Periodic Reporting for period 1 - anti-CRISPR (Selection and evolution of phage-encoded anti-CRISPR genes)
Reporting period: 2019-05-01 to 2021-04-30
The bacterial adaptive immune systems CRISPR-Cas are of pivotal importance in nature where they protect bacteria against their viral predators (phages). In response, some phages evolved a sophisticated strategy by encoding anti-CRISPR proteins (Acrs). While our molecular understanding of Acr mechanisms has raced ahead, a fundamental question remains unexplored: what is the impact of acr genes on the ecological and evolutionary dynamics of phages and their hosts? This research project explored the costs and benefits associated with Acrs, their consequences on the composition and evolution of phage populations as well as on the evolution of their host. The main results of this project are important for society as they can be implemented in biomedical research, for the development of phage therapy to combat antibiotic-resistant bacteria, as well as in biotechnologies for a better control of Cas9-based genome editing.
The research objectives of this project were the following:
1) Determine when Acrs are beneficial. Despite their potential benefits for phage survival, acr genes are absent from many phage genomes and they have been more frequently found in temperate phages (i.e. phages that have the ability to lying dormant into host genome), which suggests that their benefits may be dependent on phage genetic background.
2) Examine how Acrs impact the dynamics of phage communities. Our previous work showed that Acr-phages need to cooperate to bypass CRISPR-based resistance: a first Acr-phage enters the cell and shuts-off the CRISPR immune system, although this process is not rapid enough to go through the infection process successfully. This “sacrifice” allows a second Acr-phage to take over the immunosuppressed cell, produce progeny and kill the host. However, in nature, phage populations are often not clonal and composed of mixed genotypes, with some that encode Acrs and some that do not. Since Acr-phages generate hosts with weakened immunity, we speculate they may enable other phages to infect bacteria with CRISPR-Cas defences.
3) Explore how Acrs impact the evolution of bacteria. Because they block some of the Cas proteins that mediate immunity acquisition, Acrs may affect the emergence of CRISPR-based resistance and this may have consequences on the evolutionary dynamics of bacteria.
These research questions were studied with a multidisciplinary approach that combined phage molecular biology, experimental evolution and mathematical modelling in the group of Prof. Westra (University of Exeter, UK).
The research objectives of this project were the following:
1) Determine when Acrs are beneficial. Despite their potential benefits for phage survival, acr genes are absent from many phage genomes and they have been more frequently found in temperate phages (i.e. phages that have the ability to lying dormant into host genome), which suggests that their benefits may be dependent on phage genetic background.
2) Examine how Acrs impact the dynamics of phage communities. Our previous work showed that Acr-phages need to cooperate to bypass CRISPR-based resistance: a first Acr-phage enters the cell and shuts-off the CRISPR immune system, although this process is not rapid enough to go through the infection process successfully. This “sacrifice” allows a second Acr-phage to take over the immunosuppressed cell, produce progeny and kill the host. However, in nature, phage populations are often not clonal and composed of mixed genotypes, with some that encode Acrs and some that do not. Since Acr-phages generate hosts with weakened immunity, we speculate they may enable other phages to infect bacteria with CRISPR-Cas defences.
3) Explore how Acrs impact the evolution of bacteria. Because they block some of the Cas proteins that mediate immunity acquisition, Acrs may affect the emergence of CRISPR-based resistance and this may have consequences on the evolutionary dynamics of bacteria.
These research questions were studied with a multidisciplinary approach that combined phage molecular biology, experimental evolution and mathematical modelling in the group of Prof. Westra (University of Exeter, UK).
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.
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.
This research project characterized the impact of Acr-CRISPR interactions on the ecology and evolution of phages and bacteria and helped understanding how natural selection acts on CRISPR and acr genes. Crucially, this work led to two fundamental discoveries: (1) Bacteria may lose their CRISPR-Cas systems because of the maladaptive effects they cause during infection with temperate phages, unless phages carry an acr gene and (2) the cooperative behavior of Acr(+) phages can be exploited by Acr(-) phages.
Overall, this project has allowed to make progress beyond the state of the art and have several applications. The knowledge generated during this project will help to improve the design of therapeutic phage cocktails to be used for the treatment of bacterial infections as an alternative to the use of antibiotics. Notably our results suggest that including Acr(+) phages in such cocktails may have limited advantages, and future research is needed to determine if and which combinations with other phages enhance their benefits. CRISPR-Cas systems have important applications in gene editing, ranging from the correction of genetic abnormalities (e.g. Cystic Fibrosis, Duchenne muscular dystrophy) to the removal of antibiotic resistance genes from pathogenic bacteria. However, for these applications, CRISPR-Cas activity needs to be tightly controlled and using Acrs is a way to achieve this. This work provided valuable insight that will help their development of Acrs in biotechnologies.
Overall, this project has allowed to make progress beyond the state of the art and have several applications. The knowledge generated during this project will help to improve the design of therapeutic phage cocktails to be used for the treatment of bacterial infections as an alternative to the use of antibiotics. Notably our results suggest that including Acr(+) phages in such cocktails may have limited advantages, and future research is needed to determine if and which combinations with other phages enhance their benefits. CRISPR-Cas systems have important applications in gene editing, ranging from the correction of genetic abnormalities (e.g. Cystic Fibrosis, Duchenne muscular dystrophy) to the removal of antibiotic resistance genes from pathogenic bacteria. However, for these applications, CRISPR-Cas activity needs to be tightly controlled and using Acrs is a way to achieve this. This work provided valuable insight that will help their development of Acrs in biotechnologies.