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Antimicrobial resistance (AMR) genes are widely disseminated on plasmids. Therefore, interventions aimed at blocking plasmid uptake and transfer may curb the spread of AMR. Previous studies have used CRISPR-Cas-based technology to remove plasmids encoding AMR genes from target bacteria, using either phage- or plasmid-based delivery vehicles that typically have narrow host ranges. To make this technology feasible for removal of AMR plasmids from multiple members of complex microbial communities, an efficient, broad host-range delivery vehicle is needed. We engineered the broad host-range IncP1-plasmid pKJK5 to encode cas9 programmed to target an AMR gene. We demonstrate that the resulting plasmid pKJK5::csg has the ability to block the uptake of AMR plasmids and to remove resident plasmids from Escherichia coli . Furthermore, due to its broad host range, pKJK5::csg successfully blocked AMR plasmid uptake in a range of environmental, pig- and human-associated coliform isolates, as well as in isolates of two species of Pseudomonas . This study firmly establishes pKJK5::csg as a promising broad host-range CRISPR-Cas9 delivery tool for AMR plasmid removal, which has the potential to be applied in complex microbial communities to remove AMR genes from a broad range of bacterial species.

SummaryThe constant arms race between bacteria and their phages has resulted in a large diversity of bacterial defence systems1,2, with many bacteria carrying several systems3,4. In response, phages often carry counter-defence genes5–9. If and how bacterial defence mechanisms interact to protect against phages with counter-defence genes remains unclear. Here, we report the existence of a novel defence system, coined MADS (Methylation Associated Defence System), which is located in a strongly conserved genomic defence hotspot inPseudomonas aeruginosaand distributed across Gram-positive and Gram-negative bacteria. We find that the natural co-existence of MADS and a Type IE CRISPR-Cas adaptive immune system in the genome ofP. aeruginosaSMC4386 provides synergistic levels of protection against phage DMS3, which carries an anti-CRISPR (acr) gene. Previous work has demonstrated that Acr-phages need to cooperate to overcome CRISPR immunity, with a first sacrificial phage causing host immunosuppression to enable successful secondary phage infections10,11. Modelling and experiments show that the co-existence of MADS and CRISPR-Cas provides strong and durable protection against Acr-phages by disrupting their cooperation and limiting the spread of mutants that overcome MADS. These data reveal that combining bacterial defences can robustly neutralise phage with counter-defence genes, even if each defence on its own can be readily by-passed, which is key to understanding how selection acts on defence combinations and their coevolutionary consequences.

We commonly acknowledge that bacterial viruses (phages) shape the composition and evolution of bacterial communities in nature and therefore have important roles in ecosystem functioning. This view stems from studies in the 1990s to the first decade of the twenty-first century that revealed high viral abundance, high viral diversity and virus-induced microbial death in aquatic ecosystems as well as an association between collapses in bacterial density and peaks in phage abundance. The recent surge in metagenomic analyses has provided deeper insight into the abundance, genomic diversity and spatio-temporal dynamics of phages in a wide variety of ecosystems, ranging from deep oceans to soil and the mammalian digestive tract. However, the causes and consequences of variations in phage community compositions remain poorly understood. In this Review, we explore current knowledge of the composition and evolution of phage communities, as well as their roles in controlling the population and evolutionary dynamics of bacterial communities. We discuss the need for greater ecological realism in laboratory studies to capture the complexity of microbial communities that thrive in natural environments.

It is becoming increasingly clear that antibiotics can both positively and negatively impact the infectivity of bacteriophages (phage), but the underlying mechanisms often remain unclear. Here we demonstrate that antibiotics that target the protein translation machinery can fundamentally alter the outcome of bacteria-phage interactions by interfering with the production of phage-encoded counter-defence proteins. Specifically, using Pseudomonas aeruginosa PA14 and phage DMS3vir as a model, we show that bacteria with CRISPR-Cas immune systems have elevated levels of immunity against phage that encode anti-CRISPR (acr) genes when translation inhibitors are present in the environment. CRISPR-Cas are highly prevalent defence systems that enable bacteria to detect and destroy phage genomes in a sequence-specific manner. In response, many phages encode acr genes that are expressed immediately following infection to inhibit key steps of the CRISPR-Cas immune response. Our data show that while phage carrying acr genes can amplify efficiently on bacteria with CRISPR-Cas immune systems in the absence of antibiotics, the presence of antibiotics that act on protein translation prevents phage amplification, while protecting bacteria from lysis. These results help to understand how antibiotics-phage synergy and antagonism depend on the molecular interactions that define phage infectivity and host immunity.

