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Complex parasite communities as drivers of bacterial immunity

Periodic Reporting for period 1 - PHAGECOM (Complex parasite communities as drivers of bacterial immunity)

Reporting period: 2016-01-01 to 2017-12-31

CRISPR-Cas is one of several distinct bacterial defence mechanisms that provide immunity against viruses (phages) and other mobile genetic elements (MGEs), including plasmids. While the biochemistry of CRISPR-Cas is now well understood, its importance in host-pathogen co-evolution is unclear. Although CRISPR-Cas is clearly important for evolving resistance against MGE in nature, my previous work has shown that under laboratory conditions other resistance mechanisms are more important. This raises the question: under what ecological conditions is CRISPR-Cas-mediated resistance important for evolving MGE resistance? This research project studied how MGE community complexity affects CRISPR-Cas evolution, revealing conditions that increase or decrease CRISPR-Cas efficacy (e.g. the use of multiple phage species and the role of anti-CRISPR genes encoded on phage genomes). This knowledge has important consequences for society as it can be directly implemented in phage therapy to combat pathogenic bacteria and in the dairy industry to protect bacterial starter cultures. In addition, CRISPR-Cas is now widely used as a tool in genome editing, and this has massive potential for the treatment of genetic diseases and crop improvement.
The overall aim of this project was to study the role of CRISPR-Cas in the face of a complex MGE community consisting of phages and plasmids. I had the following research objectives:

i. Study the evolution and efficacy of CRISPR-Cas-mediated immunity.

ii. Study the fitness consequences of CRISPR-Cas over surface modification by competing bacteria in the presence or absence of MGE.

iii. Analyse molecular evolution of bacteria and MGE throughout co-evolution experiments. This will be done by deep sequencing of bacterial and phage genomes isolated at different time points.

iv. Analyse the co-evolutionary dynamics associated with CRISPR-Cas.

Main conclusions:

1. PA14 CRISPR-Cas systems do not co-evolve with phages, but instead drive phages extinct due to the generation of population-level CRISPR diversity.
2. Metapopulation dynamics (i.e. immigration of sensitive bacteria) leads to higher levels of phage persistence, but not co-evolution with CRISPR-Cas.
3. Population-level CRISPR diversity limits the ability for phage to locally adapt to their hosts.
4. Phages carrying anti-CRISPRs can cause epidemics on CRISPR-resistant bacteria when phage densities are high enough.
Evolution of CRISPR-Cas immunity was experimentally tested by infecting Pseudomonas aeruginosa bacteria with phage DMS3vir. Data from these experiments revealed that initially sensitive bacteria rapidly drive phages extinct when these bacteria can evolve CRISPR immunity. These findings were very surprising given that the consensus was that CRISPR-Cas and phages would undergo arms race dynamics coevolution, whereby host and phage constantly adapt to each other. Further analysis revealed that this extinction was due to the generation of high diversity at the population level in CRISPR loci, which limits the evolution of escape phages. This study has led to a first author publication in Nature in 2016. Based on these data I carried out follow-up experiments that show that diversity in CRISPR loci limits phage local adaptation to their hosts, due to the fact that this diversity limits mean phage infectivity. These results were published in Molecular Ecology in 2017 (senior author). In addition, this work led to follow-up research centered around the question: How do phage escape CRISPR immunity, given that they can be driven extinct so quickly? One paper tests the hypothesis whether metapopulation dynamics may allow phages to persist in the face of CRISPR-immunity. This is based on the idea that immigration of sensitive bacteria may help phage to persists, because it would provide a reservoir for phage to amplify on. I therefore performed experiments where I studied CRISPR evolution in the absence of presence of migration. The data showed that phages can persists when migration is higher, and that surface modification becomes more important when migration is higher, because of the higher fitness costs of CRISPR immunity. However, phage still does not co-evolve with their hosts. This work was published as shared first author in the journal Proceedings of the Royal Society B in 2016. Another follow-up project was to look at the effect of phages carrying anti-CRISPR (Acr) genes. Even though phage is normally driven extinct when bacteria evolve CRISPR immunity (see above), when bacteria are infected with an Acr phage the phage is not driven extinct. I performed experiments to understand the epidemiological consequences of Acr phages, and this project is now being finalized. I will be senior author on this paper, which we aim to submit to Nature.
During this project I have set up a new collaboration with Prof. Gandon, CNRS France. This collaboration aimed at understanding the effect of host heterogeneity on evolutionary emergence of parasites, and together with colleagues I used the P. aeruginosa – phage as a model system to study predictions from theoretical models build by Prof. Gandon. This manuscript is currently in preparation, expected submission April 2018.
In addition, I have set up an entirely new model system in the lab to study CRISPR evolution and the interactions with phages and other MGEs. For this, I set up the Streptococcus thermophilus system, which showed high levels of CRISPR evolution (i.e. acquisition of spacers) upon phage infection. However, I observed that spacer diversity was much lower than what I typically observed with the P. aeruginosa system, and frequently phage was not driven extinct. I therefore pursued further experiments with this system to study the co-evolutionary dynamics, which is still ongoing. The data so far indicate that in S. thermophilus bacteria co-evolve with a dynamics that most closely resembles an arms race, while their phages co-evolve according to either arms race or fluctuating selection dynamics. I am now performing molecular typing of these clones as well by deep sequencing of phage and host genomes to genetically underpin these observations. I expect to submit a manuscript on this around June 2018.
During this fellowship I have also obtained follow-up funding to start my own independent research line aimed at understanding how CRISPR-Cas interacts with MGE, in particular plasmids. I will use this project to understand how CRISPR-cas can be used to remove antimicrobial resistance plasmids from bacterial communities.
Overall, the scientific outcomes of this project and the progress made beyond the state of the art have important implications for society. The knowledge generated during this project is important for understanding the risks, potential and consequences of phage therapy for the treatment of bacterial infections as an alternative to the use of antibiotics. In addition it is important for the optimal use of CRISPR-Cas in dairy industry to protect bacterial starter cultures (e.g. for production of yoghurt and cheese) against lethal phage infections. Furthermore, this project has paved the way towards further funding aimed at understanding whether CRISPR-Cas can be used to remove antimicrobial resistance, which is one of the biggest threats to our health of our time.