Interaction of the Burkholderia type VI secretion system with innate host defenses

伯克霍尔德杆菌 VI 型分泌系统与先天宿主防御的相互作用

• 批准号: 9751108
• 负责人: Sandra Schwarz
• 金额: $ 21.74万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-01-04 至 2020-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9751108
• 关键词: 3-Dimensional; Alveolar Macrophages; Alveolus; Antibiotic Therapy; Antibiotics; Bacteria; Bacterial Infections; Burkholderia; Burkholderia pseudomallei; Carbapenems; Case Fatality Rates; Ceftazidime; Cell fusion; Cells; Clinical Trials; Cytometry; Cytoplasm; Cytosol; Development; Disease; Endosomes; Epithelial Cells; Future; Host Defense; Human; Image; In Vitro; Infection; Ingestion; Inhalation; Knowledge; Life Style; Lung; Mediating; Melioidosis; Organism; Parasites; Patients; Phenotype; Pneumonia; Population; Process; Public Health; Reporter; Reproducibility; Research Personnel; Resistance; Resolution; Role; Septic Shock; Signal Transduction; Soil; System; Testing; Thailand; Treatment Efficacy; Type III Secretion System Pathway; Virulence; Virulence Factors; cell type; effective therapy; high resolution imaging; imaging modality; improved; in vivo; intracellular parasitism; mortality; mouse model; novel; novel therapeutics; pathogen; standard care; targeted treatment; therapeutic development; tool

Melioidosis is a tropical infection caused by inoculation, inhalation, or ingestion of the Gram-negative soil saprophyte and Tier 1 select agent Burkholderia pseudomallei (Bps). Pneumonia is a common manifestation of infection, is frequently associated with septic shock, and confers a two-fold increase in mortality. Bps is intrinsically resistant to many common antibiotics. The standard therapies to which Bps is susceptible are ceftazidime and carbapenems yet patients may take days to defervesce once on treatment and need extended courses of therapy. Treatments for melioidosis have not changed substantially in decades and the case fatality rate in northeast Thailand has remained at 40% since 1989. New therapies are urgently needed; yet to develop effective therapies it is essential to advance understanding of the pathogen's ability to persist, especially in the lung. The multiple PIs of this application offer expertise in pulmonary host defenses (West) and in Burkholderia virulence factors (Schwarz). Together, these investigators have studied both Bps and the closely related bacterium Burkholderia thailandensis (Bt), a surrogate organism to Bps that is avirulent in humans and exempt from select agent restrictions. Bt/Bps, facultative intracellular bacteria, enter a wide variety of cell types and can escape the endosome into the cytosol. The type III secretion system (T3SS) is involved in cellular invasion and endosomal escape. Bt/Bps can induce cell-cell fusions in a type VI secretion system (T6SS)-mediated fashion that may facilitate intercellular spread. Bps can enter primary alveolar macrophages and bronchial epithelial cells in vitro, yet replication in alveolar macrophages is contained whereas replication in bronchial epithelial cells is unrestricted. However, the extent of intracellular parasitism by Bt/Bps, the cells that Bt/Bps infects in the lung, and the effect of T3SS and T6SS on these host-pathogen interactions have not been studied in vivo. The central hypothesis of this project is that Bps differentially parasitizes, replicates, and spreads intercellularly in specific pulmonary cell types in vivo; these processes are variably dependent on T3SS or T6SS, and they impede successful treatment. It is critical to test this hypothesis in order to develop new cell- or pathogen-targeted therapies for this infection. This will be accomplished using a novel dual fluorescent Bt reporter construct that signals when the bacterium is located within the cytoplasm of the host cell. The aims of the project are: 1) Determine whether specific cell types are differentially parasitized by Bt in the lung in vivo; 2) Determine whether T3SS and T6SS alter intracellular localization and the specific cells parasitized by Bt in the lung in vivo; 3) Determine whether standard treatment for melioidosis successfully kills intracellular Bt in the lung in vivo. The scientific value of this project is that it will advance understanding of the specific host cells parasitized by Bps in the lung in vivo and the role of T3SS and T6SS virulence factors on this interaction, evaluate how this intracellular lifestyle impacts current antibiotic treatment efficacy, and identify cellular or bacterial targets for future novel therapeutic development.

Melioidosis是由接种，吸入或摄入革兰氏阴性土壤引起的热带感染 Saprophyte和Tier 1 Select Agent Burkholderia pseudomallei（BPS）。肺炎是 感染通常与化粪池休克有关，并赋予死亡率增加两倍。 BPS是 对许多常见抗生素的本质上具有抗性。 BPS易感的标准疗法是 头孢嗪和碳青霉烯术，但患者可能需要几天的时间才能进行治疗，并且需要延长 治疗课程。几十年来，黑梅利病的治疗尚未发生实质性变化，病例死亡 自1989年以来，泰国东北部的比率一直保持在40％。迫切需要新的疗法；尚未发展 有效的疗法必须提高对病原体持续存在能力的理解，尤其是在 肺。该申请的多个PI提供了肺部宿主防御（西）和伯克霍尔德里亚的专业知识 毒力因子（Schwarz）。这些研究人员一起研究了BP和密切相关的 伯克霍尔德细菌泰国人（BT），一种替代生物的BPS，在人类中是无毒的和豁免的 从选择代理限制。 bt/bps，辅助细胞内细菌，进入多种细胞类型， 可以将内体逃脱到细胞质中。 III型分泌系统（T3SS）参与细胞侵袭 和内体逃生。 bt/bps可以在VI型分泌系统（T6SS）介导的细胞电池融合 可能有助于细胞间扩散的时尚。 BPS可以进入原发性肺泡巨噬细胞和支气管 体外上皮细胞，但肺泡巨噬细胞中的复制含有，而支气管中的复制 上皮细胞不受限制。但是，BT/BP的细胞内寄生虫的程度，BT/BPS的细胞 肺中的感染以及T3SS和T6S对这些宿主病原体相互作用的影响尚未 在体内学习。该项目的核心假设是BP差异化，重复和 在体内特定的肺部细胞类型中散布；这些过程都取决于 T3SS或T6SS，它们阻碍了成功的治疗。检验这一假设以发展至关重要 该感染的新细胞或病原体靶向疗法。这将使用新颖的双重 当细菌位于宿主的细胞质内时，荧光BT报告基因构造 细胞。该项目的目的是：1）确定BT在 体内肺； 2）确定T3SS和T6S是否改变了细胞内定位和特定细胞 由BT在体内肺部寄生； 3）确定黑梅利病的标准治疗是否成功杀死 肺部体内的细胞内BT。该项目的科学价值是它将提高对 由BPS在体内寄生的特定宿主细胞以及T3SS和T6SS毒力因子在体内的作用 这种相互作用，评估这种细胞内生活方式如何影响当前的抗生素治疗功效，并确定 未来新型治疗发育的细胞或细菌靶标。