SummaryBackgroundE. coli is a highly diverse species that generates a huge global burden of antimicrobial-resistant infections. Although it is one of the most well-studied model organisms, we lack a synthesis of the genomic distribution of antimicrobial resistance (AMR) genes according to phylogenetic and ecological variables.MethodsWe implemented Mash-based phylogrouping on ∼16,000 RefSeq E. coli genomes, and statistically modelled the link between phylogroups, host species categories, geographic subregions, climate, and AMR genes. We predict the burden of AMR across these categories using several metrics including overall counts of resistance genes, multidrug- and extensively-drug resistant (MDR and XDR) classifications, and frequencies of extended-spectrum β-lactamase and carbapenemase genes.FindingsAMR burden was highly variable across all measures between phylogroups, host species, geographic locations and climates. Phylogroups spanning the commensal-pathogen spectrum were found with high levels of AMR. MDR genomes were highly prevalent in North Africa and South-Eastern Asia, while XDR isolates were more common in Eastern Asia. Wild birds were associated with the highest levels of XDR and carbapenemase-encoding isolates. Carbapenemases, MDR genomes and most blaCTX-M groups were positively associated with temperature.InterpretationThis study synthesises data on clinical, ecological and anthropogenic variables impacting AMR in E. coli on an unprecedented scale, modelling its burden across clinically-relevant measures. It highlights key findings with direct relevance to managing the global spread of AMR and understanding its ecology. Finally, it emphasizes knowledge gaps in public databases that, if investigated, would further develop our understanding of AMR in this important pathogen and the risk posed across contexts.FundingEU Horizon 2020 ProgrammeBiotechnology and Biological Sciences Research Council (BBSRC)

Plasmids are key disseminators of antimicrobial resistance genes and virulence factors, and it is therefore critical to predict and reduce plasmid spread within microbial communities. The cost of plasmid carriage is a key metric that can be used to predict plasmids’ ecological fate, and it is unclear whether plasmid costs are affected by growth partners in a microbial community. We carried out competition experiments and tracked plasmid maintenance using a synthetic and stable 5-species community and a broad host-range plasmid as a model. We report that both the cost of plasmid carriage and its long-term maintenance in a focal strain depended on the presence of competitors, and that these interactions were species-specific. Addition of growth partners increased the plasmid cost to a focal strain, and accordingly plasmid loss from the focal species occurred over a shorter time frame in these species combinations. We propose that the destabilising effect of interspecific competition on plasmid maintenance may be leveraged in clinical and natural environments to cure plasmids from focal strains.

The acquisition of antibiotic resistance (ABR) genes via horizontal gene transfer (HGT) is a key driver of the rise in multidrug resistance amongst bacterial pathogens. Bacterial defence systems per definition restrict the influx of foreign genetic material, and may therefore limit the acquisition of ABR. CRISPR-Cas adaptive immune systems are one of the most prevalent defences in bacteria, found in roughly half of bacterial genomes, but it has remained unclear if and how much they contribute to restricting the spread of ABR. We analysed approximately 40 000 whole genomes comprising the full RefSeq dataset for 11 species of clinically important genera of human pathogens, including Enterococcus , Staphylococcus , Acinetobacter and Pseudomonas . We modelled the association between CRISPR-Cas and indicators of HGT, and found that pathogens with a CRISPR-Cas system were less likely to carry ABR genes than those lacking this defence system. Analysis of the mobile genetic elements (MGEs) targeted by CRISPR-Cas supports a model where this host defence system blocks important vectors of ABR. These results suggest a potential ‘immunocompromised’ state for multidrug-resistant strains that may be exploited in tailored interventions that rely on MGEs, such as phages or phagemids, to treat infections caused by bacterial pathogens. This article is part of the theme issue ‘The secret lives of microbial mobile genetic elements’.

We commonly acknowledge that bacterial viruses (phages) shape the composition and evolution of bacterial communities in nature and therefore have important roles in ecosystem functioning. This view stems from studies in the 1990s to the first decade of the twenty-first century that revealed high viral abundance, high viral diversity and virus-induced microbial death in aquatic ecosystems as well as an association between collapses in bacterial density and peaks in phage abundance. The recent surge in metagenomic analyses has provided deeper insight into the abundance, genomic diversity and spatio-temporal dynamics of phages in a wide variety of ecosystems, ranging from deep oceans to soil and the mammalian digestive tract. However, the causes and consequences of variations in phage community compositions remain poorly understood. In this Review, we explore current knowledge of the composition and evolution of phage communities, as well as their roles in controlling the population and evolutionary dynamics of bacterial communities. We discuss the need for greater ecological realism in laboratory studies to capture the complexity of microbial communities that thrive in natural environments.

