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它们在特定环境中的适应性，但这些病原体在特定环境中的适应性是不稳定的。 变化往往与广泛的生理重编程相一致。Mycobacteroides pesticessus（MAB）就是这样一种 病原体，在环境和宿主中表现为光滑菌落（MABS），可转变为粗糙菌落 （MABR）形态型在感染过程中的未知刺激。MAB是一种新兴的病原体， 免疫功能低下和免疫功能正常的个体引起各种感染，包括 呼吸道、皮肤脓肿、软组织感染和菌血症。几个研究小组表明，这两个 菌落形态在体外和宿主中的表型不同，但潜在的遗传成分 导致它们行为差异的原因仍不清楚。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 在生理学基础的分子遗传机制方面仍然存在着重大的知识空白， 控制着表型的差异。为了解决这个问题，在目标1中，我将使用转座子插入（Tn-seq）文库 在MABS和MABR中检测小鼠脓肿模型和7种感染中生存所需的基因 相关条件（例如，低氧、一氧化氮、金属限制、脓肿感染）。收集的数据 然后将利用个体感染相关条件来确定 两种形态型面对着宿主，如果它们使用相似的途径来响应。我已经生成了一个 有序的转座子文库，这将使我们能够确认我们选择突变体的发现。在目标2中，我将研究 独特必需基因MAB_2726c（MABS特有）和MAB_3329c（MABR特有） 在被CRISPRi击倒之后。这些基因是转录调节因子，可能具有广泛的 因此，我将使用CHiPSeq定义它们的调节组，并使用RNAseq确认我们的发现， 通过CRISPRi抑制和使用恒化器控制生长差异。假设M。 当暴露于压力、感染过程中， 转录调节因子的不同重要性有助于广泛的生理变化， 表型差异由于缺乏有效的疗法和疫苗，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提供了一个 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，也有利于我作为独立人士的研究 科学家和整个领域。