Identifying factors that drive CRISPR-Cas-dependent phage resistance in bacteria

All organisms, including humans, plants, insects and even bacteria, experience infections by viruses. Understanding how bacteria protect themselves against their viruses is important for several reasons. First, some bacteria cause disease in humans, and viruses are recognised as a promising method to cure patients, known as phage therapy. Second, bacteria are widely used in industry, for example for the production of yoghurt, and virus infections during these industrial processes cause product downgrades and large financial losses. It is therefore of key importance to understand the factors that help to protect or sensitize bacteria to virus infections. For example, in the food industry, bacteria need to be protected against viruses, whereas pathogenic bacteria that infect patients, animals or crops need to be sensitised to virus infections. CRISPR-Cas are widespread prokaryotic adaptive immune systems that can protect bacteria against infections by viruses. However, we have a limited understanding of the factors that cause CRISPR-Cas-mediated immunity to evolve in response to viruses. We hypothesise that the level of CRISPR-Cas immunity evolution may be increased when bacteria are exposed to chemical or environmental factors that trigger expression (activity) of the CRISPR-Cas adaptive immune system. In our research we will therefore first examine how CRISPR-Cas immune systems are switched on and off. Specifically, we aim to understand the role of chemical signals that are released by bacteria to trigger synchronous expression of CRISPR-Cas in bacterial populations. Secondly, we hypothesise that aggressive (virulent) viruses may outpace the CRISPR-Cas adaptive immune system of bacteria. We will examine this hypothesis by correlating virus virulence levels with the ability of bacteria to evolve CRISPR-Cas-mediated immunity. Finally, we will use our understanding of how chemical and environmental factors switch CRISPR-Cas on and off, to manipulate the extent of CRISPR-Cas immunity evolution upon virus infection. Specifically, we will expose bacteria to factors that increase or decrease CRISPR-Cas expression. Manipulating the level of CRISPR-Cas immunity that evolves is important for combatting bacterial pathogens. We will use an important human pathogen, Pseudomonas aeruginosa, for our experimental analyses. This pathogen infects amongst others patients suffering from burn wounds, cystic fibrosis, or cancer. Currently, phage therapy trials are running where burn wound patients infected with P. aeruginosa are treated with virus to kill the pathogen. We believe that it is likely to benefit the patient if virus immune systems such as CRISPR-Cas are inhibited. Our study aims to bring the development of such inhibitors one step closer.

CRISPR-Cas are widespread prokaryotic adaptive immune systems that can protect bacterial and archaeal hosts against infections by viruses and other mobile genetic elements. Our lab was the first to identify conditions where the opportunistic pathogen Pseudomonas aeruginosa UCBPP-PA14 evolves high levels of CRISPR-Cas-mediated immunity against its virus (bacteriophage; phage) DMS3vir (Westra et al, Current Biology 2015). However, CRISPR-Cas-mediated immunity fails to evolve in response to a range of virulent phages, even when they are "primed", which is a factor known to be critically important. Since CRISPR-Cas systems are typically induced upon phage infection, we hypothesize that highly virulent phage may outpace a strictly regulated CRISPR-Cas immune response. The proposed research project aims to (i) uncover how CRISPR-Cas is regulated in Pseudomonas aeruginosa UCBPP-PA14: the cues and regulatory pathways that underly CRISPR-Cas phenotypic plasticity, (ii) examine and experimentally evolve virulence levels of a collection of phages and correlate phage virulence with the ability to evolve CRISPR-Cas-mediated immunity and (iii) expose bacteria to ecological and chemical factors that alter CRISPR-Cas expression to manipulate evolution of CRISPR immunity against virulent phages. The knowledge of factors that help to protect or sensitize bacteria to virus infections is of key importance for both the food- and pharmaceutical industry.

Apart from the scientific community, the proposed research has two additional beneficiaries: 1) the general public 2) industry involved in fermentations and phage therapy. The benefits to these groups, and how we will implement the benefits, are outlined below.

Impact on industry:
(1) Phage therapy. The problems associated with antibiotic resistant bacteria have caused a resurgence of interest in using of phages as therapeutic and prophylactic antimicrobials in clinical and agricultural contexts, and knowledge of factors that impact the evolution of phage resistance are therefore of key importance. In our proposed research, we use Pseudomonas aeruginosa as a model organism, which is an important human pathogen that infects amongst others burn wound patients. Currently, EU-funded phage therapy trials are running on burn wound patients suffering from Pseudomonas aeruginosa infections (Phagoburn), and we expect that inhibiting phage immunity would improve this therapy. In this research we will specifically test chemicals that can potentially inhibit CRISPR-Cas expression and evolution, and this could be a first step in the development of commercial inhibitors. The quorum sensing (QS) inhibitor that we use has the additional benefit that it also reduces virulence, which is QS-regulated. Hence, QS-inhibitors may have multiple benefits in the context of phage therapy, and it seems logical that this potential medicine will find its way to the pharmaceutical industry.

(2) CRISPR-Cas systems are applied to protect bacterial starter cultures that are important in industrial fermentations. A fundamental understanding of factors that trigger CRISPR immunity evolution will have clear benefits to industry. Fundamental research on CRISPR-Cas has already led to the development of a new starter culture series by DuPont based on patented CRISPR Technology: CHOOZITTM SWIFT, which has reduced issues related with phage infections resulting in better yields, optimised process time and minimisation of product downgrades. These products are on the shelves in the supermarket, an excellent example of how fundamental research can find its way to the general public. This project aims to further optimise these and other applications of CRISPR-Cas.

(3) CRISPR-Cas systems could also be applied to protect key bacterial symbionts, that promote for example plant growth (bacteria in the rhizosphere) or human health (probiotics). Protecting bacteria in these "open systems" is more difficult compared to closed fermentations. I am collaborating with academic groups in the Netherlands to use CRISPR immunity in order to improve the performance of these symbionts in real environments (i.e. soil and the gut). I will also actively seek to establish collaborations with industry in the UK in order to realise these applications.

Impact on the general public:
The development of inhibitors of CRISPR-Cas will have clear benefits for the general public, since it may improve current treatment of infectious disease. In addition, the fundamental insights that may lead to improved protection of key bacterial species in fermentations or in more complex open environments, will eventually also benefit the general public. Protection of probiotics can improve human health. Protection of fermentations can reduce losses due to phage infection and protection of plant symbionts can increase yields. All of these will benefit UK economy and therefore indirectly also the general public.