CRISPR-Cas immune systems are widespread in bacteria and archaea, but not ubiquitous. Previous work has demonstrated that CRISPR immunity is associated with an infection-induced fitness cost, which may help explain the patchy distribution observed. However, the mechanistic basis of this cost has remained unclear. Using Pseudomonas aeruginosa PA14 and its phage DMS3vir as a model, we perform a 30-day evolution experiment under phage mediated selection. We demonstrate that although CRISPR is initially selected for, bacteria carrying mutations in the phage receptor rapidly invade the population following subsequent reinfections. We then test three potential mechanisms for the observed cost of CRISPR: (1) autoimmunity from the acquisition of self-targeting spacers, (2) immunopathology or energetic costs from increased cas gene expression and (3) toxicity caused by phage gene expression prior to CRISPR-mediated cleavage. We find that phages can express genes before the immune system clears the infection and that expression of these genes can have a negative effect on host fitness. While infection does not lead to increased expression of cas genes, it does cause differential expression of multiple other host processes that may further contribute to the cost of CRISPR immunity. In contrast, we found little support for infection-induced autoimmunological and immunopathological effects. Phage gene expression prior to cleavage of the genome by the CRISPR-Cas immune system is therefore the most parsimonious explanation for the observed phage-induced fitness cost.

Spatial resource heterogeneity is expected to be a key driver for the evolution of diversity. However, direct empirical support for this prediction is limited to studies carried out in simplified laboratory environments. Here, we investigate how altering spatial heterogeneity of potting compost—by the addition of water and mixing—affects the evolutionary diversification of a bacterial species, Pseudomonas fluorescens, that is naturally found in the environment. There was a greater propensity of resource specialists to evolve in the unmanipulated compost, while more generalist phenotypes dominated the compost–water mix. Genomic data were consistent with these phenotypic findings. Competition experiments strongly suggest these results are due to diversifying selection as a result of resource heterogeneity, as opposed to other covariables. Overall, our findings corroborate theoretical and in vitro findings, but in semi‐natural, more realistic conditions.

Host behavioural manipulation is a common strategy used by parasites to enhance their survival and/or transmission. Baculoviruses induce hyperactivity and tree-top disease (pre-death climbing behaviour) in their caterpillar hosts. However, little is known about the underlying mechanisms of this behavioural manipulation. A previous study showed that the baculovirus Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) induced tree-top disease at 3 days post infection in third instar S. exigua larvae and that light plays a key role in triggering this behaviour. Here we investigated the temporal requirements for the presence of light to trigger this behaviour and found that light from above was needed between 43 and 50 h post infection to induce tree-top disease. Infected larvae that were not exposed to light from above in this period finally died at low positions. Exposure to light prior to this period did not affect the final positions where larvae died. Overall we conclude that light in a particular time frame is needed to trigger SeMNPV-induced tree-top disease in S. exigua larvae.

Phages play a major role in shaping the composition, evolution and function of bacterial communities. While bacteria and phages coexist in many natural environments, their coexistence is often short-lived in the lab due to the evolution of phage resistance. However, fitness costs associated with resistance and mutational loss of resistance alleles may limit the durability of acquired resistances, potentially allowing phages to re-invade the population. Here, we explore this idea in the context of bacteria that evolve CRISPR-based immunity against their phages. Consistent with previous studies, we found that the bacterium Pseudomonas aeruginosa PA14 evolved high levels of CRISPR-based immunity and low levels of surface-based resistance following infection with phage DMS3vir, which led to rapid phage extinction. However, when these pre-immunized bacterial populations were subsequently challenged with the same phage, they failed to clear the infection and instead stably coexisted with the phage. Analysis of bacterial genotypes and phenotypes over time explained why CRISPR-Cas immunity provides only a transient advantage: in the absence of phage (i.e. following the initial phage extinction) formerly CRISPR-immune bacteria regain sensitivity due to evolutionary loss of spacers, whereas in the presence of phage (i.e. upon reinfection) selection favours surface-based resistance over CRISPR immunity. The latter results from an infection-induced fitness cost of CRISPR-immunity that is due to phage gene expression prior to target DNA cleavage by the immune system. Together, these results show that CRISPR-Cas immune systems provide only a transient benefit to bacteria upon phage infection and help to explain why bacteria and phages can coexist in natural environments even when bacteria carry CRISPR-Cas adaptive immune systems that allow for rapid acquisition of immunity against phages.

Bacteriophages encoding anti-CRISPR proteins (Acrs) must cooperate to overcome phage resistance mediated by the bacterial immune system CRISPR-Cas, where the first phage blocks CRISPR-Cas immunity in order to allow a second Acr phage to successfully replicate. However, in nature, bacteria are frequently not pre-immunized, and phage populations are often not clonal, exhibiting variations in Acr presence and strength. We explored how interactions between Acr phages and initially sensitive bacteria evolve, both in the presence and absence of competing phages lacking Acrs. We find that Acr phages benefit "Acr-negative" phages by limiting the evolution of CRISPR-based resistance and helping Acr-negative phages to replicate on resistant host sub-populations. These benefits depend on the strength of CRISPR-Cas inhibitors and result in strong Acrs providing smaller fitness advantages than weaker ones when Acr phages compete with Acr-negative phages. These results indicate that different Acr types shape the evolutionary dynamics and social interactions of phage populations in natural communities.

