Mechanisms and functions of CRISPR-Casautoregulation in bacterial immunity and pathogenesis
CRISPR-Casautoregulation在细菌免疫和发病机制中的机制和功能
基本信息
- 批准号:10624292
- 负责人:
- 金额:$ 3.17万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2021
- 资助国家:美国
- 起止时间:2021-05-15 至 2023-11-10
- 项目状态:已结题
- 来源:
- 关键词:Adaptive Immune SystemAddressAffectAntibiotic ResistanceAutoimmunityBacteriaBacteriophagesBindingBiochemicalBiologicalBiological AssayBiologyCampylobacter jejuniCellsClustered Regularly Interspaced Short Palindromic RepeatsCuesCytolysisDNADataDevelopmentEnvironmentExclusionGene ExpressionGene SilencingGenesGeneticGenetic TranscriptionGenome engineeringGoalsGrowthGuide RNAHomeostasisHorizontal Gene TransferHumanImmune systemImmunityInfectionKnowledgeLaboratoriesMediatingMemoryMetabolicMicrobial BiofilmsMobile Genetic ElementsMolecularMolecular GeneticsNeisseria meningitidisOperonPathogenesisPathway interactionsPhasePhysiologicalPlasmidsProteinsRNARegulationRepressionReproducibilityResearchRouteStimulusStreptococcus Group BStreptococcus pyogenesSystemTestingUntranslated RNAVariantVirulenceVirulence FactorsWorkderepressionenvironmental changeexperimental studygene repressionhuman pathogenimprovedin vivoinsightmortalitynew therapeutic targetnovelnucleasepathogenic bacteriapathogenic microbepreventpromoterresistance genescaffoldtoolvirulence gene
项目摘要
Project Summary
The goal of my research is to investigate how bacterial CRISPR-Cas immune systems are regulated and how
this regulation contributes to bacterial immunity and virulence. CRISPR-Cas systems protect bacteria from
bacteriophages and other mobile genetic elements and thereby prevent cell lysis but also limit the potential for
horizontal gene transfer, a major route for the dissemination of antibiotic-resistance genes. Despite all that is
known about CRISPR-Cas biology from a mechanistic standpoint, much remains to be understood about how
these systems function in their native bacterial hosts. In particular, we lack an understanding of the ways by
which bacteria regulate CRISPR-Cas expression to maximize immunity while mitigating autoimmunity and the
metabolic burden of constitutive system expression. Furthermore, Cas9 is itself a virulence factor in many
human pathogens, including S. pyogenes, S. agalactiae, F. novicida, N. meningitidis, and C. jejuni although it
is unclear how Cas9 contributes to pathogenesis. For these reasons, it is critical to understand whether and
how CRISPR-Cas expression is regulated to prepare bacteria for an impending phage infection or for growth in
a human host.
To address this gap in knowledge, we performed a screen to identify regulators of CRISPR-Cas
immunity. Interestingly, we discovered that trL, a noncoding RNA within the S. pyogenes CRISPR-Cas locus, is
capable of folding into a natural single-guide RNA that directs Cas9 to transcriptionally silence the Cas operon
promoter (Workman et al., 2020). While a trL deletion enhances Cas gene expression by ~50-fold and
stimulates CRISPR-Cas immunity by 3000-fold, it remains unknown how trL de-repression occurs under
physiological conditions. In this proposal, I will identify the conditions and genetic pathways that mediate trL de-
repression and assess the impact of this regulation on bacterial immunity and virulence. We have obtained
preliminary data demonstrating that CRISPR RNAs (crRNAs) and growth-phase specific cues modulate Cas9
expression; however, the mechanisms and consequences of Cas9 induction remain to be tested. In Aim 1, we
test the hypothesis that crRNAs and trL form an integrated genetic circuit that controls CRISPR-Cas
expression, providing a novel mechanism through which spacers, the molecular “memories” of infection,
differentially affect immunity. In Aim 2, we investigate the physiological cues and genetic networks that cause
Cas9 to accumulate in late stationary phase, and we probe whether this regulation affects immunity and
virulence in S. pyogenes. The proposed studies will help us understand how pathogenic bacteria regulate
CRISPR-Cas expression in order to survive in hostile environments. Finally, our work will inform the
development of regulatable Cas9 tools and new therapeutic targets and strategies for human pathogens.
项目摘要
我研究的目标是研究细菌CRISPR-Cas免疫系统是如何被调节的,以及
这种调节有助于细菌免疫和毒性。CRISPR-Cas系统保护细菌免受
噬菌体和其他移动的遗传元件,从而防止细胞溶解,但也限制了
水平基因转移,这是抗性基因传播的主要途径。尽管这一切
从机械学的角度了解CRISPR-Cas生物学,但关于CRISPR-Cas生物学如何发挥作用仍有许多问题有待了解。
这些系统在它们的天然细菌宿主中起作用。特别是,我们缺乏对
哪些细菌调节CRISPR-Cas表达以最大化免疫力,同时减轻自身免疫,
组成型系统表达的代谢负担。此外,Cas9本身在许多疾病中是毒力因子。
人类病原体,包括S. pyogenes、S.无乳假单胞菌F. novicida、新单胞菌N. meningitidis和C.空肠虽然它
目前尚不清楚Cas9如何参与发病机制。由于这些原因,了解是否和
如何调控CRISPR-Cas表达以准备细菌用于即将发生的噬菌体感染或用于在
一个人类宿主
为了解决这一知识缺口,我们进行了一项筛选,以确定CRISPR-Cas的调控因子。
免疫力有趣的是,我们发现trL,一个在S。化脓性链球菌CRISPR-Cas基因座,
能够折叠成指导Cas9转录沉默Cas操纵子的天然单向导RNA
启动子(Workman等,2020年)。而trL缺失使Cas基因表达增强约50倍,
尽管CRISPR-Cas刺激3000倍的免疫力,但trL去阻遏在CRISPR-Cas刺激下如何发生仍然是未知的。
生理条件。在这个提案中,我将确定条件和遗传途径,介导的trL de-
抑制,并评估这种调节对细菌免疫力和毒力的影响。我们所获得
初步数据表明,CRISPR RNA(crRNA)和生长期特异性信号调节Cas9
然而,Cas9诱导的机制和后果仍有待测试。目标1:
测试crRNA和trL形成控制CRISPR-Cas的集成遗传电路的假设
表达,提供了一种新的机制,通过这种机制,间隔区,感染的分子“记忆”,
对免疫力有不同的影响。在目标2中,我们研究了导致
Cas9在稳定期后期积累,我们探索这种调节是否影响免疫力,
毒力S.化脓拟议的研究将帮助我们了解致病菌如何调节
CRISPR-Cas表达,以便在恶劣的环境中生存。最后,我们的工作将告知
开发可调控的Cas9工具以及针对人类病原体的新治疗靶标和策略。
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)
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Rachael Workman其他文献
Rachael Workman的其他文献
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{{ truncateString('Rachael Workman', 18)}}的其他基金
Mechanisms and functions of CRISPR-Casautoregulation in bacterial immunity and pathogenesis
CRISPR-Casautoregulation在细菌免疫和发病机制中的机制和功能
- 批准号:
10402801 - 财政年份:2021
- 资助金额:
$ 3.17万 - 项目类别:
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