Bacterial alginate regulators and phage homologs repress CRISPR–Cas immunity

Borges, Adair L.; Castro, Bardo; Govindarajan, Sutharsan; Solvik, Tina; Escalante, Veronica; Bondy‐Denomy, Joseph

doi:10.1038/s41564-020-0691-3

Cited by 44 publications

(40 citation statements)

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“…Thus, the regulation of defense systems including CRISPR-Cas on the one hand and new DNA acquisition mechanisms including natural competence systems on the other hand is extremely important for bacterial adaptation to changing environments with fitness cost considerations. Recent studies provided new examples of such regulatory mechanisms involving sRNAs under the control of TCS in major pathogens [ 64 , 157 , 158 , 159 , 160 , 161 ].…”

Section: Srnas Regulating Host-pathogen Interactionsmentioning

confidence: 99%

Interplay between Regulatory RNAs and Signal Transduction Systems during Bacterial Infection

Piattelli

Peltier

Soutourina

2020

Genes

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The ability of pathogenic bacteria to stably infect the host depends on their capacity to respond and adapt to the host environment and on the efficiency of their defensive mechanisms. Bacterial envelope provides a physical barrier protecting against environmental threats. It also constitutes an important sensory interface where numerous sensing systems are located. Signal transduction systems include Two-Component Systems (TCSs) and alternative sigma factors. These systems are able to sense and respond to the ever-changing environment inside the host, altering the bacterial transcriptome to mitigate the impact of the stress. The regulatory networks associated with signal transduction systems comprise small regulatory RNAs (sRNAs) that can be directly involved in the expression of virulence factors. The aim of this review is to describe the importance of TCS- and alternative sigma factor-associated sRNAs in human pathogens during infection. The currently available genome-wide approaches for studies of TCS-regulated sRNAs will be discussed. The differences in the signal transduction mediated by TCSs between bacteria and higher eukaryotes and the specificity of regulatory RNAs for their targets make them appealing targets for discovery of new strategies to fight against multi-resistant bacteria.

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Section: Srnas Regulating Host-pathogen Interactionsmentioning

confidence: 99%

Interplay between Regulatory RNAs and Signal Transduction Systems during Bacterial Infection

Piattelli

Peltier

Soutourina

2020

Genes

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“…Of note, cells had to be grown to high cell density, which is important for Cas protein expression in P. aeruginosa (Fig. 1b) (Høyland-Kroghsbo et al, 2017;Borges et al, 2020). Analysis of fluorescent signal from single cells indicated that Csy1 and Csy4 protein levels are higher than Cas3 (Fig.…”

Section: Establishing Reporter Systems For Live Cell Visualization Ofmentioning

confidence: 99%

“…Endogenous Csy1-sfCherry and Cas3-sfCherry reporters were previously described (Borges et al, 2020). Csy4-sfCherry was constructed in a similar way.…”

Section: Construction Of Plasmids and Strainsmentioning

confidence: 99%

“…The type I-F CRISPR-Cas system of P. aeruginosa has emerged as a powerful model for understanding various aspects of CRISPR-Cas biology, including a mechanistic understanding of type I systems (Wiedenheft et al, 2011;Guo et al, 2017;Rollins et al, 2019), the discovery (Bondy-Denomy et al, 2013) and in vivo characterization (Borges et al, 2018;Landsberger et al, 2018) of phage-encoded anti-CRISPR proteins (Acrs), and identification of regulatory pathways governing CRISPR-Cas expression (Høyland-Kroghsbo et al, 2017;Lin et al, 2019a;Lin et al, 2019b;Ahator et al, 2020;Borges et al, 2020). The I-F system of P. aeruginosa PA14 consists of two CRISPR loci and six Cas proteins, namely Csy1-4, which form the Csy complex (surveillance complex of the I-F system), Cas3 (a fusion of Cas2-3), a trans-acting nuclease/helicase protein, and Cas1, which drives spacer acquisition (Wiedenheft et al, 2011;Rollins et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

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Distinct subcellular localization of a Type I CRISPR complex and the Cas3 nuclease in bacteria

