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Phages are the most abundant biological entities on earth and pose a constant challenge to their bacterial hosts. Thus, bacteria have evolved numerous ‘innate’ mechanisms of defense against phage, such as abortive infection or restriction/modification systems. In contrast, the clustered regularly interspaced short palindromic repeats (CRISPR) systems provide acquired, yet heritable, sequence-specific ‘adaptive’ immunity against phage and other horizontally-acquired elements, such as plasmids. Resistance is acquired following viral infection or plasmid uptake when a short sequence of the foreign genome is added to the CRISPR array. CRISPRs are then transcribed and processed, generally by CRISPR associated (Cas) proteins, into short interfering RNAs (crRNAs), which form part of a ribonucleoprotein complex. This complex guides the crRNA to the complementary invading nucleic acid and targets this for degradation. Recently, there have been rapid advances in our understanding of CRISPR/Cas systems. In this review, we will present the current model(s) of the molecular events involved in both the acquisition of immunity and interference stages and will also address recent progress in our knowledge of the regulation of CRISPR/Cas systems.

Bacteria have evolved diverse defense mechanisms that allow them to fight viral attacks. One such mechanism, the clustered, regularly interspaced, short palindromic repeat (CRISPR) system, is an adaptive immune system consisting of genetic loci that can take up genetic material from invasive elements (viruses and plasmids) and later use them to reject the returning invaders. It remains an open question how, despite the ongoing evolution of attack and defense mechanisms, bacteria and viral phages manage to coexist. Using a simple mathematical model and a two-dimensional numerical simulation, we found that CRISPR adaptive immunity allows for robust phage-bacterium coexistence even when the number of virus species far exceeds the capacity of CRISPR-encoded genetic memory. Coexistence is predicted to be a consequence of the presence of many interdependent species that stress but do not overrun the bacterial defense system.

Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated genes are linked to a mechanism of acquired resistance against bacteriophages. Bacteria can integrate short stretches of phage-derived sequences (spacers) within CRISPR loci to become phage resistant. In this study, we further characterized the efficiency of CRISPR1 as a phage resistance mechanism in Streptococcus thermophilus. First, we show that CRISPR1 is distinct from previously known phage defense systems and is effective against the two main groups of S. thermophilus phages. Analyses of 30 bacteriophage-insensitive mutants of S. thermophilus indicate that the addition of one new spacer in CRISPR1 is the most frequent outcome of a phage challenge and that the iterative addition of spacers increases the overall phage resistance of the host. The added new spacers have a size of between 29 to 31 nucleotides, with 30 being by far the most frequent. Comparative analysis of 39 newly acquired spacers with the complete genomic sequences of the wild-type phages 2972, 858, and DT1 demonstrated that the newly added spacer must be identical to a region (named proto-spacer) in the phage genome to confer a phage resistance phenotype. Moreover, we found a CRISPR1-specific sequence (NNAGAAW) located downstream of the proto-spacer region that is important for the phage resistance phenotype. Finally, we show through the analyses of 20 mutant phages that virulent phages are rapidly evolving through single nucleotide mutations as well as deletions, in response to CRISPR1.

In basic terms, the immune system has two lines of defense: innate immunity and adaptive immunity. Innate immunity is the first immunological, non-specific (antigen-independent) mechanism for fighting against an intruding pathogen. It is a rapid immune response, occurring within minutes or hours after aggression, that has no immunologic memory. Adaptive immunity, on the other hand, is antigen-dependent and antigen-specific; it has the capacity for memory, which enables the host to mount a more rapid and efficient immune response upon subsequent exposure to the antigen. There is a great deal of synergy between the adaptive immune system and its innate counterpart, and defects in either system can provoke illness or disease, such as autoimmune diseases, immunodeficiency disorders and hypersensitivity reactions. This article provides a practical overview of innate and adaptive immunity, and describes how these host defense mechanisms are involved in both health and illness.

