The fitness costs associated with acr genes were experimentally measured through direct competition of phage DMS3vir with or without an acr gene (referred to as Acr(+) and Acr(-) phages, respectively) on their host Pseudomonas aeruginosa PA14, in which the functional CRISPR-Cas system was knock-out. Our data revealed that acr genes were not associated with measurable fitness cost. These results therefore do not explain why not all phages carry acr genes if they are cost-free (other than occupying space on phage genome).
An alternative explanation is that the benefits they provide might not be as large as previously assumed. Analyses of 10-days infection experiment, where wildtype PA14 bacteria were infected with either Acr(-) or Acr(+) phages, revealed that Acrs provide phages with transient benefits. Even though they persisted longer than Acr(-)phages, they were eventually driven towards extinction due to the emergence of a bacterial subpopulation that acquired phage resistance through surface modification (sm). Based on this data, we carried out additional experiments which suggested that the benefits associated with acr genes may be more important when phages are temperate. Indeed, CRISPR-Cas can target temperate phages when they integrate into the bacterial genome causing severe immunopathological effects (autoimmunity) which drive the evolutionary loss of the CRISPR-Cas locus. The presence of acr genes protects both the phage and the host and is therefore highly beneficial in this context.
This work led to a follow-up question: can Acr(+) phages also benefit other mobile genetic elements against CRISPR-Cas activity? Previous work in our lab showed that Acr(+) phages can generate immunosuppressed bacteria, which could potentially be exploited by “cheater” Acr(-) phages. Based on this hypothesis, we demonstrated that Acr(-) phages can indeed benefit from the presence of Acr(+) phages, although these benefits were observed with Acrs that strongly inhibit CRISPR-Cas, but not with weaker ones.
Investigating the mechanism underlying this exploitation, we showed that Acr(+) phages benefit Acr(-) phages through two mechanisms: First, they directly help the replication of Acr(-) phages by generating immunosuppressed sub-populations of hosts. Second, Acr(+) phages indirectly protect Acr(-) phages by limiting the capacity of the hosts to evolve CRISPR-resistance. Indeed, using experimental evolution coupled with deep amplicon sequencing, we established that initially sensitive bacteria exposed to Acr(+) phages evolved CRISPR-based resistance at very low frequencies and acquired resistance through the modification of the phage-receptor instead.
The dissemination of the results was achieved through the publication of two articles in 2020, one in Nature and the other in Cell Host & Microbes. A third article is currently in preparation and will be submitted mid-2021 as well as a review article.