CRISPR-Cas immune systems are present in around half of bacterial genomes. Given the specificity and adaptability of this immune mechanism, it is perhaps surprising that they are not more widespread. Recent insights into the requirement for specific host factors for the function of some CRISPR-Cas subtypes, as well as the negative epistasis between CRISPR-Cas and other host genes, have shed light on potential reasons for the partial distribution of this immune strategy in bacteria. In this study, we examined how mutations in the bacterial mismatch repair system, which are frequently observed in natural and clinical isolates and cause elevated host mutation rates, influence the evolution of CRISPR-Cas–mediated immunity. We found that hosts with a high mutation rate very rarely evolved CRISPR-based immunity to phage compared to wild-type hosts. We explored the reason for this effect and found that the higher frequency at which surface mutants pre-exist in the mutator host background causes them to rapidly become the dominant phenotype under phage infection. These findings suggest that natural variation in bacterial mutation rates may, therefore, influence the distribution of CRISPR-Cas adaptive immune systems. This article is part of a discussion meeting issue ‘The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems’.

Over the past decade, the field of CRISPR-Cas research has received a lot of attention from the scientific community. While initially, this mostly concerned microbiologists who were fascinated by the discovery that some bacteria encode RNA-guided adaptive immune systems, this rapidly spread to other scientific disciplines following the development of groundbreaking molecular biology tools [1] and, more recently, to the public domain where the societal and ethical implications and legislation surrounding CRISPR applications are being debated. Some of the potential CRISPR applications that are currently being explored in the laboratory would involve the release of CRISPR genes into confined or open environments—for example, when CRISPR would be used to protect focal bacterial species against phage infections, when it is applied to suppress the spread of antimicrobial resistance or to control vectors of disease [2–4]. One component of the debate surrounding the societal impacts of these applications entails an assessment of the potential risks associated with these strategies (e.g. [5–7]), which requires an understanding of how CRISPR-Cas behaves in an ecological context. In this special issue, we explore this question by examining the evolutionary history of CRISPR-Cas immune systems, where they occur naturally, when they evolve and how this impacts the spread and evolution of other DNA elements. Finally, we return to the question how CRISPR-Cas may be exploited in an ecological context for the benefit of human health, and the ethical challenges that are associated with this. (a) CRISPR-Cas adaptive immune systems: a brief overview CRISPR-Cas adaptive immune systems were discovered around 15 years ago [8–11] and are estimated to exist in approximately 50% of all bacterial genomes and roughly 90% of all archaeal genomes [12]. A CRISPR immunity phenotype is genetically encoded by a so-called CRISPR locus (clustered regularly interspaced short palindromic repeats)—an array of repetitive and unique sequences (repeats and spacers, respectively), both of which are typically around 30 nt in length. Spacers are derived from (foreign) genetic elements, such as plasmids and viruses, and provide immunity to re-infection based on recognition of the cognate sequence (known as ‘protospacer’) [13–15]. Bacteria or archaea may carry a single linked array of spacers interspersed with repeats (one CRISPR locus) or multiple loci. CRISPR loci can evolve very rapidly owing to insertion of new spacers and the occasional loss of spacers or deletion of the CRISPR locus itself, which can cause very closely related strains to carry unique combinations of spacer sequences, known as a CRISPR allele. The overall length of CRISPR loci will increase and decrease with the acquisition and loss of spacers, and can vary from as little as a single spacer flanked by two repeats to hundreds of spacers and repeats [16]. As new spacers are added at the so-called leader-end of the locus, which is the sequence that contains the CRISPR promoter, CRISPR loci form an inverse chronological record of previous infections from the leader to the trailer end of the locus [17]. The extent to which different strains share the same spacer sequences in the same order (usually at the trailer end of the CRISPR locus) is commonly used to define related allele groups as a measure of their evolutionary relatedness. Apart from the genetic CRISPR memory, a functional CRISPR-Cas immune system also requires a set of CRISPR-associated genes (cas genes), which encode the protein machinery required for carrying out the immune response [18]. Cas operons vary in their cas gene composition and gene synteny, resulting in a classification of CRISPR-Cas systems into two classes, six types and 33 subtypes [19–22]. These diverse CRISPR-Cas variants differ in many of their mechanistic details, which have been discussed elsewhere [23,24], yet also have commonalities in the basic steps of the immune pathway. For example, two Cas proteins—Cas1 and Cas2—are almost invariably part of CRISPR-Cas immune systems and are responsible for inserting new spacer sequences into CRISPR arrays, sometimes assisted by other Cas proteins (reviewed in [15,25]). CRISPR transcripts are processed by either Cas proteins or housekeeping RNases [26], and the resulting processed CRISPR RNAs (crRNAs) are bound by Cas proteins to form a ribonucleoprotein complex that serves to detect and cleave complementary nucleic acid sequences [23].

