CRISPR-Cas provides limited phage immunity to a prevalent gut bacterium in gnotobiotic mice

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Many bacteria and archaea harbor the adaptive CRISPR-Cas system, which stores small nucleotide fragments from previous invasions of nucleic acids via viruses or plasmids. This molecular archive blocks further invaders carrying identical or similar nucleotide sequences. However, few of these systems have been confirmed experimentally to be active in gut bacteria. Here, we demonstrate experimentally that the type I-C CRISPR-Cas system of the prevalent gut bacterium Eggerthella lenta can specifically target and cleave foreign DNA in vitro 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 using traditional phage assays in vitro but also in vivo using gnotobiotic (GB) mice. Both high phage titer and an increased number of spacer acquisition events were observed when E. lenta was exposed to a low multiplicity of infection in vitro, and three phage genes were found to contain protospacer hotspots. Fewer new spacer acquisitions were detected in vivo than in vitro. Longitudinal analysis of phage-bacteria interactions showed sustained coexistence in the gut of GB mice, with phage abundance being approximately one log higher than the bacteria. Our findings show that while the type I-C CRISPR-Cas system is active in vitro and in vivo, a highly virulent phage in vitro was still able to co-exist with its bacterial host in vivo. Taken altogether, our results suggest that the CRISPR-Cas defense system of E. lenta provides only partial immunity in the gut.

TidsskriftISME Journal
Sider (fra-til)432–442
StatusUdgivet - 2023

Bibliografisk note

Funding Information:
Funding was provided by the Danish Council for Independent Research with grant ID: DFF-6111-00316 (PhageGut). This work is supported by the Joint Programming Initiative “Healthy Diet for a Healthy Life”, specifically here, the Danish Agency for Science and Higher Education. S.M. acknowledges funding from the Canadian Institutes of Health Research (Team grant on Intestinal Microbiomics, Institute of Nutrition, Metabolism, and Diabetes). S.M. holds a Tier 1 Canada Research Chair in Bacteriophages.

Publisher Copyright:
© 2023, The Author(s), under exclusive licence to International Society for Microbial Ecology.

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