Alternative Roles for CRISPR/Cas Systems in Bacterial Pathogenesis

1Department of Microbiology and Immunology, Microbiology and Molecular Genetics Program, Emory University School of Medicine, Atlanta, Georgia, United States ofAmerica, 2Emory Vaccine Center, Emory University, Atlanta, Georgia, United States of America, 3Yerkes National Primate Research Center, Emory University, Atlanta,Georgia, United States of America, 4Division of Infectious Diseases, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia, United States ofAmerica


CRISPR/Cas Systems Play Critical Roles in Pathogenesis
We recently demonstrated that components of the Type II CRISPR/Cas system in Francisella novicida are necessary for this intracellular bacterial pathogen to evade detection by a host pattern recognition receptor and cause disease [5]. Cas9, in conjunction with tracrRNA and a novel small RNA termed scaRNA (small, CRISPR/Cas-associated RNA), target an endogenous transcript encoding an immunostimulatory bacterial lipoprotein (BLP), leading to mRNA degradation and decreased transcript levels ( Figure 1A) [5]. Surprisingly, this action does not rely on any of the crRNAs, but instead is predicted to utilize tracrRNA to target mRNA. In the absence of this regulation, increased BLP levels trigger the activation of a Toll-like Receptor 2 (TLR2)-dependent proinflammatory response, and result in complete attenuation of the bacteria during infection ( Figure 1B). These CRISPR/Cas components are therefore critical to the ability of F. novicida to cause disease.
Cas9 is also required for the ability of both Neisseria meningitidis and Campylobacter jejuni to attach to, invade, and replicate within epithelial cells, traits that are essential to their virulence [5,8]. Currently, the mechanism of Cas9-mediated pathogenesis by these organisms is unknown. It is likely that Cas9, in conjunction with one or more small RNAs, acts to alter the stability of a transcript and that this is important for virulence, similar to its function in F. novicida. Interestingly, the role of Cas9 in C. jejuni virulence correlated with strains producing a sialylated lipooligosaccharide structure in the outer envelope [8]. Coupled with the fact that Cas9 regulates production of a membrane BLP in Francisella, it is tempting to speculate that CRISPR/Cas systems that control mRNA stability may be widely involved in regulation of envelope structure.
In addition to the role of Cas9 in the virulence of bacterial pathogens, Cas2 has been implicated in the ability of Legionella pneumophila to survive within amoebae [9]. Since amoebae are essential to L. pneumophila survival in the environment [10], the role of Cas2 in this process may be critical to environmental persistence and subsequent transmission to other hosts. Exactly how Cas2 mediates Legionella intracellular survival is unknown. Due to its endonuclease activity involved in CRISPR adaptation, Cas2 is hypothesized to also function in either the processing of small RNA regulators during intracellular infection, or to be a direct mediator of mRNA degradation [9]. Interestingly, Cas9 plays no role in the intracellular survival of L. pneumophila in amoebae [9]. Additionally, Cas2 has no currently observed role in F. novicida gene regulation or virulence [5], demonstrating that while Type II CRISPR/Cas systems are similar across species, different organisms may have co-opted distinct components for pathogenesis. Funding: This work was supported by National Institutes of Health (NIH) grants U54-AI057157 from the Southeastern Regional Center of Excellence for Emerging Infections and Biodefense and R56-AI87673 to DSW, who is also supported by a Burroughs Wellcome Fund Investigator in the Pathogenesis of Infectious Disease award (http://www.bwfund.org/grant-programs/infectious-diseases/ investigators-pathogenesis-infectious-disease). TRS was supported by the NSF Graduate Research Fellowship, as well as the ARCS Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