Many bacteria encode CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats; CRISPR-associated) adaptive immune systems to protect themselves against their viruses (phages)1. To overcome resistance, phages have evolved anti-CRISPR proteins (Acr), which naturally vary in their potency to suppress the host immune system and avoid phage extinction2,3,4,5. However, these Acr-phages need to cooperate in order to overcome CRISPR-based resistance4,5: while many initial infections by Acr-phages are unsuccessful, they nonetheless lead to the production of Acr proteins, which generate immunosuppressed cells that can be successfully exploited by other Acr-phages in the population4,5. Here we test the prediction that phages lacking acr genes (Acr-negative phages) may exploit this cooperative behaviour6. We demonstrate that Acr-negative phages can indeed benefit from the presence of Acr-positive phages during pairwise competitions, but the extent of this exploitation depends on the potency of the Acr protein. Specifically, “strong” Acr proteins are more exploitable and benefit both phage types, whereas “weak” Acr proteins predominantly benefit Acr-positive phages only and therefore provide a greater fitness advantage during competition with Acr-negative phages. This work further helps to explain what defines the strength of an Acr protein, how selection acts on different Acr types in a phage community context, and how this can shape the dynamics of phage populations in natural communities.

Of the numerous approaches to counteracting the spread of antimicrobial resistance (AMR), one that is receiving increased attention is the use of CRISPR-Cas-based gene editing technology to remove resistance-conferring sequences from bacteria. Proof-of-principle studies have shown that CRISPR-Cas is able to successfully remove AMR genes from monocultures or very simple bacterial communities in the laboratory, but these constructs and their delivery must be adapted to be used in real-world medical settings. One area where such a technology may be implemented is tackling healthcare-associated infections. Development of a CRISPR-Cas9 cassette that is able to target particular resistance genes would be a powerful tool in addressing these infections. CRISPR-Cas9 will therefore be implemented to target AMR genes in clinical isolates of vancomycin-resistant Enterococcus faecium, and extended-spectrum β-lactamase (CTX-M) or carbapenemase (Oxa-48)-producing Klebsiella pneumoniae and Escherichia coli, by utilising a broad host range conjugative plasmid to deliver this construct via conjugation. Preliminary work using E. coli MG1655 as plasmid donor have shown that all strains can receive the plasmid, and there is significant variation in conjugation frequency between E. coli recipients.

Antimicrobial resistance (AMR) is a growing threat to healthcare. Many AMR-encoding genes are carried on accessory genetic elements like plasmids, which spread between phylogenetically distant members of bacterial communities. CRISPR-Cas based technologies may help combat the spread of AMR genes by removal of such plasmids. To optimise this technology, implementation of a delivery method which can reach a diverse range of bacteria is needed. Therefore, we engineered broad host-range conjugative plasmid pKJK5 to express Cas9. pKJK5::Cas[GmR] encodes a guide RNA which targets Gentamicin resistance gene aacC1, while pKJK5::Cas[nt] encodes a non-targeting guide RNA. After confirming its Ca9 activity by electroporation of targeted and untargeted plasmids, we set up a proof-of-concept experiment to conjugatively deliver pKJK5::Cas from a donor Escherichia coli strain DH5α to unrelated recipient E. coli K12, and to remove Gentamicin-resistance encoding target plasmid pHERD30T. For these means, we mixed donors and recipients in liquid media and incubated them overnight; proportions of the total population were enumerated by differential plating. We found that Gentamicin-resistant recipients were reduced by 33 % when treated with pKJK5::Cas[GmR] compared to treatment with pKJK5::Cas[nt], indicating that pKJK5::Cas[GmR] has the ability to remove AMR plasmids from recipient cells. This proof-of-concept experiment shows how an engineered broad host-range conjugative plasmid is an effective means of removing AMR-encoding plasmids and may be a viable approach to remove resistance genes from complex bacterial communities. To make this method more effective, community experiments as well as optimisation of Cas9 activity are needed.

Primer sequences used for protospacer analysis. Annealing position indicates where the primer binds to the phage 2972 genome.

The immunity profile for each host after a 3 day coevolution experiment with DMS3vir in nutrient rich LB medium, showing the proportion of bacterial clones that evolved resistance by surface modification (sm) or CRISPR-Cas as well as those that did not evolve resistance (Sensitive). Error bars represent 95 % CI.

