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Surprising results from two independent research groups, recently published in the journal Cell, show that phage join forces to overcome bacteria’s CRISPR-based immune defenses. Improved understanding of the interactions between phage and their bacterial hosts could help advance phage-based therapies and stimulate viral research.



As part of their lifecycle, phage predate bacteria, injecting their genetic material and hijacking the bacteria’s replication machinery. To defend themselves against phage attack, many bacterial species have evolved defensive systems, with over half of sequenced bacterial genomes found to encode a CRISPR-Cas “immune system”1,2. In response, many phage have acquired anti-CRISPR (acr) genes whose products inhibit this system. However, previous evidence had shown that some Acrs are more effective than others and the details of how they are deployed and utilized by a phage during infection were unclear.



With antibiotic resistance being an ever-growing concern, increasing attention has been given to understanding the mechanisms that underly phage function as well as their potential use as a therapy for targeted bacterial killing. The concept of phage therapy has been around for decades, however effective treatments employing this technique are still not available, which could be atributed in part to knowledge gaps like these.



Teams from the University of California, San Francisco (UCSF) and the University of Exeter were both working on phage infection of Pseudomonas aeruginosa (P. aeruginosa), a Gram-negative bacterium often associated with respiratory and sepsis-related infections, especially in immunocompromised patients. P. aeruginosa also played host to the first phage in which acr genes were identified3.



Using phage naturally encoding diverse acr genes as well as an isogenic phage panel in which the acr genes were swapped, the UCSF team were able to show that Acr proteins were unable to fully protect their associated phage, suggesting that Acr deployment and action is an imperfect process.



Phage maintained in the lytic phase by removal of the C repressor gene, gp1, showed that a critical concentration of Acr protein was required in individual cells for successful lytic replication. The Acr protein could be contributed by multiple phage so even where a specific phage failed to replicate, the Acr it contributed to that bacterial cell left it immunosuppressed and therefore more vulnerable to successful attack by other phage. Furthermore, using phage with and without CRISPR repression capabilities, they demonstrated that Acr-mediated immune suppression was required to establish lysogeny. Cooperation between multiple phage therefore enables them to overcome bacterial immunity.



Likewise, the team at the University of Exeter found that bacteria may retain partial immunity to Acr-encoding phage and that in these circumstances cooperation between multiple phage is required for successful bacterial killing. Using equal quantities of starting phage in differing concentrations of bacterial host cells, they showed that the initial density of Acr-producing phage to bacterial host cells determines whether phage go extinct or are amplified. They referred to this as the epidemiological tipping point, which was influenced by the strength of CRISPR-Cas immunity and effectiveness of Acr activity.



The authors acknowledge the inevitable deficiencies of laboratory-based culture experiments that lack the variation in conditions and bacterial populations that would be seen in a “field” situation. However, the data provide useful insights that will help scientists continue to tease apart phage mechanisms and direct future work. Metagenomic analyses of the interactions between bacteria and infecting phage in natural populations will expand the understanding of the population dynamics and help to understand the foundations of this cooperative action.



References



1. Grissa, I., Vergnaud, G., and Pourcel, C. (2007). The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats. BMC Bioinformatics 8, 172.



2. Makarova, K.S., Wolf, Y.I., Alkhnbashi, O.S., Costa, F., Shah, S.A., Saunders, S.J., Barrangou, R., Brouns, S.J., Charpentier, E., Haft, D.H., et al. (2015). An updated evolutionary classification of CRISPR-Cas systems. Nat. Rev. Microbiol. 13, 722–736.



3. Bondy-Denomy, J., Pawluk, A., Maxwell, K.L., and Davidson, A.R. (2013). Bacteriophage genes that inactivate the CRISPR/Cas bacterial immune system. Nature 493, 429–432.