Prokaryotes have developed several strategies to defend themselves against foreign genetic elements. One of those defense mechanisms is the recently identified CRISPR/Cas system, which is used by approximately half of all bacterial and almost all archaeal organisms. The CRISPR/Cas system differs from the other defense strategies because it is adaptive, hereditary and it recognizes the invader by a sequence specific mechanism. To identify the invading foreign nucleic acid, a crRNA that matches the invader DNA is required, as well as a short sequence motif called protospacer adjacent motif (PAM). We recently identified the PAM sequences for the halophilic archaeon Haloferax volcanii, and found that several motifs were active in triggering the defense reaction. In contrast, selection of protospacers from the invader seems to be based on fewer PAM sequences, as evidenced by comparative sequence data. This suggests that the selection of protospacers has stricter requirements than the defense reaction. Comparison of CRISPR-repeat sequences carried by sequenced haloarchaea revealed that in more than half of the species, the repeat sequence is conserved and that they have the same CRISPR/Cas type.

CRISPR systems, as bacterial defenses against phages, logically must display in their functioning a sequence of at least three major steps. These, in order of occurrence, are “facilitation,” adaptation and interference, where the facilitation step is the main issue considered in this commentary. Interference is the blocking of phage infections as mediated in part by CRISPR spacer sequences. Adaptation, at least as narrowly defined, is the acquisition of these spacer sequences by CRISPR loci. Facilitation, in turn and as defined here, corresponds to phage-naïve bacteria avoiding death follow first-time exposure to specific phages, where bacterial survival of course is necessary for subsequent spacer acquisition. Working from a variety of perspectives, I argue that a requirement for facilitation suggests that CRISPR systems may play secondary rather than primary roles as bacterial defenses, particularly against more virulent phages. So considered, the role of facilitation in CRISPR functioning could be viewed as analogous to the building, in vertebrate animals, of adaptive immunity upon an immunological foundation comprised of mechanisms that are both more generally acting and innate.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and their associated proteins (Cas; CRISPR associated) are a bacterial defense mechanism against extra-chromosomal elements. CRISPR/Cas systems are distinct from other known defense mechanisms insofar as they provide acquired and heritable immunity. Resistance is accomplished in multiple stages in which the Cas proteins provide the enzymatic machinery. Importantly, subtype-specific proteins have been shown to form complexes in combination with small RNAs, which enable sequence-specific targeting of foreign nucleic acids. We used Pectobacterium atrosepticum, a plant pathogen that causes soft-rot and blackleg disease in potato, to investigate protein-protein interactions and complex formation in the subtype I-F CRISPR/Cas system. The P. atrosepticum CRISPR/Cas system encodes six proteins: Cas1, Cas3, and the four subtype specific proteins Csy1, Csy2, Csy3 and Cas6f (Csy4). Using co-purification followed by mass spectrometry as well as directed co-immunoprecipitation we have demonstrated complex formation by the Csy1-3 and Cas6f proteins, and determined details about the architecture of that complex. Cas3 was also shown to co-purify all four subtype-specific proteins, consistent with its role in targeting. Furthermore, our results show that the subtype I-F Cas1 and Cas3 (a Cas2-Cas3 hybrid) proteins interact, suggesting a protein complex for adaptation and a role for subtype I-F Cas3 proteins in both the adaptation and interference steps of the CRISPR/Cas mechanism.

Piwi-interacting RNAs (piRNAs) and CRISPR RNAs (crRNAs) are two recently discovered classes of small noncoding RNA that are found in animals and prokaryotes, respectively. Both of these novel RNA species function as components of adaptive immune systems that protect their hosts from foreign nucleic acids—piRNAs repress transposable elements in animal germlines, whereas crRNAs protect their bacterial hosts from phage and plasmids. The piRNA and CRISPR systems are nonhomologous but rather have independently evolved into logically similar defense mechanisms based on the specificity of targeting via nucleic acid base complementarity. Here we review what is known about the piRNA and CRISPR systems with a focus on comparing their evolutionary properties. In particular, we highlight the importance of several factors on the pattern of piRNA and CRISPR evolution, including the population genetic environment, the role of alternate defense systems and the mechanisms of acquisition of new piRNAs and CRISPRs.

