Probing Differences in Gene Essentiality Between Mycobacteroides abscessus Smooth and Rough Morphotypes During Infection

探讨脓肿分枝杆菌感染过程中光滑形态和粗糙形态之间基因重要性的差异

• 批准号: 10679727
• 负责人: Brittany Nicole Ross
• 金额: $ 7.43万
• 依托单位: GEORGIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10679727
• 关键词: Abscess; Address; Anabolism; Appearance; Bacillus subtilis; Bacteremia; Bacteria; Behavior; Behavioral; Biological Assay; CRISPR interference; Cell Wall; ChIP-seq; Chronic; Communities; Data; Educational process of instructing; Environment; Essential Genes; Exposure to; Face; Fellowship; Gene Library; Genes; Genetic; Genetic Transcription; Genome; Growth; Human; Immunocompetent; Immunocompromised Host; In Vitro; Individual; Infection; Infectious Skin Diseases; Knowledge; Libraries; Lung; Metals; Methods; Modeling; Molecular; Molecular Genetics; Morbidity - disease rate; Morphology; Mus; Mutation; Mycobacterium abscessus; Mycobacterium smegmatis; Nitric Oxide; Oxygen; Pathogenicity; Pathway interactions; Patients; Phenotype; Physiological; Physiology; Postdoctoral Fellow; Pseudomonas aeruginosa; Regulon; Repression; Research; Research Training; Respiratory Tract Infections; Scientist; Soft Tissue Infections; Stimulus; Stress; Surface; Technical Expertise; Technology; Testing; Training; Vaccines; Vibrio cholerae; antimicrobial; behavior in vitro; effective therapy; emerging pathogen; fitness; improved; in vivo; insight; knock-down; mutant; mycobacterial; opportunistic pathogen; pathogen; respiratory; skin abscess; transcriptome sequencing; transposon sequencing

PROJECT SUMMARY Several pathogens utilize morphological changes to improve their fitness in a particular environment, but these changes often coincide with broad physiological reprogramming. Mycobacteroides abscessus (MAB) is one such pathogen, presenting as a smooth colony (MABS) in the environment and host, which can transition to a rough (MABR) morphotype following an unknown stimulus during infection. MAB is an emerging pathogen among immunocompromised and immunocompetent individuals causing a wide variety of infections, including respiratory, skin abscesses, soft tissue infection, and bacteremia. Several groups have shown that these two colony morphotypes are phenotypically distinct in vitro and in the host, yet the underlying genetic components contributing to their behavioral differences are still unknown. We have found that MABS and MABR require unique genes for survival indicating that they are molecularly distinct despite having a nearly identical genome. There still remains a significant gap in knowledge in the molecular genetic mechanisms underlying physiology that controls the phenotypic differences. To address this, in Aim 1, I will use transposon insertion (Tn-seq) libraries in both MABS and MABR to examine genes required for survival in a murine abscess model and seven infection relevant conditions (e.g., low oxygen, nitric oxide, metal limitation, abscess infection). The data gathered from individual infection relevant conditions will then be leveraged to determine which antimicrobial mechanisms the two morphotypes face in the host and if they use similar pathways to respond. I have already generated an ordered transposon library which will allow us to confirm our findings of select mutants. In Aim 2, I will investigate uniquely essential genes, MAB_2726c (unique to MABS) and MAB_3329c (unique to MABR) by assaying survival following knocked down with CRISPRi. These genes are transcriptional regulators which likely have broad effects; therefore, I will define their regulome using CHiPSeq and confirm our findings using RNAseq following repression by CRISPRi and controlling for differences in growth using a chemostat. I hypothesize that M. abscessus MABS and MABR have different essential genes when exposed to stress, during infection, and differential essentiality of transcriptional regulators contribute to broad physiological changes that confer phenotypic differences. Due to the lack of effective therapies and a vaccine, this fellowship aims to build a research portfolio that will address the urgent need for further understanding of this pathogen. Tn-seq offers a method for quick and broad identification of genes essential for survival in various environments and public availability of the data generated not only benefits my study of MAB as a postdoc but also as an independent scientist and the field at large.

项目摘要 几种病原体利用形态学变化来改善其在特定环境中的适应性，但是这些 变化通常与广泛的生理重编程相吻合。分枝杆菌的演（mab）就是这样的 病原体，在环境和宿主中以光滑的菌落（mAb）表示，可以过渡到粗糙 （MABR）在感染过程中未知刺激后的形态型。 mab是一种新兴的病原体 免疫功能低下和免疫能力的个体引起了各种各样的感染，包括 呼吸道，皮肤脓肿，软组织感染和菌血症。几个小组表明这两个 菌落形态型在体外和宿主上是表型截然不同的，但是潜在的遗传成分 导致其行为差异的贡献仍然未知。我们发现mabs和mabr需要独特 生存的基因表明，尽管基因组几乎相同，但它们的分子很明显。那里 仍然在生理学的分子遗传机制方面仍然存在显着差距 控制表型差异。为了解决这个问题，在AIM 1中，我将使用Transposon插入（TN-Seq）库 在mAb和MABR中，检查了鼠脓肿模型中生存所需的基因和七个感染 相关条件（例如，低氧，一氧化氮，金属限制，脓肿感染）。从中收集的数据 然后，将利用各个感染相关条件，以确定哪些抗菌机制 两种形态型面对宿主，如果他们使用类似的途径进行响应。我已经生成了 订购的转座子库，这将使我们能够确认所选突变体的发现。在AIM 2中，我将调查 通过分析生存的生存 在被克里斯普里击倒之后。这些基因是转录调节因子，可能具有广泛 效果；因此，我将使用chipseq定义他们的规范，并使用rnaseq确认我们的发现。 CRISPRI的抑制并控制化学固定剂的生长差异。我假设M。 腹肌mAB和MABR在受到压力，感染期间和 转录调节剂的差异本质有助于赋予广泛的生理变化 表型差异。由于缺乏有效的疗法和疫苗，该研究金旨在建立一个 研究组合将迫切需要进一步了解这种病原体。 TN-Seq提供a 快速，广泛识别基因在各种环境和公众中必不可少的基因的方法 生成的数据的可用性不仅有利于我对MAB作为博士后的研究，而且有益于独立 科学家和整个领域。