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.

AbstractIt 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.

AbstractThe acquisition of antibiotic resistance genes via horizontal gene transfer 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 antibiotic resistance. 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 antibiotic resistance. We analysed ~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 horizontal gene transfer, and found that pathogens with a CRISPR-Cas system were less likely to carry antibiotic resistance genes than those lacking this defence system. Analysis of the mobile genetic elements targeted by CRISPR-Cas supports a model where this host defence system blocks important vectors of antibiotic resistance. These results suggest a potential “immunocompromised” state for multidrug-resistant strains that may be exploited in tailored interventions that rely on mobile genetic elements, such as phage or phagemids, to treat infections caused by bacterial pathogens.

Widespread antibiotic resistance in commensal bacteria creates a persistent challenge for human health. Resident drug-resistant microbes can prevent clinical interventions, colonize wounds post-surgery, pass resistance traits to pathogens or move to more harmful niches following routine interventions such as catheterization. Accelerating the removal of resistant bacteria or actively decolonizing particular lineages from hosts could therefore have a number of long-term benefits. However, removing resident bacteria via competition with probiotics, for example, poses a number of ecological challenges. Resident microbes are likely to have physiological and numerical advantages and competition based on bacteriocins or other secreted antagonists is expected to give advantages to the dominant partner, via positive frequency dependence. Since a narrow range of E. coli genotypes (primarily those belonging to the clonal group ST131) cause a significant proportion of multi-drug resistant infections, this group presents a promising target for decolonization with bacteriophage, as narrow host range viral predation could lead to selective removal of particular genotypes. In this study we tested how a combination of a ST131-specific phage and competition from the well-known probiotic E. coli Nissle strain could displace E. coli ST131 under aerobic and anaerobic growth conditions in vitro. We showed that the addition of phage could break the frequency-dependent advantage of a numerically dominant ST131 isolate. Moreover, the addition of competing E. coli Nissle could improve the ability of phage to suppress ST131 by two orders of magnitude. Low-cost phage resistance evolved readily in these experiments and was not inhibited by the presence of a probiotic competitor. Nevertheless, combinations of phage and probiotic produced stable long-term suppression of ST131 over multiple transfers and under both aerobic and anaerobic growth conditions. Combinations of phage and probiotic therefore have real potential for accelerating the removal of drug resistant commensal targets.

AbstractDiversity in host resistance often associates with reduced pathogen spread. This may result from ecological and evolutionary processes, likely with feedback between them. Theory and experiments on bacteria-phage interactions have shown that genetic diversity of the bacterial adaptive immune system can limit phage evolution to overcome resistance. Using the CRISPR-Cas bacterial immune system and lytic phage, we engineered a host-pathogen system where each bacterial host genotype could be infected by only one phage genotype. With this model system, we explored how CRISPR diversity impacts the spread of phage when they can overcome a resistance allele, how immune diversity affects the evolution of the phage to increase its host range, and if there was feedback between these processes. We show that increasing CRISPR diversity benefits susceptible bacteria via a dilution effect, which limits the spread of the phage. We suggest that this ecological effect impacts the evolution of novel phage genotypes, which then feeds back into phage population dynamics.

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.

AbstractPlasmids 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.

AbstractUnderstanding plasmid transfer dynamics remains a key knowledge gap in the mitigation of antibiotic resistance gene spread. Direct effects of exposure to stressors on plasmid uptake are well monitored. However, it remains untested whether evolution of strains under stress conditions modulates subsequent plasmid uptake. Here, we evolved a compost derived microbial community for six weeks under copper stress and non-exposed control conditions. We then tested the ability of isolated clones from both treatments to take up the broad host range plasmid pKJK5 from anE.colidonor strain. Clones pre-adapted to copper displayed a significantly increased probability to be permissive towards the plasmid compared to those isolated from the control treatment. Further, increased phylogenetic distance to the donor strain was significantly and negatively correlated with plasmid uptake probabilities across both treatments.

AbstractE. coliis a highly diverse bacterial species that generates a huge global burden of antimicrobial-resistant infections. A wealth of whole genome sequence data is available on public databases for this species, presenting new opportunities to analyse the distribution of antimicrobial resistance (AMR) genes across its genetic and ecological diversity. We extracted and categorised metadata on host species and geographic location and combined this within silicophylogrouping to describe the characteristics of ∼16,000 assembledE. coligenomes from the NCBI RefSeq database. We estimated AMR carriage using various metrics: counts of overall genes, multidrug- and extensively drug-resistant categories, and selected β-lactamases of current global concern –blaCTX-Mand carbapenemase genes. We present estimates of AMR carriage for these metrics by species type (human, agricultural/domestic animal, wild birds and other wild animals), geographic subregion, and across phylogroups. In addition, we describe the distribution of phylogroups within host types and geographic subregions. Our findings show high AMR carriage in commensal-associated phylogroups, agricultural and wild animal hosts and in many subregions. However, we also quantify large biases in sequencing data, the substantial gaps in our knowledge of AMR in many hosts, regions and environmental settings, and the need for systematic sampling to gain a more accurate picture.

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-defense proteins. Specifically, using Pseudomonas aeruginosa PA14 and phage DMS3vir as a model, we show that bacteria with Clustered Regularly Interspaced Short Palindromic Repeat, CRISPR associated (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 defense 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 the 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.