Streptococcus thermophilus, similar to other Bacteria and Archaea, has developed defense mechanisms to protect cells against invasion by foreign nucleic acids, such as virus infections and plasmid transformations. One defense system recently described in these organisms is the CRISPR-Cas system (Clustered Regularly Interspaced Short Palindromic Repeats loci coupled to CRISPR-associated genes). Two S. thermophilus CRISPR-Cas systems, CRISPR1-Cas and CRISPR3-Cas, have been shown to actively block phage infection. The CRISPR1-Cas system interferes by cleaving foreign dsDNA entering the cell in a length-specific and orientation-dependant manner. Here, we show that the S. thermophilus CRISPR3-Cas system acts by cleaving phage dsDNA genomes at the same specific position inside the targeted protospacer as observed with the CRISPR1-Cas system. Only one cleavage site was observed in all tested strains. Moreover, we observed that the CRISPR1-Cas and CRISPR3-Cas systems are compatible and, when both systems are present within the same cell, provide increased resistance against phage infection by both cleaving the invading dsDNA. We also determined that overall phage resistance efficiency is correlated to the total number of newly acquired spacers in both CRISPR loci.

Background: CRISPR/Cas systems allow archaea and bacteria to resist invasion by foreign nucleic acids.

Results: The CRISPR/Cas system in Haloferax recognized six different PAM sequences that could trigger a defense response.

Conclusion: The PAM sequence specificity of the defense response in type I CRISPR systems is more relaxed than previously thought.

Significance: The PAM sequence requirements for interference and adaptation appear to differ markedly.

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system provides adaptive and heritable immunity against foreign genetic elements in most archaea and many bacteria. Although this system is widespread and diverse with many subtypes, only a few species have been investigated to elucidate the precise mechanisms for the defense of viruses or plasmids. Approximately 90% of all sequenced archaea encode CRISPR/Cas systems, but their molecular details have so far only been examined in three archaeal species: Sulfolobus solfataricus, Sulfolobus islandicus, and Pyrococcus furiosus. Here, we analyzed the CRISPR/Cas system of Haloferax volcanii using a plasmid-based invader assay. Haloferax encodes a type I-B CRISPR/Cas system with eight Cas proteins and three CRISPR loci for which the identity of protospacer adjacent motifs (PAMs) was unknown until now. We identified six different PAM sequences that are required upstream of the protospacer to permit target DNA recognition. This is only the second archaeon for which PAM sequences have been determined, and the first CRISPR group with such a high number of PAM sequences. Cells could survive the plasmid challenge if their CRISPR/Cas system was altered or defective, e.g. by deletion of the cas gene cassette. Experimental PAM data were supplemented with bioinformatics data on Haloferax and Haloquadratum.

Despite lacking the adaptive immunity that is found in higher vertebrates, insects are able to defend themselves from a large battery of pathogens by multiple innate immune responses using molecular mechanisms that are strikingly similar to the innate immune responses of other multicellular organisms, including humans. The fruit fly Drosophila melanogaster is therefore an excellent model organism for studying the basic principles of innate immunity using genetic and molecular biology techniques. In Drosophila, invading pathogens that pass through the epithelial barriers (a first line of self-defense) can encounter humoral and cellular responses that utilize pattern-recognition receptors to identify pathogen-associated molecular patterns in the hemolymph or on the immune cell surface. Some pathogens escape recognition and elimination in the hemolymph by invading the host cytoplasm. Some intracellular pathogens such as Listeria monocytogenes are, nevertheless, eliminated by immune reactions such as autophagy through intracellular identification by pattern-recognition receptors.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loci, together with cas (CRISPR–associated) genes, form the CRISPR/Cas adaptive immune system, a primary defense strategy that eubacteria and archaea mobilize against foreign nucleic acids, including phages and conjugative plasmids. Short spacer sequences separated by the repeats are derived from foreign DNA and direct interference to future infections. The availability of hundreds of shotgun metagenomic datasets from the Human Microbiome Project (HMP) enables us to explore the distribution and diversity of known CRISPRs in human-associated microbial communities and to discover new CRISPRs. We propose a targeted assembly strategy to reconstruct CRISPR arrays, which whole-metagenome assemblies fail to identify. For each known CRISPR type (identified from reference genomes), we use its direct repeat consensus sequence to recruit reads from each HMP dataset and then assemble the recruited reads into CRISPR loci; the unique spacer sequences can then be extracted for analysis. We also identified novel CRISPRs or new CRISPR variants in contigs from whole-metagenome assemblies and used targeted assembly to more comprehensively identify these CRISPRs across samples. We observed that the distributions of CRISPRs (including 64 known and 86 novel ones) are largely body-site specific. We provide detailed analysis of several CRISPR loci, including novel CRISPRs. For example, known streptococcal CRISPRs were identified in most oral microbiomes, totaling ∼8,000 unique spacers: samples resampled from the same individual and oral site shared the most spacers; different oral sites from the same individual shared significantly fewer, while different individuals had almost no common spacers, indicating the impact of subtle niche differences on the evolution of CRISPR defenses. We further demonstrate potential applications of CRISPRs to the tracing of rare species and the virus exposure of individuals. This work indicates the importance of effective identification and characterization of CRISPR loci to the study of the dynamic ecology of microbiomes.

