Roles of Phosphate Uptake in Pneumococcal Antibiotic Resistance and Virulence

描述（由申请人提供）：肺炎链球菌（肺炎球菌）是一种主要的人类呼吸道病原体，在世界范围内仍然是严重的健康威胁。肺炎链球菌对多种抗生素的耐药性正以惊人的速度增加，面临肺炎球菌侵袭性疾病风险的免疫功能低下个体的人数继续增加。关于肺炎球菌的生理和代谢在促进其人类宿主的定植和毒力方面所起的作用，人们知之甚少。该资助的总体目标是探索无机磷酸盐（Pi）摄取及其调控在肺炎链球菌的发病机制、抗生素耐药性和金属离子稳态中的作用。Pi摄取已经与肺炎球菌侵袭性疾病有关，但对这种参与的机制知之甚少。一些证据表明，肺炎链球菌对Pi转运的调节与之前确定的模式细菌（如大肠杆菌和枯草芽孢杆菌）的机制根本不同。因此，这项资助将挑战目前基于这些模型细菌系统的Pi摄取范式，并首次将Pi摄取与肺炎链球菌的发病机制联系起来。此外，这项工作将验证和探索一种未知的肺炎球菌¿-内酰胺耐药性的新机制。最后，该资助汇集了科学文献中的几个最新观点，以形成细胞Pi摄取和Pi细胞数量与几种关键二价阳离子的可用性和毒性有关的假设，包括锌（Zn2+）和锰（Mn2+）。此奖助金将满足四个具体目标。目的1将确定两个预测的Pi ABC转运蛋白泵（指定为Pst1和Pst2）是否在肺炎链球菌中单独摄取Pi，以及这种摄取是否协调。Aims 1的另一个目标是开发一个强大的遗传工具箱和一个关于Pi摄取及其调控的概念框架，这将允许对Aims 2-4中的主题进行有效的探索。目的2将确定Pst1和Pst2 Pi转运体以及可能的调节因子（指定为PnpRS， PhoU1和PhoU2）在肺炎球菌定植和毒力中的作用。Aim 2还将确定在感染过程中，除了Pst1和Pst2之外，是否还有其他途径摄取Pi。Aim 3将确定Pst1转运体的表达增加是否与对内酰胺类抗生素的耐药性有关，以及Pst2转运体是否也起作用。目的4将探讨Pi摄取与细胞数量和二价金属离子稳态之间是否存在联系。从这项工作中获得可发表数据的可能性很大，这将作为未来RO1应用的基础，并为肺炎链球菌开辟一个重要的新研究领域。此外，这笔拨款有可能为抗生素和疫苗的开发提供新的靶点，并提供有关这一重要机会性病原体中未表征的¿-内酰胺抗生素耐药机制的信息。

DESCRIPTION (provided by applicant): Streptococcus pneumoniae (pneumococcus) is a major human respiratory pathogen that remains a serious health threat worldwide. Resistance of S. pneumoniae to multiple antibiotics is increasing at an alarming rate, and the population of immunocompromised individuals who are at risk for pneumococcal invasive diseases continues to grow. Relatively little is known about the roles played by pneumococcal physiology and metabolism in promoting colonization and virulence of its human host. The overall goal of this grant is to explore the roles played by inorganic phosphate (Pi) uptake and its regulation in the pathogenesis, antibiotic resistance, and metal ion homeostasis of S. pneumoniae. Pi uptake has already been implicated in pneumococcal invasive disease, but little is known about the mechanisms underlying this involvement. Several pieces of evidence show that the regulation of Pi transport in S. pneumoniae is fundamentally different from the mechanisms determined previously for model bacteria, like E. coli and B. subtilis. Therefore, this grant will challenge te current paradigms of Pi uptake that are based on these model bacterial systems and relate Pi uptake to the pathogenesis of S. pneumoniae for the first time. In addition, this work will validat and explore a new mechanism of pneumococcal ¿-lactam resistance about which nothing is known. Finally, this grant draws together several recent ideas from the scientific literature to formulate the hypothesis that cellular Pi uptake and Pi cellular amount are tied to the availabilit and toxicity of several key divalent cations, including zinc (Zn2+) and manganese (Mn2+). Four Specific Aims will be met by this grant. Aim 1 will determine whether two predicted Pi ABC transporter pumps (designated as Pst1 and Pst2) separately uptake Pi in S. pneumoniae and whether this uptake is coordinated. Another goal of Aim 1 is to develop a powerful genetic toolbox and a conceptual framework about Pi uptake and its regulation that will allow effective exploration of the topics in Aims 2-4. Aim 2 will determine the roles of the Pst1 and Pst2 Pi transporters and the likely regulators (designated as PnpRS, PhoU1, and PhoU2) in pneumococcal colonization and virulence. Aim 2 will also determine whether there are additional routes of Pi uptake during infection, besides Pst1 and Pst2. Aim 3 will determine whether increased expression of the Pst1 transporter is responsible for resistance to ¿-lactam antibiotics and whether the Pst2 transporter also plays a role. Aim 4 will explore whether there are links between Pi uptake and cellular amount and divalent metal ion homeostasis. There is a high likelihood of obtaining publishable data from this work that will serve as the basis for a future RO1 application and open up a significant new research area in S. pneumoniae. In addition, this grant has the potential to provide new targets for antibiotic and vaccine development and to provide information about an uncharacterized mechanism of ¿-lactam antibiotic resistance in this important opportunistic pathogen.