Supplementary Figures S1-S3

The emergence and re-emergence of pathogens remains a major public health concern. Unfortunately, when and where pathogens will (re-)emerge is notoriously difficult to predict, as the erratic nature of those events is reinforced by the stochastic nature of pathogen evolution during the early phase of an epidemic. For instance, mutations allowing pathogens to escape host resistance may boost pathogen spread and promote emergence. Yet, the ecological factors that govern such evolutionary emergence remain elusive because of the lack of ecological realism of current theoretical frameworks and the difficulty of experimentally testing their predictions. Here, we develop a theoretical model to explore the effects of the heterogeneity of the host population on the probability of pathogen emergence, with or without pathogen evolution. We show that evolutionary emergence and the spread of escape mutations in the pathogen population is more likely to occur when the host population contains an intermediate proportion of resistant hosts. We also show that the probability of pathogen emergence rapidly declines with the diversity of resistance in the host population. Experimental tests using lytic bacteriophages infecting their bacterial hosts containing Clustered Regularly Interspaced Short Palindromic Repeat and CRISPR-associated (CRISPR-Cas) immune defenses confirm these theoretical predictions. These results suggest effective strategies for cross-species spillover and for the management of emerging infectious diseases., Author summary The probability that an epidemic will break out is highly dependent on the ability of the pathogen to acquire new adaptive mutations and to induce evolutionary emergence. Forecasting pathogen emergence thus requires a good understanding of the interplay between the epidemiology and evolution taking place at the onset of an outbreak. Here, we provide a comprehensive theoretical framework to analyze the impact of host population heterogeneity on the probability of pathogen evolutionary emergence. We use this model to predict the impact of the fraction of susceptible hosts, the inoculum size of the pathogen, and the diversity of host resistance on pathogen emergence. Our experiments using lytic bacteriophages and CRISPR-resistant bacteria support our theoretical predictions and demonstrate that manipulating the diversity of resistance alleles in a host population may be an effective way to limit the emergence of new pathogens.

SummarySome phages encode anti-CRISPR (acr) genes, which antagonize bacterial CRISPR-Cas immune systems by binding components of its machinery, but it is less clear how deployment of these acr genes impacts phage replication and epidemiology. Here we demonstrate that bacteria with CRISPR-Cas resistance are still partially immune to Acr-encoding phage. As a consequence, Acr-phages often need to cooperate in order to overcome CRISPR resistance, with a first phage taking down the host CRISPR-Cas immune system to allow a second Acr- phage to successfully replicate. This cooperation leads to epidemiological tipping points in which the initial density of Acr-phage tips the balance from phage extinction to a phage epidemic. Furthermore, both higher levels of CRISPR-Cas immunity and weaker Acr activities shift the tipping points towards higher phage densities. Collectively these data help to understand how interactions between phage-encoded immune suppressors and the CRISPR systems they target shape bacteria-phage population dynamics.HighlightsBacteria with CRISPR immunity remain partially resistant to Acr-phageSequentially infecting Acr phages cooperate to overcome CRISPR resistanceAcr-phage epidemiology depends on the initial phage densityCRISPR resistant bacteria can drive Acr phages extincteTOC blurbSome phages encode Acr proteins that block bacterial CRISPR-Cas immune systems. Although CRISPR-Cas can clear the first infection, this Acr-phage still suppresses the host immune system, which can be exploited by other Acr-phages. This is critical for Acr-phage amplification, but this “cooperation” only works beyond a critical Acr-phage density threshold.

The family Baculoviridae encompasses a large number of invertebrate viruses, mainly infecting caterpillars of the order Lepidoptera. The baculovirus Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) induces physiological and behavioral changes in its host Spodoptera exigua, as well as immunological responses, which may affect virus transmission. Here we show that the SeMNPV-encoded protein tyrosine phosphatase 2 (PTP2) induces mild apoptosis in Spodoptera frugiperda (Sf) 21 cells upon transient expression. Transient expression of a catalytic-site mutant of ptp2 did not lead to apoptosis, indicating that the phosphatase activity of PTP2 is needed to induce apoptosis. We also found that the caspase level (indicator of apoptosis) was higher in cells transfected with the ptp2 gene than in cells transfected with the catalytic mutant. Adding a caspase inhibitor reduced the level of ptp2-induced apoptosis. Moreover, deletion of the ptp2 gene from the viral genome prevented the induction of apoptosis in S. exigua hemocytes. The virus titer and virulence indices (the viral infectivity and the time to death) were not affected by deletion of the ptp2 gene. However, the viral occlusion body yield from S. exigua larvae infected with the mutant virus lacking the ptp2 gene was much lower than the yield from larvae infected with the wild-type (WT) virus. We hypothesize that the observed pro-apoptotic effects of PTP2 are the result of PTP2-mediated immune suppression in larvae, which consequently leads to higher viral occlusion body yields.

Cystatin B (CSTB) is an anti-protease frequently mutated in progressive myoclonus epilepsy (EPM1), a devastating degenerative disease. This work shows that rat CSTB is an unstable protein that undergoes structural changes following the interaction with a chaperone, either prokaryotic or eukaryotic. Both the prokaryotic DnaK and eukaryotic HSP70 promote CSTB polymerization. Denaturated CSTB is polymerized by the chaperone alone. Native CSTB monomers are more stable than denatured monomers and require Cu(2+) for chaperone-dependent polymerization. Cu(2+) interacts with at least two conserved histidines, at positions 72 and 95 modifying the structure of native monomeric CSTB. Subsequently, CSTB becomes unstable and readily responds to the addition of DnaK or HSP70, generating polymers. This reaction depends strictly on the presence of this divalent metal ion and on the presence of one cysteine in the protein chain. The cysteine deletion mutant does not polymerize. We propose that Cu(2+) modifies the redox environment of the protein, allowing the oxidation of the cysteine residue of CSTB that triggers polymerization. These polymers are sensitive to reducing agents while polymers obtained from denatured CSTB monomers are DTT resistant. We propose that the Cu(2+)/HSP70 dependent polymers are physiological and functional in eukaryotic cells. Furthermore, while monomeric CSTB has anti-protease function, it seems likely that polymeric CSTB fulfils different function(s).

