Investigating Cell-Wall Synthesis in Mycobacterium abscessus
研究脓肿分枝杆菌细胞壁的合成
基本信息
- 批准号:10116157
- 负责人:
- 金额:$ 5.1万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2020
- 资助国家:美国
- 起止时间:2020-03-01 至 2022-02-28
- 项目状态:已结题
- 来源:
- 关键词:Active SitesAffectAllelesAmino Acid SequenceAmino AcidsAntibioticsBacteriaBindingBiogenesisBiological AssayBiological ProcessBiologyCRISPR interferenceCell WallCell divisionCellsCellular StructuresCo-ImmunoprecipitationsComplexCoupledDataDefectDevelopmentDiseaseDrug TargetingEnzymesFoundationsGeneticGenus MycobacteriumGrowthHigh-Throughput Nucleotide SequencingHomologous GeneImmunocompromised HostImmunoprecipitationImpairmentIncidenceInfectionKnowledgeLife Cycle StagesLipoprotein BindingLiquid substanceLungLung diseasesMicroscopyMorphologyMutagenesisMycobacterium abscessusMycobacterium tuberculosisPathogenicityPatientsPenicillinsPeptidoglycanPeptidyltransferasePhysiologyPlayProcessProteinsRepressionResistanceResourcesRoleSkin TissueSoft Tissue InfectionsSolidSpecificityStructure of parenchyma of lungTestingTimeTransmembrane DomainWorkbasecell growthcrosslinkexperimental studygene synthesisgenome-wideinsightknock-downmycobacterialnew therapeutic targetnon-tuberculosis mycobacterianovel therapeuticsoverexpressionprotein complexprotein functiontime use
项目摘要
Project Summary/Abstract
Mycobacterium abscessus (Mab) is a rapidly growing non-tuberculous mycobacterium (NTM) that causes
a wide range of illnesses including lung, skin and soft-tissue infections, as well as disseminated disease.
Treatment of Mab infections is difficult because the bacterium is intrinsically resistant to many classes of
antibiotics. Thus, there is a need to develop new therapies against Mab infection. The mycobacterial cell wall is
a popular target for antibiotics as its biogenesis is essential for bacterial growth. While cell wall synthesis has
been extensively studied in M. tuberculosis (Mtb), relatively little is known about how Mab builds its cell wall and
how this process differs from Mtb’s. To identify components of cell wall synthesis with unique roles in Mab
physiology, I performed genome-wide transposon mutagenesis coupled with high throughput-sequencing on
Mab. I then compared the essentiality of cell wall enzymes between Mab and Mtb. The data reveals Mab3167c,
a predicted penicillin-binding-lipoprotein (PBP-lipo), as being essential in Mab, while its homolog in Mtb is non-
essential. Mab3167c is predicted to be a transpeptidase that cross-links segments of the foundational
peptidoglycan (PG) layer of the cell wall. My preliminary data shows that repressing PBP-lipo impairs bacterial
growth and leads to gross morphological abnormalities in the cell. Given that PG synthesis has not been studied
in Mab, nor has the function of PBP-lipo, this proposal seeks to answer two central questions: 1) What is the role
of PBP-lipo in Mab PG synthesis? and 2) What cell wall enzymes genetically and physically interact with PBP-
lipo in Mab? Aim 1 interrogates the localization and function of PBP-lipo during PG synthesis using time-lapse
microscopy. With this approach, I will determine in real time where PBP-lipo localizes in the cell and assess how
its depletion influences PG synthesis. Aim 2 seeks to identify the functional genetic and physical network of
PBP-lipo in Mab. Previous work from our lab demonstrated that Mtb cell wall enzymes have unique sets of
genetic interactions and work in protein complexes to coordinate PG synthesis in a spatially and temporarily
coordinated manner. Using CRISPR-interference, I will knock down cell wall enzymes in combination with PBP-
lipo to determine which pairs genetically interact. I will also perform immunoprecipitation assays to identify
putative binding partners of PBP-lipo. All together, this work will elucidate when and where PBP-lipo functions in
the cell as well as illuminate how this enzyme contributes to PG synthesis. Moreover, this work will identify cell
wall synthesis genes that genetically interact with PBP-lipo as well as uncover proteins that function in complex
with the enzyme. These experiments will help uncover the specificities of Mab PG synthesis and cell wall
construction. Ultimately, my findings will not only advance the knowledge of cell wall biology in NTMs, but also
provide key insights into new drug targets and inform the development of successful treatments for Mab infection.
