Restriction-Modification and CRISPR-Cas Systems: Cooperation Between Innate and Adaptive Immunity in Prokaryotes

Bacteria have evolved numerous mechanisms to resist the constant assault of viruses (called bacteriophages, or simply phages) that can infect and kill them. Restriction-modification (RM) systems represent one such strategy. Generally, these systems provide defense by coordinating the activities of two distinct enzymes: a restriction endonuclease and a methyltransferase. Both enzymes recognize the same short DNA sequences. The methyltransferase modifies these target sites in the host chromosome, which prevents the restriction endonuclease from cleaving the host’s own DNA. In contrast, foreign phage DNA is usually not methylated at these sequences. Consequently, upon injection into the host, the viral DNA is recognized and cleaved by the restriction endonuclease, preventing the progression of the phage’s life cycle. Therefore, RM systems are considered a part of the innate immune response because they can provide defense against any phage, including ones that have never been encountered previously, as long as they harbor RM target sites. Clustered regularly interspaced short palindromic repeats (CRISPR) loci and their associated genes (cas) form another defense system that destroys foreign DNA. The CRISPR array consists of a series of repetitive DNA sequences separated by unique DNA sequences known as spacers. During phage infection, short DNA fragments are taken from the viral DNA and integrated into the CRISPR locus to form new spacers. These sequences are then transcribed into CRISPR RNAs (crRNAs). In type II-A CRISPRCas systems, the crRNAs guide the Cas9 nuclease to a matching viral DNA target for cleavage. As such, unlike RM systems, CRISPR-Cas systems represent an adaptive immune response because they require an initial exposure to a virus in order to become successfully immunized through the acquisition of new spacer sequences. CRISPR-Cas and RM are two of the most prevalent types of defense systems found in bacteria and often co-exist together in a single host. Yet, how they may interact with each other in the context of immunity during bacteriophage infection is poorly understood. Here, in my thesis work, I investigate the interplay between RM and type II-A CRISPR-Cas systems. First, I demonstrate that RM systems provide a weak and temporary protection that stimulates CRISPR spacer acquisition, enabling the cells to survive the viral infection. Then, I go on to show that the restriction activity of the RM system is critical for this process and that the rate of spacer acquisition is correlated to the number of RM target sites in the phage genome. To further uncover the mechanistic link between restriction and the acquisition of new spacers, I implement next-generation sequencing to demonstrate that spacers are preferentially extracted at the dsDNA breaks (DSBs) generated by the restriction endonuclease. Additionally, I show that the host DNA repair complex, AddAB, can process these breaks, which further enhances spacer acquisition. Finally, I follow the dynamics between RM and CRISPR-Cas during the chain of events that occur upon viral infection. I demonstrate that although the RM system provides an immediate line of defense due to its ability to recognize a broad range of foreign invaders, it is ultimately overcome by the rapid emergence of methylated phages, resulting in the death of much of the bacterial population. However, the early RM immune response creates substrates for spacer acquisition by the CRISPR-Cas system in a subset of cells. By using these newly acquired spacers which specify the viral sequences for lethal cleavage by Cas9, these cells can now extinguish the methylated phages, resulting in the survival and regrowth of the population. Collectively, my thesis reveals the molecular mechanisms connecting RM and CRISPR-Cas systems in providing a synergistic anti-phage defense. Reminiscent of eukaryotic immunity, I demonstrate that RM systems provide an initial, short-lived innate immune response, which stimulates a secondary, more robust adaptive immune response by CRISPR-Cas. This work highlights an example of cooperation between RM and CRISPR-Cas, which are two of the most common bacterial defense systems. However, prokaryotes have been shown to harbor a multitude of other putative antiphage defense systems, which can often exist together in a single host. I predict that future studies will likely uncover many more fascinating instances of immune interaction among other sets of defense systems