14967104-a274-4c1c-96fb-e8ca958c63d7
CRISPR-Cas provides limited phage immunity to a prevalent gut bacterium in gnotobiotic mice
Many prokaryotes harbor an adaptive immune system, CRISPR-Cas, that store small nucleotide fragments from previous invasions of nucleic acids. This molecular archive is then used to block invaders carrying the same or similar nucleotide sequences. However, very few of these systems have been experimentally found to be active in gut bacteria. Here, we demonstrate that the type I-C CRISPR-Cas system of the prevalent gut bacterium Eggerthella lenta can specifically target and cleave foreign DNA by using a plasmid transformation assay. We also show that the CRISPR-Cas system acquires new immunities (spacers) from the genome of a virulent E. lenta phage (PMBT5) using traditional phage assays but also in vivo using gnotobiotic (GB) mice. Interestingly, much less new spacer acquisitions were detected in vivo than in vitro. An increased number of spacer acquisition events were observed when E. lenta was exposed to a low multiplicity of infection (MOI), and three genes were detected on the phage genome as protospacer hotspots in vitro. The protospacer adjacent motif (PAM) was predicted to be 5'-TTC based on the protospacers on the phage genome perfectly matching the acquired spacers. Longitudinal analysis of bacterial and phage abundance showed sustained coexistence in the gut of the GB mice, with phage abundance being approximately one log higher than the bacteria. Our findings showed that while the type I-C CRISPR-Cas system of E. lenta is active in vitro and in vivo, a highly virulent phage in vitro was still able co-exist with its bacterial host in vivo, suggesting that this defense only provides partial immunity in the gut.
Many prokaryotes harbor an adaptive immune system, CRISPR-Cas, that store small nucleotide fragments from previous invasions of nucleic acids. This molecular archive is then used to block invaders carrying the same or similar nucleotide sequences. However, very few of these systems have been experimentally found to be active in gut bacteria. Here, we demonstrate that the type I-C CRISPR-Cas system of the prevalent gut bacterium Eggerthella lenta can specifically target and cleave foreign DNA by using a plasmid transformation assay. We also show that the CRISPR-Cas system acquires new immunities (spacers) from the genome of a virulent E. lenta phage (PMBT5) using traditional phage assays but also in vivo using gnotobiotic (GB) mice. Interestingly, much less new spacer acquisitions were detected in vivo than in vitro. An increased number of spacer acquisition events were observed when E. lenta was exposed to a low multiplicity of infection (MOI), and three genes were detected on the phage genome as protospacer hotspots in vitro. The protospacer adjacent motif (PAM) was predicted to be 5'-TTC based on the protospacers on the phage genome perfectly matching the acquired spacers. Longitudinal analysis of bacterial and phage abundance showed sustained coexistence in the gut of the GB mice, with phage abundance being approximately one log higher than the bacteria. Our findings showed that while the type I-C CRISPR-Cas system of E. lenta is active in vitro and in vivo, a highly virulent phage in vitro was still able co-exist with its bacterial host in vivo, suggesting that this defense only provides partial immunity in the gut.