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

项目总结 一些病原体利用形态变化来改善它们在特定环境中的适应性，但这些 变化往往与广泛的生理重新编程相吻合。脓肿分枝杆菌(MAB)就是这样的一种 病原菌，在环境和寄主中以光滑菌落(MABS)的形式出现，可以过渡到粗糙的 (MABR)感染期间未知刺激后的形态类型。单抗是一种新出现的病原体 导致多种感染的免疫受损和免疫能力正常的人，包括 呼吸道、皮肤脓肿、软组织感染和菌血症。几个小组已经证明，这两个人 在体外和宿主中，菌落形态是不同的表型，但潜在的遗传成分 造成他们行为差异的原因尚不清楚。我们发现MABS和MABR需要唯一的 生存基因表明，尽管它们的基因组几乎相同，但它们在分子上是不同的。那里 在生理学基础上的分子遗传机制方面仍然存在着显著的知识差距 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)文库 在MABS和MABR中检查在小鼠脓肿模型和七种感染中生存所需的基因 相关条件(如低氧、一氧化氮、金属受限、脓肿感染)。从以下来源收集的数据 然后，将利用个别感染相关条件来确定哪些抗菌机制 在宿主中有两种形态，如果它们使用相似的途径做出反应。我已经生成了一个 有序的转座子文库，这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 独有必需基因MAB_2726c(MABS独有)和MAB_3329c(MABR独有) 紧随其后的是CRISPRI。这些基因是转录调节基因，可能具有广泛的 效果；因此，我将使用CHiPSeq定义它们的调节组，并使用RNAseq确认我们的发现如下 CRISPRi的抑制和使用恒化器控制生长的差异。我假设M。 脓肿MABS和MABR在暴露于压力、感染期间和 转录调控因子的不同重要性有助于产生广泛的生理变化 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 将解决进一步了解这种病原体的迫切需要的研究组合。TN-SEQ提供 一种快速而广泛地识别在各种环境和公共环境中生存所必需的基因的方法 所产生的数据的可用性不仅有利于我作为博士后研究MAB，而且作为一个独立的 科学家和整个领域。

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.