Author Summary

Human bodies are complex ecological systems in which various microbial organisms and viruses interact with each other and with the human host. The Human Microbiome Project (HMP) has resulted in >700 datasets of shotgun metagenomic sequences, from which we can learn about the compositions and functions of human-associated microbial communities. CRISPR/Cas systems are a widespread class of adaptive immune systems in bacteria and archaea, providing acquired immunity against foreign nucleic acids: CRISPR/Cas defense pathways involve integration of viral- or plasmid-derived DNA segments into CRISPR arrays (forming spacers between repeated structural sequences), and expression of short crRNAs from these single repeat-spacer units, to generate interference to future invading foreign genomes. Powered by an effective computational approach (the targeted assembly approach for CRISPR), our analysis of CRISPR arrays in the HMP datasets provides the very first global view of bacterial immunity systems in human-associated microbial communities. The great diversity of CRISPR spacers we observed among different body sites, in different individuals, and in single individuals over time, indicates the impact of subtle niche differences on the evolution of CRISPR defenses and indicates the key role of bacteriophage (and plasmids) in shaping human microbial communities.

Background

All archaeal and many bacterial genomes contain Clustered Regularly Interspaced Short Palindrome Repeats (CRISPR) and variable arrays of the CRISPR-associated (cas) genes that have been previously implicated in a novel form of DNA repair on the basis of comparative analysis of their protein product sequences. However, the proximity of CRISPR and cas genes strongly suggests that they have related functions which is hard to reconcile with the repair hypothesis.

Results

The protein sequences of the numerous cas gene products were classified into ~25 distinct protein families; several new functional and structural predictions are described. Comparative-genomic analysis of CRISPR and cas genes leads to the hypothesis that the CRISPR-Cas system (CASS) is a mechanism of defense against invading phages and plasmids that functions analogously to the eukaryotic RNA interference (RNAi) systems. Specific functional analogies are drawn between several components of CASS and proteins involved in eukaryotic RNAi, including the double-stranded RNA-specific helicase-nuclease (dicer), the endonuclease cleaving target mRNAs (slicer), and the RNA-dependent RNA polymerase. However, none of the CASS components is orthologous to its apparent eukaryotic functional counterpart. It is proposed that unique inserts of CRISPR, some of which are homologous to fragments of bacteriophage and plasmid genes, function as prokaryotic siRNAs (psiRNA), by base-pairing with the target mRNAs and promoting their degradation or translation shutdown. Specific hypothetical schemes are developed for the functioning of the predicted prokaryotic siRNA system and for the formation of new CRISPR units with unique inserts encoding psiRNA conferring immunity to the respective newly encountered phages or plasmids. The unique inserts in CRISPR show virtually no similarity even between closely related bacterial strains which suggests their rapid turnover, on evolutionary scale. Corollaries of this finding are that, even among closely related prokaryotes, the most commonly encountered phages and plasmids are different and/or that the dominant phages and plasmids turn over rapidly.

