBP2X of Streptococcus pneumoniae bound to a cephalosporin antibiotic. Recently the PASTA domain of a Ser/Thr kinase of B. subtilis was shown to bind both intact and hydrolyzed PGN. Incubation of B. subtilis spores with PGN stimulated spore germination and increased Ser/Thr phosphorylation. Some specificity with respect to the source of PGN and these functional effects was observed, suggesting a preference for meso-diaminopimelic acid (m-DAP)-containing PGN in stimulating spore germination. The M. tuberculosis genome encodes two proteins that contain PASTA domains, the Ser/Thr protein kinase PknB (Rv0014c) whose extracellular region comprises four PASTA domains, and the bifunctional penicillin binding protein PBP2 (PonA2, Rv3682), which has a single PASTA domain at the extreme carboxy-terminus of protein distal to the extracellular transpeptidase and transglycosylase-containing domains. In this work we determined that PknB is localized to septum and cell poles.