Mechanisms and functions of CRISPR-Casautoregulation in bacterial immunity and pathogenesis

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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.