Govindarajan

Borges

Bondy‐Denomy

2020

Preprint

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CRISPR-Cas systems are prokaryotic adaptive immune systems that target mobile genetic elements including phages. While CRISPR-Cas systems have been well characterized biochemically and structurally, in vivo spatiotemporal regulation and cell biology remains largely unaddressed. Here, we studied the localization of native Type I-F CRISPR-Cas system of the pathogen Pseudomonas aeruginosa via fluorescent fusions to Csy1 (Cas8), Csy4 (Cas6) and Cas3 which are involved in PAM recognition, crRNA processing, and nuclease activity, respectively. Chromosomally tagged proteins were fully functional for phage targeting and inhibited by anti-CRISPR proteins. When targeted to an integrated prophage, the Csy complex and Cas3 generated a single cellular focus, however when lacking a protospacer target, the Csy complex is broadly nucleoid bound, while Cas3 is diffuse in the cytoplasm. Nucleoid association for the Csy proteins is crRNA-dependent, but crRNA-sequence independent, suggesting formation of the Csy complex engenders genome surveillance. Expression of anti-CRISPR AcrIF2, known to prevent the Csy complex from binding to DNA via protospacer adjacent motif (PAM) competition, abolished nucleoid surveillance, while AcrIF1, which blocks crRNA:DNA hybridization downstream of the PAM, did not. These results suggest the genome surveillance is a result of direct PAM interactions, independent of other host factors. The Cas9 nuclease, when heterologously expressed in P. aeruginosa, also appears nucleoid localized when its gRNA is provided, which is abolished by PAM mimic, AcrIIA4. Finally, by employing fluorescently labeled phages, we observe that phage infection does not significantly modulate the distribution of the Csy complex and Cas3, despite doing so as a prophage. In summary, our findings demonstrate that a Type I CRISPR-Cas complex in its natural environment is localized in the nucleoid, separated from nuclease-helicase, Cas3.

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“…How are CRISPR systems regulated within their bacterial hosts to prevent autoimmunity and enhance targeting of foreign agents? Several studies have demonstrated transcriptional and post-transcriptional CRISPR-Cas regulation in response to extracellular and intracellular cues 20 , including phage infection [21][22][23][24][25] , quorum sensing 26,27 , membrane stress [28][29][30][31] , metabolic status 21,32,33 and surface association 34 . In many cases where the regulatory mechanisms are known, host-encoded transcription factors interact with CRISPR-Cas promoters 32,33,35,36 , although several studies have shown that archaeal type I-A systems can be intrinsically controlled by dedicated Cas-encoded transcription factors 25,[37][38][39][40] .…”

Section: Introductionmentioning

confidence: 99%

A natural single-guide RNA repurposes Cas9 to autoregulate CRISPR-Cas expression

Workman

Pammi

Nguyen

et al. 2020

Preprint

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CRISPR-Cas systems provide their prokaryotic hosts with acquired immunity against viruses and other foreign genetic elements, but how these systems are regulated to prevent auto-immunity is poorly understood. In type II CRISPR-Cas systems, a transactivating CRISPR RNA (tracrRNA) scaffold functions together with a CRISPR RNA (crRNA) guide to program Cas9 for the recognition and cleavage of foreign DNA targets. Here, we show that a long-form tracrRNA performs an unexpected second function by folding into a natural single guide that directs Cas9 to transcriptionally repress its own promoter. Further, we demonstrate that Pcas9 serves as a critical regulatory node; de-repression causes a dramatic induction of Cas genes, crRNAs and tracrRNAs resulting in a 3,000-fold increase in immunization rates against unrecognized viruses. Heightened immunity comes at the cost of increased auto-immune toxicity, demonstrating the critical importance of the controller. Using a bioinformatic analysis, we provide evidence that tracrRNA-mediated autoregulation is widespread in type II CRISPR-Cas systems. Collectively, we unveil a new paradigm for the intrinsic regulation of CRISPR-Cas systems by natural single guides, which may facilitate the frequent horizontal transfer of these systems into new hosts that have not yet evolved their own regulatory strategies.

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Bacterial alginate regulators and phage homologs repress CRISPR–Cas immunity

Cited by 44 publications

References 50 publications

Interplay between Regulatory RNAs and Signal Transduction Systems during Bacterial Infection

Interplay between Regulatory RNAs and Signal Transduction Systems during Bacterial Infection

Distinct subcellular localization of a Type I CRISPR complex and the Cas3 nuclease in bacteria

A natural single-guide RNA repurposes Cas9 to autoregulate CRISPR-Cas expression

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