Parasitic infections are often followed by changes in host behaviour. Numerous and exquisite examples of such behavioural alterations are known, covering a broad spectrum of parasites and hosts. Most descriptions of such parasite-induced changes in host behaviour are observational reports, while experimentally confirmed examples of parasite genes inducing these changes are limited. In this study, we review changes in invertebrate host behaviour observed upon infection by parasites and discuss such changes in an evolutionary context. We then explore possible mechanisms involved in parasite-induced changes in host behaviour. Genes and pathways known to play a role in invertebrate behaviour are reviewed, and we hypothesize how parasites (may) affect these pathways. This review provides the state of the art in this exciting, interdisciplinary field by exploring possible pathways triggered in hosts, suggesting methodologies to unravel the molecular mechanisms that lead to changes in host behaviour.

Prokaryotic CRISPR-Cas adaptive immune systems insert spacers derived from viruses and other parasitic DNA elements into CRISPR loci to provide sequence-specific immunity1,2. This frequently results in high within-population spacer diversity3,4,5,6, but it is unclear if and why this is important. Here we show that, as a result of this spacer diversity, viruses can no longer evolve to overcome CRISPR-Cas by point mutation, which results in rapid virus extinction. This effect arises from synergy between spacer diversity and the high specificity of infection, which greatly increases overall population resistance. We propose that the resulting short-lived nature of CRISPR-dependent bacteria–virus coevolution has provided strong selection for the evolution of sophisticated virus-encoded anti-CRISPR mechanisms7.

Some phages encode anti-CRISPR (acr) genes, which antagonize bacterial CRISPR-Cas immune systems by binding components of its machinery, but it is less clear how deployment of these acr genes impacts phage replication and epidemiology. Here, we demonstrate that bacteria with CRISPR-Cas resistance are still partially immune to Acr-encoding phage. As a consequence, Acr-phages often need to cooperate in order to overcome CRISPR resistance, with a first phage blocking the host CRISPR-Cas immune system to allow a second Acr-phage to successfully replicate. This cooperation leads to epidemiological tipping points in which the initial density of Acr-phage tips the balance from phage extinction to a phage epidemic. Furthermore, both higher levels of CRISPR-Cas immunity and weaker Acr activities shift the tipping points toward higher initial phage densities. Collectively, these data help elucidate how interactions between phage-encoded immune suppressors and the CRISPR systems they target shape bacteria-phage population dynamics.

CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems are prokaryotic adaptive immune systems that provide protection against infection by parasitic mobile genetic elements, such as viruses and plasmids. CRISPR-Cas systems are found in approximately half of all sequenced bacterial genomes and in nearly all archaeal genomes. In this review, we summarize our current understanding of the evolutionary ecology of CRISPR-Cas systems, highlight their value as model systems to answer fundamental questions concerning host–parasite coevolution, and explain how CRISPR-Cas systems can be useful tools for scientists across virtually all disciplines.

Migration of hosts and parasites can have a profound impact on host–parasite ecological and evolutionary interactions. Using the bacterium Pseudomonas aeruginosa UCBPP-PA14 and its phage DMS3vir, we here show that immigration of naive hosts into coevolving populations of hosts and parasites can influence the mechanistic basis underlying host defence evolution. Specifically, we found that at high levels of bacterial immigration, bacteria switched from clustered regularly interspaced short palindromic repeats (CRISPR-Cas) to surface modification-mediated defence. This effect emerges from an increase in the force of infection, which tips the balance from CRISPR to surface modification-based defence owing to the induced and fixed fitness costs associated with these mechanisms, respectively.

Many parasites manipulate host behaviour to enhance parasite transmission and survival. A fascinating example is baculoviruses, which often induce death in caterpillar hosts at elevated positions ('tree-top' disease). To date, little is known about the underlying processes leading to this adaptive host manipulation. Here, we show that the baculovirus Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) triggers a positive phototactic response in S. exigua larvae prior to death and causes the caterpillars to die at elevated positions. This light-dependent climbing behaviour is specific for infected larvae, as movement of uninfected caterpillars during larval development was light-independent. We hypothesize that upon infection, SeMNPV captures a host pathway involved in phototaxis and/or light perception to induce this remarkable behavioural change.

Increasing evidence shows that host behaviour often changes following infection by a variety of parasites, including viruses. The altered behaviour is either induced by the parasites to enhance parasite survival and transmission, or is a response of the host to avoid spread of infection in the host population. Given the high prevalence of viruses among insects, in a virus-host interaction or in a virus-vector relationship, viruses might have a huge impact on insect behaviour. This review first describes known examples of changes in insect behaviour upon virus infection. Although scarce, any known information on the underlying mechanism is also included. Special attention is given to baculoviruses and the hyperactivity and tree-top disease that they induce in their caterpillar hosts, so far the best studied systems in this research field. Subsequently, we discuss the virus-induced changes in insect behaviour from an ecological and evolutionary point of view.