CRISPR/Cas Systems Can Control Bacterial Physiology
Different types of CRISPR/Cas systems have also been observed to contribute to bacterial physiology beyond defense against foreign nucleic acids. The CRISPR/Cas system in Pseudomonas aeruginosa plays a role in modulating biofilm formation [11,12]. While the precise mechanism is unknown, the data suggest that when P. aeruginosa is lysogenized by a specific bacteriophage, the CRISPR/Cas system interacts with a particular gene in the chromosomally integrated prophage to inhibit the creation of biofilms [11,12]. It is unclear if the CRISPR/Cas system targets DNA or mRNA, but it is known that the interaction requires the Cas proteins involved in both crRNA maturation and targeting/degradation, as well as a specific targeting crRNA with sequence similarity to the prophage gene [11,12]. Given the importance of biofilm formation in the pathogenic life cycle of P. aeruginosa [12], it is likely that this intricate CRISPR/Cas regulatory schema plays an important role in infection.
Additionally, Cas1 and the crRNA array in the CRISPR/Cas system of Escherichia coli K-12 (also present in EHEC and UPEC strains [1]) play a role in mediating DNA repair [13]. Given the universality of Cas1 in all known CRISPR/Cas systems, it is intriguing to speculate that it may broadly function in this regard. Further, since DNA damage may occur as a product of host defenses during infection (i.e., production of reactive nitrogen and oxygen species) [14], it is interesting to consider that CRISPR/Cas systems may provide pathogens another layer of redundancy in their ability to resist and repair damage incurred during infection.

Are Self-Targeting CRISPR/Cas Systems Involved in ''Autoimmunity'' or Gene Regulation?
Another potential example of noncanonical functionality of CRISPR/Cas systems in gene regulation and virulence may involve self-targeting crRNAs with spacer sequences complementary to chromosomally encoded genes [15,16]. For example, selftargeting crRNAs are predicted to target hypothetical proteins in the pathogens Clostridium botulinum, N. meningitidis, and Yersinia pestis, two sporulation genes and a gene involved in S-layer biosynthesis in Clostridium tetani, and the fdrA gene involved in protein transport in Enterobacter spp. [15]. Since crRNAs are known to target DNA, their specificity for chromosomal genes has been suggested to likely result in detrimental chromosomal cleavage, which can result in large chromosomal deletions, and thus has been termed ''autoimmunity'' [17]. It has been hypothesized that self-targeting crRNAs can be tolerated if CRISPR/Cas systems in bacteria encoding them are either nonfunctional or are significantly degenerated, thereby preventing ''autoimmune'' recognition and cleavage of the chromosome [15]. Indeed, CRISPR/Cas systems encoding selftargeting crRNAs often have degenerated Cas proteins [15,16]. However, this could nonetheless be consistent with a role in gene regulation for at least some self-targeting crRNAs [16]. Inactive Cas1 and Cas2 proteins would not necessarily inhibit self-targeting abilities, but instead prevent acquisition of new crRNAs [3,4]. Since acquisition of new crRNAs can lead to loss of previously acquired crRNAs [18], degeneration of Cas1 and Cas2 may actually be favored to prevent loss of regulatory crRNAs.
Additionally, it has been demonstrated that a catalytically inactive Cas9 is still capable of binding DNA targets and inhibiting transcription, resulting in repression of the targeted gene [19]. Therefore, degenerated Cas proteins could theoretically still participate in gene regulation. Furthermore, the CRISPR/Cas system in P. aeruginosa, capable of targeting a chromosomally integrated element without causing chromosomal degradation, is fully functional against bacteriophage infection, suggesting that chromosomal targeting by an active CRISPR/Cas system does not necessarily lead to ''autoimmune'' events [11,12,20]. Finally, as observed in F. novicida, if mRNA but not DNA is targeted (i.e., FTN_1103) [5], there would be no negative selection against targeting endogenous genes in the chromosome. It is therefore tempting to speculate that self-targeting crRNAs may act as regulatory elements in at least some of the aforementioned and other pathogens.

Perspectives
While well established to play roles in defending bacteria from bacteriophages and other foreign genetic elements, the critical roles that CRISPR/Cas systems play in the ability of pathogenic organisms to evade host defenses and replicate within the host are just now being appreciated. Given that CRISPR/Cas systems are widely distributed among prokaryotes (,50% of bacteria and 99% of Archaea) and are present in both pathogenic and commensal organisms [1], as well as their specificity and adaptability, it is very likely that more examples of their alternative functions in gene regulation controlling virulence, commensalism, and broader physiology will be revealed. Future work elucidating how CRISPR/Cas systems contribute to bacterial virulence will allow for the identification of novel host defense evasion strategies that bacterial pathogens utilize during infection.