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。
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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Brittany Nicole Ross的其他文献
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