Conclusion

We proposed previously that Cas proteins comprise a novel DNA repair system. The association of the cas genes with CRISPR and, especially, the presence, in CRISPR units, of unique inserts homologous to phage and plasmid genes make us abandon this hypothesis. It appears most likely that CASS is a prokaryotic system of defense against phages and plasmids that functions via the RNAi mechanism. The functioning of this system seems to involve integration of fragments of foreign genes into archaeal and bacterial chromosomes yielding heritable immunity to the respective agents. However, it appears that this inheritance is extremely unstable on the evolutionary scale such that the repertoires of unique psiRNAs are completely replaced even in closely related prokaryotes, presumably, in response to rapidly changing repertoires of dominant phages and plasmids.

This article was reviewed by: Eric Bapteste, Patrick Forterre, and Martijn Huynen.

Open peer review

Reviewed by Eric Bapteste, Patrick Forterre, and Martijn Huynen.

For the full reviews, please go to the Reviewers' comments section.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), together with associated genes (cas), form the CRISPR–cas adaptive immune system, which can provide resistance to viruses and plasmids in bacteria and archaea. Here, we use mathematical models, population dynamic experiments, and DNA sequence analyses to investigate the host–phage interactions in a model CRISPR–cas system, Streptococcus thermophilus DGCC7710 and its virulent phage 2972. At the molecular level, the bacteriophage-immune mutant bacteria (BIMs) and CRISPR–escape mutant phage (CEMs) obtained in this study are consistent with those anticipated from an iterative model of this adaptive immune system: resistance by the addition of novel spacers and phage evasion of resistance by mutation in matching sequences or flanking motifs. While CRISPR BIMs were readily isolated and CEMs generated at high rates (frequencies in excess of 10−6), our population studies indicate that there is more to the dynamics of phage–host interactions and the establishment of a BIM–CEM arms race than predicted from existing assumptions about phage infection and CRISPR–cas immunity. Among the unanticipated observations are: (i) the invasion of phage into populations of BIMs resistant by the acquisition of one (but not two) spacers, (ii) the survival of sensitive bacteria despite the presence of high densities of phage, and (iii) the maintenance of phage-limited communities due to the failure of even two-spacer BIMs to become established in populations with wild-type bacteria and phage. We attribute (i) to incomplete resistance of single-spacer BIMs. Based on the results of additional modeling and experiments, we postulate that (ii) and (iii) can be attributed to the phage infection-associated production of enzymes or other compounds that induce phenotypic phage resistance in sensitive bacteria and kill resistant BIMs. We present evidence in support of these hypotheses and discuss the implications of these results for the ecology and (co)evolution of bacteria and phage.

Author Summary

The evidence that the CRISPR regions of the genomes of archaea and bacteria play a role in the ecology and (co)evolution of these microbes and their viruses is overwhelming: (i) the spacers (variable sequences of 26–72 bp of DNA between the repeats of this region) of these prokaryotes are homologous to the DNA of viruses in their communities; (ii) experimentally, the acquisition and incorporation of spacers of viral DNA can protect these organisms from subsequent infection by these viruses; (iii) experimentally, viruses evade this immunity by mutation in homologous protospacers or protospacer-adjacent motifs (PAMs). Not so clear are the nature and magnitude of the role CRISPR plays in this ecology and evolution. Here, we use mathematical models, experiments with Streptococcus thermophilus and the phage 2972, and DNA sequence analyses to explore the contribution of CRISPR–cas immunity to the ecology and (co)evolution of bacteria and their viruses. The results of this study suggest that the contribution of CRISPR to the ecology of bacteria and phage is more modest and limited, and the conditions for a CRISPR–mediated coevolutionary arms race between these organisms more restrictive, than anticipated from models based on the canonical view of phage infection and CRISPR–cas immunity.

