Abstract
Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems in bacteria and archaea provide adaptive immunity against invading foreign nucleic acids. Previous studies suggest that certain bacteria employ their Type II CRISPR-Cas systems to target their own genes, thus evading host immunity. However, whether other CRISPR-Cas systems have similar functions during bacterial invasion of host cells remains unknown. Here we identify a novel role for Type I CRISPR-Cas systems in evading host defenses in Pseudomonas aeruginosa strain UCBPP-PA14. The Type I CRISPR-Cas system of PA14 targets the mRNA of the bacterial quorum-sensing regulator LasR to dampen the recognition by toll-like receptor 4, thus diminishing the pro-inflammatory responses of the host in cell and mouse models. Mechanistically, this nuclease-mediated RNA degradation requires a “5′-GGN-3′” recognition motif in the target mRNA, and HD and DExD/H domains in Cas3 of the Type I CRISPR-Cas system. As LasR and Type I CRISPR-Cas systems are ubiquitously present in bacteria, our findings elucidate an important common mechanism underlying bacterial virulence.
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Acknowledgements
We thank Dr George A O'Toole from Dartmouth Medical School for providing PA14 CRISPR-Cas components deletion and complementary mutants and vector pMQ70. We thank Dr Dominique Ferrandon of University of Strasbourg, France, for providing PA14 lasR deleting mutant and are indebted to Mary Clare Rollins and Dr Blake Wiedenheft of Montana State University for providing Csy1-4 expressing E. coli strain, indebted to Dr Jin-Town Wang from National Taiwan University for providing K. pneumonia NTUH-2044. We thank S Abrahamson (University of North Dakota Imaging Core Facility) for help with confocal imaging. This work was supported by National Institute of Health (AI109317-01A1 and AI101973-01 as well as NIH INBRE P20GM103442 and COBRE grants P20GM113123).
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Supplementary information, Figure S1
RNA was isolated when PA14 WT and total CRISPR-Cas cluster deletion mutant, single element deletion mutants and complementary strains grew at an OD600 nm of 1.0 in LB medium, and transcripts of indicated cas and crispr genes were quantified by qPCR. (PDF 405 kb)
Supplementary information, Figure S2
RNA was isolated when PA14 WT, ΔTCR, ΔlasR and their complementary strains grew at an OD600 nm of 2.0 in M9 medium. (PDF 171 kb)
Supplementary information, Figure S3
(A) Coomassie-blue stained SDS-polyacrylamide gel of the affinity purified Csy complexes, WT Cas3 and its mutants. (PDF 401 kb)
Supplementary information, Figure S4
Homology comparison of Cas3 amino acid sequence in PA14, E. coli UT189, PA2192, P. mendocina and P. stuzeri. (PDF 222 kb)
Supplementary information, Figure S5
Mice infected with 5 × 106 CFU/mouse with PA14 WT, ΔTCR and its complementary strain, single ΔlasR mutant and ΔTCRΔlasR double deletion mutant, respectively. (PDF 354 kb)
Supplementary information, Figure S6
Depletion of LasR alters TLR4-related host defense in mice and macrophages. (PDF 407 kb)
Supplementary information, Figure S7
Data of human AMs collaborate murine macrophage results. (PDF 399 kb)
Supplementary information, Figure S8
(A) PA14 WT, ΔTCR and its complementary strains were cultured in LB medium for 24 h shaking at 220 rpm, and then static grew in tubes for 72 h. (PDF 247 kb)
Supplementary information, Figure S9
MH-S cells were infected with K. pneumoniae NTUH-2044 and E. coli K12 respectively at an MOI of 20:1. (PDF 221 kb)
Supplementary information, Table S1
Relative normalized expression of host innate and adaptive immune response factors. (PDF 417 kb)
Supplementary information, Table S2
CRISPR-Cas and QS systems in the pathogen species1,# (PDF 598 kb)
Supplementary information, Table S3
Strains, plasmids and primers used in this study (PDF 584 kb)
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Li, R., Fang, L., Tan, S. et al. Type I CRISPR-Cas targets endogenous genes and regulates virulence to evade mammalian host immunity. Cell Res 26, 1273–1287 (2016). https://doi.org/10.1038/cr.2016.135
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DOI: https://doi.org/10.1038/cr.2016.135
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