!
!
项目总结/摘要
结核分枝杆菌(Mycobacterium canceressus,Mab)是一种快速生长的非结核分枝杆菌(NTM),
一系列疾病,包括肺部、皮肤和软组织感染以及传播性疾病。
Mab感染的治疗是困难的,因为该细菌对许多种类的抗生素具有内在抗性。
抗生素因此,需要开发针对Mab感染的新疗法。分枝杆菌的细胞壁
它是抗生素的一个受欢迎的靶标,因为它的生物合成对于细菌生长是必不可少的。虽然细胞壁的合成
在M.对于结核病(Mtb),关于Mab如何构建其细胞壁以及
这个过程与结核分枝杆菌有何不同鉴定在单克隆抗体中具有独特作用的细胞壁合成组分
生理学,我进行了全基因组转座子诱变加上高通量测序,
mAb.然后比较了单抗和结核分枝杆菌细胞壁酶的重要性。数据显示Mab 3167 c,
一种预测的青霉素结合脂蛋白(PBP-lipo),在Mab中是必需的,而其在Mtb中的同系物是非必需的。
具有本质意义Mab 3167 c被预测为是一种转肽酶,其交联基础蛋白的片段。
细胞壁的肽聚糖(PG)层。我的初步数据显示,抑制PBP-lipo会损害细菌
生长并导致细胞中的总体形态异常。鉴于PG合成尚未被研究
在Mab,也没有PBP-lipo的功能,这项建议试图回答两个中心问题:1)什么是作用
PBP-lipo在单克隆抗体PG合成中的应用2)什么样的细胞壁酶在遗传和物理上与PBP相互作用-
在Mab做抽脂目的1利用延时摄影技术研究PG合成过程中PBP-lipo的定位和功能
显微镜通过这种方法,我将在真实的时间内确定PBP-lipo在细胞中的定位位置,并评估其如何定位。
其消耗影响PG合成。目标2旨在确定功能遗传和物理网络,
Mab中的PBP-lipo。我们实验室以前的工作表明,结核分枝杆菌细胞壁酶具有独特的
遗传相互作用和蛋白质复合物的工作,以协调PG合成在空间和时间上,
协调的方式。使用CRISPR干扰,我将结合PBP敲除细胞壁酶-
通过抽脂来确定哪对基因会相互作用我还将进行免疫沉淀分析,
PBP-lipo的推定结合配偶体。总之,这项工作将阐明何时何地PBP-lipo功能,
细胞以及阐明这种酶如何有助于PG合成。此外,这项工作将确定细胞
与PBP-lipo基因相互作用的壁合成基因,以及发现在复合物中起作用的蛋白质。
用酶。这些实验将有助于揭示单克隆抗体PG合成和细胞壁的特异性
建设最终,我的发现不仅将推进NTMs中细胞壁生物学的知识,
为新药靶点提供关键见解,并为Mab感染的成功治疗方法的开发提供信息。
!
!
项目成果
期刊论文数量(1)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
Transposon mutagenesis in Mycobacterium abscessus identifies an essential penicillin-binding protein involved in septal peptidoglycan synthesis and antibiotic sensitivity.
- DOI:10.7554/elife.71947
- 发表时间:2022-06-06
- 期刊:
- 影响因子:7.7
- 作者:Akusobi, Chidiebere;Benghomari, Bouchra S.;Zhu, Junhao;Wolf, Ian D.;Singhvi, Shreya;Dulberger, Charles L.;Ioerger, Thomas R.;Rubin, Eric J.;Kana, Bavesh D.
- 通讯作者:Kana, Bavesh D.
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Chidiebere Akusobi其他文献
Chidiebere Akusobi的其他文献
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{{ truncateString('Chidiebere Akusobi', 18)}}的其他基金
Investigating Cell-Wall Synthesis in Mycobacterium abscessus
研究脓肿分枝杆菌细胞壁的合成
- 批准号:
9905835 - 财政年份:2020
- 资助金额:
$ 5.1万 - 项目类别:
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