The CRISPR/Cas adaptive immune system provides resistance against phages and plasmids in Archaea and Bacteria. CRISPR loci integrate short DNA sequences from invading genetic elements that provide small RNA-mediated interference in subsequent exposure to matching nucleic acids. In Streptococcus thermophilus, it was previously shown that the CRISPR1/Cas system can provide adaptive immunity against phages and plasmids by integrating novel spacers following exposure to these foreign genetic elements that subsequently direct the specific cleavage of invasive homologous DNA sequences. Here, we show that the S. thermophilus CRISPR3/Cas system can be transferred into Escherichia coli and provide heterologous protection against plasmid transformation and phage infection. We show that interference is sequence-specific, and that mutations in the vicinity or within the proto-spacer adjacent motif (PAM) allow plasmids to escape CRISPR-encoded immunity. We also establish that cas9 is the sole cas gene necessary for CRISPR-encoded interference. Furthermore, mutation analysis revealed that interference relies on the Cas9 McrA/HNH- and RuvC/RNaseH-motifs. Altogether, our results show that active CRISPR/Cas systems can be transferred across distant genera and provide heterologous interference against invasive nucleic acids. This can be leveraged to develop strains more robust against phage attack, and safer organisms less likely to uptake and disseminate plasmid-encoded undesirable genetic elements.

It has been speculated that the rise of the adaptive immune system in jawed vertebrates some 400 million years ago gave them a superior protection to detect and defend against pathogens that became more elusive and/or virulent to the host that had only innate immune system. First, this line of thought implies that adaptive immune system was a new, more sophisticated layer of host defense that operated independently of the innate immune system. Second, the natural consequence of this scenario would be that pathogens would have exercised so strong an evolutionary pressure that eventually no host could have afforded not to have an adaptive immune system. Neither of these arguments is supported by the facts. First, new experimental evidence has firmly established that operation of adaptive immune system is critically dependent on the ability of the innate immune system to detect invader-pathogens and second, the absolute majority of animal kingdom survive just fine with only an innate immune system. Thus, these data raise the dilemma: If innate immune system was sufficient to detect and protect against pathogens, why then did adaptive immune system develop in the first place? In contrast to the innate immune system, the adaptive immune system has one important advantage, precision. By precision I mean the ability of the defense system to detect and remove the target, for example, infected cells, without causing unwanted bystander damage of surrounding tissue. While the target precision per se is not important for short-term immune response, it becomes a critical factor when the immune response is long lasting, as during chronic infection. In this paper I would like to propose new, “toxic index” hypothesis where I argue that the need to reduce the collateral damage to the tissue during chronic infection(s) was the evolutionary pressure that led to the development of the adaptive immune system.

All cellular systems evolve ways to combat predators and genomic parasites. In bacteria and archaea, numerous resistance mechanisms have developed against phage. Our understanding of this defensive repertoire has recently been expanded to include the CRISPR system of Clustered, Regularly Interspaced Short Palindromic Repeats. In this remarkable pathway, short sequence tags from invading genetic elements are actively incorporated into the host's CRISPR locus, to be transcribed and processed into a set of small RNAs that guide the destruction of foreign genetic material. Here, we review the inner workings of this adaptable and heritable immune system and draw comparisons to small RNA-guided defense mechanisms in eukaryotic cells.

Clustered regularly interspaced short palindromic repeats (CRISPRs) comprise a family of short DNA repeat sequences that are separated by non repetitive spacer sequences and, in combination with a suite of Cas proteins, are thought to function as an adaptive immune system against invading DNA. The number of CRISPR arrays in a bacterial chromosome is variable, and the content of each array can differ in both repeat number and in the presence or absence of specific spacers. We utilized a comparative sequence analysis of CRISPR arrays of the plant pathogen Erwinia amylovora to uncover previously unknown genetic diversity in this species. A total of 85 E. amylovora strains varying in geographic isolation (North America, Europe, New Zealand, and the Middle East), host range, plasmid content, and streptomycin sensitivity/resistance were evaluated for CRISPR array number and spacer variability. From these strains, 588 unique spacers were identified in the three CRISPR arrays present in E. amylovora, and these arrays could be categorized into 20, 17, and 2 patterns types, respectively. Analysis of the relatedness of spacer content differentiated most apple and pear strains isolated in the eastern U.S. from western U.S. strains. In addition, we identified North American strains that shared CRISPR genotypes with strains isolated on other continents. E. amylovora strains from Rubus and Indian hawthorn contained mostly unique spacers compared to apple and pear strains, while strains from loquat shared 79% of spacers with apple and pear strains. Approximately 23% of the spacers matched known sequences, with 16% targeting plasmids and 5% targeting bacteriophage. The plasmid pEU30, isolated in E. amylovora strains from the western U.S., was targeted by 55 spacers. Lastly, we used spacer patterns and content to determine that streptomycin-resistant strains of E. amylovora from Michigan were low in diversity and matched corresponding streptomycin-sensitive strains from the background population.

