<p dir="ltr">Anti-CRISPR (Acr) proteins are produced by phages to deactivate CRISPR–Cas systems in bacteria and archaea, thus expanding the CRISPR toolbox for genome editing. In this study, we present the structure and function of AcrIF24, an Acr protein that inhibits the type I-F CRISPR–Cas system in Pseudomonas aeruginosa. AcrIF24 forms a homodimer that binds to two surveillance complexes (Csy), preventing CRISPR RNA from hybridizing with target DNA. Additionally, AcrIF24 acts as an anti-CRISPR-associated (Aca) protein, suppressing the transcription of the acrIF23-acrIF24 operon. Whether alone or in complex with Csy, AcrIF24 binds to the acrIF23-acrIF24 promoter DNA with nanomolar affinity. The 2.7 Å structure of the Csy–AcrIF24–promoter DNA complex reveals how transcriptional suppression occurs. Our findings demonstrate that AcrIF24 functions as an Acr-Aca fusion protein and enhance our understanding of the varied mechanisms employed by Acr proteins.</p><p dir="ltr">In the ongoing evolutionary struggle between bacteria and bacteriophages, the emergence of CRISPR and anti-CRISPR systems has shaped host-pathogen interactions significantly. Bacteriophages exert intense selective pressure on bacteria, driving the evolution of defense mechanisms such as restriction enzymes and the CRISPR-Cas system. Conversely, bacteriophages have evolved anti-CRISPR proteins (Acrs) to counteract CRISPR-Cas-mediated targeting. Here, we investigate the interactions and regulatory mechanisms within co-encoded Acrs, focusing on AcrVA1-5 from a prophage within Moraxella bovoculi. Our findings reveal that AcrVA1 and AcrVA2 form a stable complex capable of inhibiting Cas12a-mediated DNA cleavage, with AcrVA1 regulating the activity of AcrVA2. Additionally, AcrVA4 and AcrVA5 form a complex that modulates Cas12a activity by inhibiting DNA binding and lysin acetylation, respectively. Structural and biochemical analyses uncover a complex regulatory network governing the function of co-encoded Acrs, highlighting their role in downregulating DNA targeting in response to Cas12a presence and aiding the survival of both phage and host bacteria during infection.</p>
| Identifer | oai:union.ndltd.org:purdue.edu/oai:figshare.com:article/26360374 |
| Date | 26 July 2024 |
| Creators | Indranil Arun Mukherjee (19202494) |
| Source Sets | Purdue University |
| Detected Language | English |
| Type | Text, Thesis |
| Rights | CC BY 4.0 |
| Relation | https://figshare.com/articles/thesis/_b_Insights_into_the_Molecular_Interactions_of_Anti-CRISPR_Proteins_in_Bacteria_to_Evade_CRISPR-Cas_Immunity_b_/26360374 |
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