Many parasites alter host behaviour to enhance their chance of transmission. Recently, the ecdysteroid UDP-glucosyl transferase (egt) gene from the baculovirus Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV) was identified to induce tree-top disease in L. dispar larvae. Infected gypsy moth larvae died at elevated positions (hence the term tree-top disease), which is thought to promote dissemination of the virus to lower foliage. It is, however, unknown whether egt has a conserved role among baculoviruses in inducing tree-top disease. Here, we studied tree-top disease induced by the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) in two different host insects, Trichoplusia ni and Spodoptera exigua, and we investigated the role of the viral egt gene therein. AcMNPV induced tree-top disease in both T. ni and S. exigua larvae, although in S. exigua a moulting-dependent effect was seen. Those S. exigua larvae undergoing a larval moult during the infection process died at elevated positions, while larvae that did not moult after infection died at low positions. For both T. ni and S. exigua, infection with a mutant AcMNPV lacking egt did not change the position where the larvae died. We conclude that egt has no highly conserved role in inducing tree-top disease in lepidopteran larvae. The conclusion that egt is a 'gene for an extended phenotype' is therefore not generally applicable for all baculovirus-host interactions. We hypothesize that in some baculovirus-host systems (including LdMNPV in L. dispar), an effect of egt on tree-top disease can be observed through indirect effects of egt on moulting-related climbing behaviour.

Although many parasites are known to manipulate the behavior of their hosts, the mechanisms underlying such manipulations are largely unknown. Baculoviruses manipulate the behavior of caterpillar hosts by inducing hyperactivity and by inducing climbing behavior leading to death at elevated positions (tree-top disease or Wipfelkrankheit). Whether hyperactivity and tree-top disease are independent manipulative strategies of the virus is unclear. Recently, we demonstrated the involvement of the protein tyrosine phosphatase (ptp) gene of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) in the induction of hyperactivity in Spodoptera exigua larvae. Here we show that AcMNPV ptp is not required for tree-top disease, indicating that in S. exigua baculovirus-induced hyperactivity and tree-top disease are independently induced behaviors that are governed by distinct mechanisms.

Many parasites manipulate host behaviour to enhance parasite transmission and survival. A fascinating example is baculoviruses, which often induce death in caterpillar hosts at elevated positions (‘tree-top’ disease). To date, little is known about the underlying processes leading to this adaptive host manipulation. Here, we show that the baculovirus Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) triggers a positive phototactic response in S. exigua larvae prior to death and causes the caterpillars to die at elevated positions. This light-dependent climbing behaviour is specific for infected larvae, as movement of uninfected caterpillars during larval development was light-independent. We hypothesize that upon infection, SeMNPV captures a host pathway involved in phototaxis and/or light perception to induce this remarkable behavioural change.

SUMMARY Bacteria have a range of distinct immune strategies that provide protection against bacteriophage (phage) infections. While much has been learned about the mechanism of action of these defense strategies, it is less clear why such diversity in defense strategies has evolved. In this review, we discuss the short- and long-term costs and benefits of the different resistance strategies and, hence, the ecological conditions that are likely to favor the different strategies alone and in combination. Finally, we discuss some of the broader consequences, beyond resistance to phage and other genetic elements, resulting from the operation of different immune strategies.

SummaryIn the face of infectious disease, organisms evolved a range of defense mechanisms, with a clear distinction between those that are constitutive (always active) and those that are inducible (elicited by parasites) [1]. Both defense strategies have evolved from each other [2], but we lack an understanding of the conditions that favor one strategy over the other. While it is hard to generalize about their degree of protection, it is possible to make generalizations about their associated fitness costs, which are commonly detected [3–5]. By definition, constitutive defenses are always “on,” and are therefore associated with a fixed cost, independent of parasite exposure [4, 5]. Inducible defenses, on the other hand, may lack costs in the absence of parasites but become costly when defense is elicited [6] through processes such as immunopathology [7]. Bacteria can evolve constitutive defense against phage by modification/masking of surface receptors [8, 9], which is often associated with reduced fitness in the absence of phage [10]. Bacteria can also evolve inducible defense using the CRISPR-Cas (clustered regularly interspaced short palindromic repeat, CRISPR associated) immune system [11], which is typically elicited upon infection [12–14]. CRISPR-Cas functions by integrating phage sequences into CRISPR loci on the host genome [15]. Upon re-infection, CRISPR transcripts guide cleavage of phage genomes [16–20]. In nature, both mechanisms are important [21, 22]. Using a general theoretical model and experimental evolution, we tease apart the mechanism that drives their evolution and show that infection risk determines the relative investment in the two arms of defense.