New and exciting insights into the importance of the innate immune system are revolutionizing our understanding of immune defense against infections, pathogenesis, and the treatment and prevention of infectious diseases. The innate immune system uses multiple families of germline-encoded pattern recognition receptors (PRRs) to detect infection and trigger a variety of antimicrobial defense mechanisms. PRRs are evolutionarily highly conserved and serve to detect infection by recognizing pathogen-associated molecular patterns that are unique to microorganisms and essential for their survival. Toll-like receptors (TLRs) are transmembrane signalling receptors that activate gene expression programs that result in the production of proinflammatory cytokines and chemokines, type I interferons and antimicrobial factors. Furthermore, TLR activation facilitates and guides activation of adaptive immune responses through the activation of dendritic cells. TLRs are localized on the cell surface and in endosomal/lysosomal compartments, where they detect bacterial and viral infections. In contrast, nucleotide-binding oligomerization domain proteins and RNA helicases are located in the cell cytoplasm, where they serve as intracellular PRRs to detect cytoplasmic infections, particularly viruses. Due to their ability to enhance innate immune responses, novel strategies to use ligands, synthetic agonists or antagonists of PRRs (also known as ‘innate immunologicals’) can be used as stand-alone agents to provide immediate protection or treatment against bacterial, viral or parasitic infections. Furthermore, the newly appreciated importance of innate immunity in initiating and shaping adaptive immune responses is contributing to our understanding of vaccine adjuvants and promises to lead to improved next-generation vaccines.

DNA viruses are a significant contributor to human morbidity and mortality. The immune system protects against viral infections through coordinated innate and adaptive immune responses. While the antigen-specific adaptive mechanisms have been extensively studied, the critical contributions of innate immunity to anti-viral defenses have only been revealed in the very recent past. Central to these anti-viral defenses is the recognition of viral pathogens by a diverse set of germ-line encoded receptors that survey nearly all cellular compartments for the presence of pathogens. In this review, we discuss the recent advances in the innate immune sensing of DNA viruses and focus on the recognition mechanisms involved.

The past is never dead. It's not even pastWilliam Faulkner (1951)

Bacteria can acquire heritable immunity to viral (phage) enemies by incorporating phage DNA into their own genome. This mechanism of anti-viral defence, known by the acronym CRISPR, simultaneously stores detailed information about current and past enemies and the evolved resistance to them. As a high-resolution genetic marker that is intimately tied with the host–pathogen interaction, the CRISPR system offers a unique, and relatively untapped, opportunity to study epidemiological and coevolutionary dynamics in microbial communities that were previously neglected because they could not be cultured in the laboratory. We briefly review the molecular mechanisms of CRISPR-mediated host–pathogen resistance, before assessing their potential importance for coevolution in nature, and their utility as a means of studying coevolutionary dynamics through metagenomics and laboratory experimentation.

Studies of the immune mechanisms of allograft rejection have predominantly focused on the adaptive immune system that includes T cells and B cells. Recent investigations into the innate immune system, which recognizes foreign antigens through more evolutionarily primitive pathways, have demonstrated a critical role of the innate immune system in the regulation of the adaptive immune system. Innate immunity has been extensively studied in its role as the host's first-line defense against microbial pathogens; however, it is becoming increasingly recognized for its ability to also recognize host-derived molecules that result from tissue damage. The capacity of endogenous damage signals acting through the innate immune system to lower immune thresholds and promote immune recognition and rejection of transplant grafts is only beginning to be appreciated. An improved understanding of these pathways may reveal novel therapeutic targets to decrease graft alloreactivity and increase graft longevity.

