TB Membrane Transporters and Intrinsic Resistance

描述（由申请人提供）：结核病继续对公众健康构成威胁，在控制这种疾病的努力中，对常用抗生素的耐药性是一个日益普遍的问题。因此，迫切需要开发新的药物和治疗策略。由于分枝杆菌细胞壁的低渗透性和抗生素的主动外排，分枝杆菌对抗生素表现出固有的耐药性。了解药物外排系统在固有抗生素耐药性中的作用及其调控将导致新的结核病治疗方法，因为抑制这些固有系统可能会提高现有药物的疗效并缩短治疗时间。先前我们发现泛素衍生肽（Ub-peptides）有助于巨噬细胞的分枝杆菌杀灭库（Alonso等，2007；Purdy等，2009）。我们的第一个目标是更好地了解这些肽的杀菌作用。初步研究表明，ub肽的行为与抗菌肽类似，可损害分枝杆菌的膜功能。结核分枝杆菌（Mtb）与人类免疫反应共同进化，可能对亚致死浓度的宿主抗微生物化合物具有耐药机制。在现代疾病中，这些系统可能对抗生素产生固有的耐药性。为了确定结核分枝杆菌的内在耐药机制，采用了两种互补的方法，以杀分枝杆菌的ub -肽为工具。1)。我们在转座子突变体筛选中直接鉴定出ABC和MFS转运体突变体对ub -肽处理更敏感。2.） 分析结核分枝杆菌暴露于ub -peptide后的表达谱。10%的ub肽调节基因编码预测膜蛋白，包括ATP结合盒（ABC）或主要促进者超家族（MFS）运输系统。这些差异表达的基因改变了细菌对抗菌肽和抗生素的反应。为了证明这一点，我们获得了缺乏ABC和MFS转运体的Mtb突变体，发现它们对抗菌肽和抗生素更敏感。我们的工作假设是，Ub-肽是宿主抗菌肽，Mtb ABC和MFS膜运输系统有助于Mtb内在的抗菌素耐药性和毒力。这项工作的长期目标是指导这些膜运输系统的治疗靶向，以增加结核分枝杆菌对宿主抗菌化合物和抗生素治疗的固有敏感性。我们的目标如下：1)我们将描述ub肽作用的机制。2)为了更好地了解结核分枝杆菌的内在耐药性，我们将确定ABC转运体Pst和Rv0986-Rv0987以及MFS转运体Rv1634和Rv3239c在结核分枝杆菌内在耐药性中的作用。3)我们将通过巨噬细胞存活试验和小鼠感染来确定这些ABC和MFS转运体是否有助于结核分枝杆菌的毒力。这些研究将扩大我们对结核分枝杆菌对抗菌b肽和抗生素固有耐药的认识，并将确定未来药物治疗方法的靶点。

DESCRIPTION (provided by applicant): Tuberculosis continues to pose a threat to public health, and resistance to commonly used antibiotics is an increasingly prevalent problem in efforts to control this disease. Thus, there is a great need to develop new drugs and therapeutic strategies. Mycobacteria display intrinsic resistance to antibiotics due to the low permeability of the mycobacterial cell wall and active efflux of antibiotics. An understanding of drug efflux systems role in intrinsic antibiotic resistance and their regulation will lead to new therapeutic approaches to TB since inhibition of these intrinsic systems may improve the efficacy of current drugs and shorten treatment time. Previously we showed that ubiquitin-derived peptides (Ub-peptides) contribute the mycobactericidal repertoire of macrophages (Alonso et al., 2007; Purdy et al., 2009). Our first objective is to better understand the bactericidal actions of these peptides. Initial studies indicate that Ub-peptides behave like antimicrobial peptides to impair mycobacterial membrane function. Mycobacterium tuberculosis (Mtb) has co-evolved with the human immune response and likely possesses resistance mechanisms to sub-lethal concentrations of host antimicrobial compounds. In modern disease, these systems may provide intrinsic resistance to antibiotics. To identify Mtb intrinsic resistance mechanisms, two complementary approaches were taken using mycobactericidal Ub-peptides as a tool. 1.) We directly identified ABC and MFS transporter mutants more susceptible to treatment with Ub-peptides in a transposon mutant screen. 2.) The expression profile of Mtb exposed to Ub-peptides was analyzed. Ten percent of Ub-peptide regulated genes encoded predicted membrane proteins including ATP Binding Cassette (ABC) or Major Facilitator Superfamily (MFS) transport systems. These differentially expressed genes alter the bacterial response to the presence of antimicrobial peptides and antibiotics. To show this we acquired Mtb mutants that lack ABC and MFS transporters and found that they are more susceptible to antimicrobial peptides and antibiotics. Our working hypothesis is that Ub- peptides are host antimicrobial peptides and that Mtb ABC and MFS membrane transport systems contribute to Mtb intrinsic antimicrobial resistance and virulence. The long-term goal of this work will direct therapeutic targeting of these membrane transport systems to increase the inherent sensitivity of Mtb to host antimicrobial compounds and antibiotic treatment. Our aims are the following: 1) We will characterize the mechanism of Ub-peptide action. 2) To better understand intrinsic resistance of Mtb we will define the role of the ABC transporters Pst and Rv0986-Rv0987, and the MFS transporters Rv1634, and Rv3239c in the intrinsic resistance of Mtb. 3) We will determine if these ABC and MFS transporters contribute to M. tuberculosis virulence through macrophage survival assays and mouse infections. These studies will expand our knowledge of M. tuberculosis intrinsic resistance to antimicrobial Ub-peptides and antibiotics, and will identify targets for future drug therapy approaches.