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A CRISPR/Cas System Mediates Bacterial Innate Immune Evasion and Virulence


A CRISPR/Cas System Mediates Bacterial Innate Immune Evasion and Virulence

Timothy R Sampson et al. Nature.

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CRISPR/Cas (clustered regularly interspaced palindromic repeats/CRISPR-associated) systems are a bacterial defence against invading foreign nucleic acids derived from bacteriophages or exogenous plasmids. These systems use an array of small CRISPR RNAs (crRNAs) consisting of repetitive sequences flanking unique spacers to recognize their targets, and conserved Cas proteins to mediate target degradation. Recent studies have suggested that these systems may have broader functions in bacterial physiology, and it is unknown if they regulate expression of endogenous genes. Here we demonstrate that the Cas protein Cas9 of Francisella novicida uses a unique, small, CRISPR/Cas-associated RNA (scaRNA) to repress an endogenous transcript encoding a bacterial lipoprotein. As bacterial lipoproteins trigger a proinflammatory innate immune response aimed at combating pathogens, CRISPR/Cas-mediated repression of bacterial lipoprotein expression is critical for F. novicida to dampen this host response and promote virulence. Because Cas9 proteins are highly enriched in pathogenic and commensal bacteria, our work indicates that CRISPR/Cas-mediated gene regulation may broadly contribute to the regulation of endogenous bacterial genes, particularly during the interaction of such bacteria with eukaryotic hosts.

Conflict of interest statement

The authors declare no competing financial interests.


Figure 1
Figure 1. Cas9, tracrRNA, and scaRNA are necessary for FTN_1103 repression
(A) Schematic of the F. novicida Type II CRISPR-CAS locus, containing cas9, cas1, cas2, and cas4, as well as the crRNA array (repeats indicated by vertical red lines), tracrRNA (blue), scaRNA (orange), and predicted promoters (black arrows). Relative expression of FTN_1103 in (B) wild-type (WT), Δcas9, Δcas1, Δcas2, and Δcas4 strains and (C) WT, Δcas9, ΔscaRNA, ΔcrRNA, and ΔtracrRNA strains (n = 4, bars represent s.d.).
Figure 2
Figure 2. Cas9, tracrRNA, and scaRNA associate and mediate FTN_1103 degradation
(A) FTN_1103 stability in the indicated strains (n = 3). (B) Schematic of Cas9 indicating five endonuclease domains (RuvC-I - RuvC-IV, HNH) and the ARM. (C) Relative expression of FTN_1103 in the indicated strains (n = 4). (D) Schematic of predicted hybridization between tracrRNA, scaRNA, and FTN_1103. Bars highlight mutated bases (green), red represents the FTN_1103 start codon. (E–G) Immunoprecipitation from WT, Cas9-FLAG, or Cas9:R59A-FLAG, and qRT-PCR for (E) scaRNA, (F) tracrRNA or (G) FTN_1103 (n = 4). (H) Relative expression of FTN_1103 in WT, ΔscaRNA, scaRNA:rc4-8 (reverse complement of bases 4–8), scaRNA:rc48–54, ΔtracrRNA, and tracrRNA:rc13-17 strains (n = 4, bars represent s.d.).
Figure 3
Figure 3. Cas9, tracrRNA, and scaRNA facilitate evasion of TLR2 signaling by temporal repression of FTN_1103
IL-6 secretion from wild-type (WT) and TLR2−/ − bone marrow-derived macrophages (BMDM) (A) unstimulated (Un) or stimulated with membrane proteins (relative MOI of 20:1 for 5 hours) from wild-type (WT), the indicated single mutants, or double deletion strains also lacking FTN_1103 (n = 3), or (B) infected with the same strains at an MOI of 20:1 for 5 hours (n =6). Relative expression of (C) FTN_1103, (D) cas9, (E) scaRNA, and (F) tracrRNA during infection of BMDM with the indicated strains (n = 3, bars represent s.d.).
Figure 4
Figure 4. Cas9, tracrRNA, and scaRNA are necessary for virulence
(A) Competitive indices of wild-type and the indicated mutant or double mutant strains from murine spleens, 48 hours post-infection. Bars represent the geometric mean. (B) Mice were infected with 107 cfu of either wild-type (black circle), Δcas9 (blue square), ΔscaRNA (yellow triangle), ΔtracrRNA (green diamond), or the corresponding cis-complemented strains (open symbols), and survival monitored over time. (C) Mice were vaccinated with 104 cfu of either Δcas9, ΔscaRNA, or ΔtracrRNA strains, or PBS. Twenty-eight days later, mice were challenged with 107 cfu wild-type.

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