CRISPR (Clustered, Regularly, Interspaced, Short, Palindromic Repeats) loci have been shown to provide prokaryotes with an adaptive immunity against viruses and plasmids. CRISPR arrays are transcribed and processed into small CRISPR RNA molecules, which base-pair with invading DNA or RNA and lead to its degradation by CRISPR-associated (Cas) protein complexes. New spacers can be acquired by active CRISPR/Cas systems, and thus the sequences of these spacers provide a record of the past “infection history” of the organism. Recently we used spacer sequences from archaeal genomes to infer gene exchange events among archaeal species and genera and to demonstrate that at least in this domain of life CRISPR indeed has an anti-viral role.

Studies of the Escherichia, Neisseria, Thermotoga, and Mycobacteria clustered regularly interspaced short palindromic repeat (CRISPR) subtypes have resulted in a model whereby CRISPRs function as a defense system against bacteriophage infection and conjugative plasmid transfer. In contrast, we previously showed that the Yersinia-subtype CRISPR region of Pseudomonas aeruginosa strain UCBPP-PA14 plays no detectable role in viral immunity but instead is required for bacteriophage DMS3-dependent inhibition of biofilm formation by P. aeruginosa. The goal of this study is to define the components of the Yersinia-subtype CRISPR region required to mediate this bacteriophage-host interaction. We show that the Yersinia-subtype-specific CRISPR-associated (Cas) proteins Csy4 and Csy2 are essential for small CRISPR RNA (crRNA) production in vivo, while the Csy1 and Csy3 proteins are not absolutely required for production of these small RNAs. Further, we present evidence that the core Cas protein Cas3 functions downstream of small crRNA production and that this protein requires functional HD (predicted phosphohydrolase) and DEXD/H (predicted helicase) domains to suppress biofilm formation in DMS3 lysogens. We also determined that only spacer 1, which is not identical to any region of the DMS3 genome, mediates the CRISPR-dependent loss of biofilm formation. Our evidence suggests that gene 42 of phage DMS3 (DMS3-42) is targeted by CRISPR2 spacer 1 and that this targeting tolerates multiple point mutations between the spacer and DMS3-42 target sequence. This work demonstrates how the interaction between P. aeruginosa strain UCBPP-PA14 and bacteriophage DMS3 can be used to further our understanding of the diverse roles of CRISPR system function in bacteria.

Lactococcus lactis is a biotechnological workhorse for food fermentations and potentially therapeutic products and is therefore widely consumed by humans. It is predominantly used as a starter microbe for fermented dairy products, and specialized strains have adapted from a plant environment through reductive evolution and horizontal gene transfer as evidenced by the association of adventitious traits with mobile elements. Specifically, L. lactis has armed itself with a myriad of plasmid-encoded bacteriophage defensive systems to protect against viral predation. This known arsenal had not included CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins), which forms a remarkable microbial immunity system against invading DNA. Although CRISPR/Cas systems are common in the genomes of closely related lactic acid bacteria (LAB), none was identified within the eight published lactococcal genomes. Furthermore, a PCR-based search of the common LAB CRISPR/Cas systems (Types I and II) in 383 industrial L. lactis strains proved unsuccessful. Here we describe a novel, Type III, self-transmissible, plasmid-encoded, phage-interfering CRISPR/Cas discovered in L. lactis. The native CRISPR spacers confer resistance based on sequence identity to corresponding lactococcal phage. The interference is directed at phages problematic to the dairy industry, indicative of a responsive system. Moreover, targeting could be modified by engineering the spacer content. The 62.8-kb plasmid was shown to be conjugally transferrable to various strains. Its mobility should facilitate dissemination within microbial communities and provide a readily applicable system to naturally introduce CRISPR/Cas to industrially relevant strains for enhanced phage resistance and prevention against acquisition of undesirable genes.