TB Membrane Transporters and Intrinsic Resistance

结核病膜转运蛋白和固有耐药性

• 批准号: 8868006
• 负责人: Georgiana E. Purdy
• 金额: $ 38.5万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-05 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8868006
• 关键词: ATP-Binding Cassette Transporters; Anti-Bacterial Agents; Antibiotic Resistance; Antibiotic Therapy; Antibiotics; Antimicrobial Cationic Peptides; Antimicrobial Resistance; Bacteria; Binding; Biological Assay; Cathepsins; Cell Membrane Permeability; Cell Wall; Circular Dichroism; Data; Development; Disease; Drug Efflux; Environment; Exposure to; Future; Genes; Genus Mycobacterium; Goals; Human; Immune response; In Vitro; Infection; Knowledge; Lead; Membrane; Membrane Proteins; Membrane Transport Proteins; Metabolic; Molecular Profiling; Mus; Mycobacterium tuberculosis; NMR Spectroscopy; Peptide Antibiotics; Peptide Hydrolases; Peptides; Permeability; Pharmaceutical Preparations; Pharmacotherapy; Play; Predisposition; Property; Public Health; Regulation; Resistance; Rest; Role; Signal Transduction; System; Therapeutic; Time; Transmembrane Transport; Tuberculosis; Ubiquitin; Virulence; Work; antibiotic efflux; antimicrobial; antimicrobial peptide; bactericide; base; differential expression; disorder control; improved; inhibitor/antagonist; killings; macrophage; mouse model; mutant; mycobacterial; novel therapeutic intervention; novel therapeutics; research study; resistance mechanism; response; therapeutic target; tool

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.

描述（由申请人提供）：结核病继续对公众健康构成威胁，而对常用抗生素的耐药性是控制这种疾病的努力中日益普遍的问题。因此，非常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流出，分枝杆菌表现出对抗生素的内在耐药性。了解药物流出系统在抗生素耐药性中的作用及其调节将带来新的结核病治疗方法，因为抑制这些内在系统可能会提高现有药物的疗效并缩短治疗时间。 之前我们表明，泛素衍生肽（Ub 肽）有助于巨噬细胞的分枝杆菌杀菌能力（Alonso 等，2007；Purdy 等，2009）。我们的首要目标是更好地了解这些肽的杀菌作用。初步研究表明，Ub 肽的行为类似于抗菌肽，会损害分枝杆菌膜功能。结核分枝杆菌 (Mtb) 与人类免疫反应共同进化，并且可能对亚致死浓度的宿主抗菌化合物具有耐药机制。在现代疾病中，这些系统可能提供对抗生素的内在耐药性。为了确定 Mtb 内在耐药机制，采用了两种互补的方法，以杀分枝杆菌 Ub 肽为工具。 1.) 我们在转座子突变体筛选中直接鉴定了对 Ub 肽处理更敏感的 ABC 和 MFS 转运蛋白突变体。 2.) 分析了暴露于 Ub 肽的 Mtb 的表达谱。百分之十的 Ub 肽调节基因编码预测的膜蛋白，包括 ATP 结合盒 (ABC) 或主要促进子超家族 (MFS) 转运系统。这些差异表达的基因改变了细菌对抗菌肽和抗生素存在的反应。为了证明这一点，我们获得了缺乏 ABC 和 MFS 转运蛋白的 Mtb 突变体，并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 膜转运系统有助于 Mtb 固有的抗菌耐药性和毒力。这项工作的长期目标将指导这些膜转运系统的治疗靶向，以提高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）我们将表征 Ub 肽的作用机制。 2) 为了更好地理解 Mtb 的内在耐药性，我们将定义 ABC 转运蛋白 Pst 和 Rv0986-Rv0987 以及 MFS 转运蛋白 Rv1634 和 Rv3239c 在 Mtb 内在耐药性中的作用。 3) 我们将通过巨噬细胞存活测定和小鼠感染来确定这些 ABC 和 MFS 转运蛋白是否对结核分枝杆菌毒力有贡献。这些研究将扩大我们对结核分枝杆菌对抗菌 Ub 肽和抗生素固有耐药性的了解，并将确定未来药物治疗方法的靶点。

项目成果

Crystal structure of the transcriptional regulator Rv1219c of Mycobacterium tuberculosis.

结核分枝杆菌转录调节因子 Rv1219c 的晶体结构。

• DOI: 10.1002/pro.2424
• 发表时间: 2014
• 期刊: Protein science : a publication of the Protein Society
• 作者: Kumar,Nitin;Radhakrishnan,Abhijith;Wright,CatherineC;Chou,Tsung-Han;Lei,Hsiang-Ting;Bolla,JaniReddy;Tringides,MariosL;Rajashankar,KanagalaghattaR;Su,Chih-Chia;Purdy,GeorgianaE;Yu,EdwardW
• 通讯作者: Yu,EdwardW

Confinement-Induced Drug-Tolerance in Mycobacteria Mediated by an Efflux Mechanism.

• DOI: 10.1371/journal.pone.0136231
• 发表时间: 2015
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Luthuli BB;Purdy GE;Balagaddé FK
• 通讯作者: Balagaddé FK

Reply to Brennan, "Biofilms and Mycobacterium tuberculosis".

回复布伦南，“生物膜和结核分枝杆菌”。

• DOI: 10.1128/iai.00436-17
• 发表时间: 2017
• 期刊: Infection and immunity
• 影响因子: 3.1
• 作者: Purdy,GeorgianaE