The evolutionary ecology of CRISPR-Cas adaptive immunity

Microbial communities play a vital role in shaping their local environment and provide many important ecosystem services. The structure and function of microbial communities is dependent on interactions with prokaryote-specific viruses and other mobile genetic elements (MGEs), but we know relatively little about these interactions in nature. The prokaryotic adaptive immune system CRISPR-Cas provides resistance to phage and other MGEs by inserting phage-derived sequences into CRISPR loci on the host genome to allow sequence specific immunological memory against re-infection. Compared to the specific mechanism of CRISPR-Cas, phage resistance via surface modification provides general defense against a range of phage by physically modifying the cell surface to prevent phage infection. CRISPR-Cas and surface modification have been shown to be the most common mechanisms for rapid evolution of de novo phage resistance and therefore likely play important roles in shaping microbial communities. It has been suggested that we may be able to manipulate CRISPR-Cas evolution to our advantage, but very little research has been done investigating the evolutionary outcome of such manipulation. In this thesis I investigate the importance of different ecological drivers on when CRISPR-Cas is favoured over phage resistance via surface modification. I find that increasing CRISPR allele diversity within a host population increases phage immunity at the population level. However, increasing genetic diversity within the phage population increases selection for generalist phage defence via surface modification over specific CRISPR-Cas resistance. I also attempt to investigate the importance of cell-cell communication in the evolution of bacterial resistance; however these experiments were hampered by secondary effects of inhibiting cell-cell communication. These results are discussed in context with recent findings with the aim of expanding our knowledge of CRISPR-Cas evolution and ecology and suggesting where further research would be beneficial

Diversity in CRISPR-based immunity protects susceptible genotypes by restricting phage spread and evolution.

This is the final version. Available from the publisher via the DOI in this record.Data deposited at dryad: https://doi.org/10.5061/dryad.66t1g1k00.Diversity in host resistance often associates with reduced pathogen spread. This may result from ecological and evolutionary processes, likely with feedback between them. Theory and experiments on bacteria-phage interactions have shown that genetic diversity of the bacterial adaptive immune system can limit phage evolution to overcome resistance. Using the CRISPR-Cas bacterial immune system and lytic phage, we engineered a host-pathogen system where each bacterial host genotype could be infected by only one phage genotype. With this model system, we explored how CRISPR diversity impacts the spread of phage when they can overcome a resistance allele, how immune diversity affects the evolution of the phage to increase its host range, and if there was feedback between these processes. We show that increasing CRISPR diversity benefits susceptible bacteria via a dilution effect, which limits the spread of the phage. We suggest that this ecological effect impacts the evolution of novel phage genotypes, which then feeds back into phage population dynamics

The effect of Quorum sensing inhibitors on the evolution of CRISPR-based phage immunity in Pseudomonas aeruginosa

This is the final version. Available from Springer Nature via the DOI in this record. Quorum sensing controls the expression of a wide range of important traits in the opportunistic pathogen Pseudomonas aeruginosa, including the expression of virulence genes and its CRISPR-cas immune system, which protects from bacteriophage (phage) infection. This finding has led to the speculation that synthetic quorum sensing inhibitors could be used to limit the evolution of CRISPR immunity during phage therapy. Here we use experimental evolution to explore if and how a quorum sensing inhibitor influences the population and evolutionary dynamics of P. aeruginosa upon phage DMS3vir infection. We find that chemical inhibition of quorum sensing decreases phage adsorption rates due to downregulation of the Type IV pilus, which causes delayed lysis of bacterial cultures and favours the evolution of CRISPR immunity. Our data therefore suggest that inhibiting quorum sensing may reduce rather than improve the therapeutic efficacy of pilus-specific phage, and this is likely a general feature when phage receptors are positively regulated by quorum sensing.Lundbeck FoundationLundbeck FoundationRoyal Societ

The diversity-generating benefits of a prokaryotic adaptive immune system

Published onlineJOURNAL ARTICLEProkaryotic CRISPR-Cas adaptive immune systems insert spacers derived from viruses and other parasitic DNA elements into CRISPR loci to provide sequence-specific immunity. This frequently results in high within-population spacer diversity, but it is unclear if and why this is important. Here we show that, as a result of this spacer diversity, viruses can no longer evolve to overcome CRISPR-Cas by point mutation, which results in rapid virus extinction. This effect arises from synergy between spacer diversity and the high specificity of infection, which greatly increases overall population resistance. We propose that the resulting short-lived nature of CRISPR-dependent bacteria-virus coevolution has provided strong selection for the evolution of sophisticated virus-encoded anti-CRISPR mechanisms.S.v.H. has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement number 660039. E.R.W. received funding from the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme (FP7/2007-2013) under Research Executive Agency grant agreement number 327606. E.R.W., A.B. and M.B. also acknowledge the Natural Environment Research Council, the Biotechnology and Biological Sciences Research Council, the Royal Society, the Leverhulme Trust, the Wellcome Trust and the AXA research fund for funding. J.M.B.-D. was supported by the University of California San Francisco Program for Breakthrough in Biomedical Research, the Sandler Foundation, and a National Institutes of Health Director’s Early Independence Award (DP5-OD021344). H.C. was funded by the Erasmus+ programme (European Union), the Explora’Sup programme (Région Rhône-Alpes) and the Centre Régional des Œuvres Universitaires et Scolaires (CROUS; French State)