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
基本信息
- 批准号:10679727
- 负责人:
- 金额:$ 7.43万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 关键词:AbscessAddressAnabolismAppearanceBacillus subtilisBacteremiaBacteriaBehaviorBehavioralBiological AssayCRISPR interferenceCell WallChIP-seqChronicCommunitiesDataEducational process of instructingEnvironmentEssential GenesExposure toFaceFellowshipGene LibraryGenesGeneticGenetic TranscriptionGenomeGrowthHumanImmunocompetentImmunocompromised HostIn VitroIndividualInfectionInfectious Skin DiseasesKnowledgeLibrariesLungMetalsMethodsModelingMolecularMolecular GeneticsMorbidity - disease rateMorphologyMusMutationMycobacterium abscessusMycobacterium smegmatisNitric OxideOxygenPathogenicityPathway interactionsPatientsPhenotypePhysiologicalPhysiologyPostdoctoral FellowPseudomonas aeruginosaRegulonRepressionResearchResearch TrainingRespiratory Tract InfectionsScientistSoft Tissue InfectionsStimulusStressSurfaceTechnical ExpertiseTechnologyTestingTrainingVaccinesVibrio choleraeantimicrobialbehavior in vitroeffective therapyemerging pathogenfitnessimprovedin vivoinsightknock-downmutantmycobacterialopportunistic pathogenpathogenrespiratoryskin abscesstranscriptome sequencingtransposon sequencing
项目摘要
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。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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