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.

项目概要 一些病原体利用形态变化来提高它们在特定环境中的适应性，但是这些病原体 变化通常与广泛的生理重编程同时发生。脓肿分枝杆菌 (MAB) 就是其中之一 病原体，在环境和宿主中呈现为光滑菌落 (MABS)，可转变为粗糙菌落 (MABR) 感染期间未知刺激后的形态类型。 MAB 是一种新出现的病原体 免疫功能低下和免疫功能正常的个体会引起多种感染，包括 呼吸道、皮肤脓肿、软组织感染和菌血症。多个研究小组已经表明，这两个 集落形态型在体外和宿主中表现型不同，但潜在的遗传成分 造成他们行为差异的原因仍然未知。我们发现 MABS 和 MABR 需要独特的 生存基因表明，尽管它们具有几乎相同的基因组，但它们在分子上是不同的。那里 在生理学的分子遗传机制方面仍然存在巨大的知识空白 控制表型差异。为了解决这个问题，在目标 1 中，我将使用转座子插入 (Tn-seq) 库 在 MABS 和 MABR 中检查小鼠脓肿模型和七种感染中生存所需的基因 相关情况（例如，低氧、一氧化氮、金属限制、脓肿感染）。收集的数据来自 然后将利用与个体感染相关的条件来确定哪种抗菌机制 宿主体内存在两种形态类型，以及它们是否使用相似的途径做出反应。我已经生成了一个 有序的转座子库将使我们能够确认我们对选定突变体的发现。在目标 2 中，我将调查 通过测定存活率来确定独特的必需基因 MAB_2726c（MABS 独有）和 MAB_3329c（MABR 独有） 用 CRISPRi 击倒后。这些基因是转录调节因子，可能具有广泛的作用 影响；因此，我将使用 CHiPSeq 定义他们的调节组，并使用 RNAseq 确认我们的发现，如下 通过 CRISPRi 抑制并使用恒化器控制生长差异。我假设 M. 脓肿 MABS 和 MABR 在暴露于压力、感染期间和 转录调节因子的不同重要性有助于广泛的生理变化，从而赋予 表型差异。由于缺乏有效的治疗方法和疫苗，该奖学金旨在建立一个 研究组合将解决进一步了解这种病原体的迫切需要。 Tn-seq 提供了 快速广泛鉴定在各种环境和公众中生存所必需的基因的方法 生成的数据的可用性不仅有利于我作为博士后对 MAB 的研究，而且也有利于我作为独立研究人员的研究 科学家和整个领域。