Bacteria have a broad array of defence mechanisms to fight bacteria-specific viruses (bacteriophages, phages) and other invading mobile genetic elements. Among those mechanisms, the 'CRISPR-Cas' (Clustered Regularly Interspaced Short Palindromic Repeats - CRISPR-associated) system keeps record of previous infections to prevent re-infection and thus provides acquired immunity. However, phages are not defenceless against CRISPR-based bacterial immunity. Indeed, they can escape CRISPR systems by encoding one or several anti-CRISPR (Acr) proteins. Acr proteins are among the earliest proteins produced upon phage infection, as they need to quickly inhibit CRISPR-Cas system before it can destroy phage genetic material. As a result, Acrs do not perfectly protect phage from the CRISPR-Cas system, and infection often fails. However, even if the infection fails, Acr can induce a lasting inactivation of the CRISPR-Cas system. The method presented here aims to assess the lasting CRISPR-Cas inhibition in Pseudomonas aeruginosa induced by Acr proteins by:•Infecting the P. aeruginosa strain with a phage carrying an acr gene.•Making the cell electrocompetent while eliminating the phage•Transforming the cells with a plasmid targeted by the CRISPR-Cas system and a non-targeted one to measure the relative transformation efficiency of the plasmids. This method can be adapted to measure which parameters influence Acr-induced immunosuppression in different culture conditions.

CRISPR-Cas are prokaryotic defence systems that provide protection against invasion by mobile genetic elements (MGE), including bacteriophages. MGE can overcome CRISPR-Cas defences by encoding anti-CRISPR (Acr) proteins. These proteins are produced in the early stages of the infection and inhibit the CRISPR-Cas machinery to allow phage replication. While research on Acr has mainly focused on their discovery, structure and mode of action, and their applications in biotechnology, the impact of Acr on the ecology of MGE as well as on the coevolution with their bacterial hosts only begins to be unravelled. In this review, we summarise our current understanding on the distribution of anti-CRISPR genes in MGE, the ecology of phages encoding Acr, and their coevolution with bacterial defence mechanisms. We highlight the need to use more diverse and complex experimental models to better understand the impact of anti-CRISPR in MGE-host interactions.

AbstractPlasmids 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 model system consisting of a synthetic and stable five-species community and a broad host-range plasmid, engineered to carry different payloads. 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 cost of a high-payload plasmid to a focal strain, and accordingly, plasmid loss from the focal species occurred over a shorter time frame. 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.

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.

Competitive bacteria-bacteriophage interactions have resulted in the evolution of a plethora of bacterial defense systems preventing phage propagation. In recent years, computational and bioinformatic approaches have underpinned the discovery of numerous novel bacterial defense systems. Anti-phage systems are frequently encoded together in genomic loci termed defense islands. Here we report the identification and characterisation of a novel anti-phage system, that we have termed Shield, which forms part of the Pseudomonas defensive arsenal. The Shield system comprises the core component ShdA, a membrane-bound protein harboring an RmuC domain. Heterologous production of ShdA alone is sufficient to mediate bacterial immunity against several phages. We demonstrate that Shield and ShdA confer population-level immunity and that they can also decrease transformation efficiency. We further show that ShdA homologues can degrade DNA in vitro and, when expressed in a heterologous host, can alter the organisation of the host chromosomal DNA. Use of comparative genomic approaches identified how Shield can be divided into four subtypes, three of which contain additional components that in some cases can negatively affect the activity of ShdA and/or provide additional lines of phage defense. Collectively, our results identify a new player within the Pseudomonas bacterial immunity arsenal that displays a novel mechanism of protection, and reveals a role for RmuC domains in phage defense.

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'.

CRISPR-Cas systems provide bacteria and archaea with adaptive, heritable immunity against their viruses (bacteriophages and phages) and other parasitic genetic elements. CRISPR-Cas systems are highly diverse, and we are only beginning to understand their relative importance in phage defense. In this review, we will discuss when and why CRISPR-Cas immunity against phages evolves, and how this, in turn, selects for the evolution of immune evasion by phages. Finally, we will discuss our current understanding of if, and when, we observe coevolution between CRISPR-Cas systems and phages, and how this may be influenced by the mechanism of CRISPR-Cas immunity.

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.

Diversity in host resistance often associates with reduced pathogen spread. This may result from ecological and evolutionary processes, likely with feedback between them. Theory and experiments on bacteria-phage interactions have shown that genetic diversity of the bacterial adaptive immune system can limit phage evolution to overcome resistance. Using the CRISPR-Cas bacterial immune system and lytic phage, we engineered a host-pathogen system where each bacterial host genotype could be infected by only one phage genotype. With this model system, we explored how CRISPR diversity impacts the spread of phage when they can overcome a resistance allele, how immune diversity affects the evolution of the phage to increase its host range and if there was feedback between these processes. We show that increasing CRISPR diversity benefits susceptible bacteria via a dilution effect, which limits the spread of the phage. We suggest that this ecological effect impacts the evolution of novel phage genotypes, which then feeds back into phage population dynamics.

AbstractCRISPR-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.

CRISPR-Cas systems provide adaptive immunity against mobile genetic elements, but employment of this resistance mechanism is often reported with a fitness cost for the host. Whether or not CRISPR-Cas systems are important barriers for the horizontal spread of conjugative plasmids, which play a crucial role in the spread of antibiotic resistance, will depend on the fitness costs of employing CRISPR-based defences and the benefits of resisting conjugative plasmids. To estimate these costs and benefits we measured bacterial fitness associated with plasmid immunity using Escherichia coli and the conjugative plasmid pOX38-Cm. We find that CRISPR-mediated immunity fails to confer a fitness benefit in the absence of antibiotics, despite the large fitness cost associated with carrying the plasmid in this context. Similar to many other conjugative plasmids, pOX38-Cm carries a CcdAB toxin-anti-toxin (TA) addiction system. These addiction systems encode long-lived toxins and short-lived anti-toxins, resulting in toxic effects following the loss of the TA genes from the bacterial host. Our data suggest that the lack of a fitness benefit associated with CRISPR-mediated defence is due to expression of the TA system before plasmid detection and degradation. As most antibiotic resistance plasmids encode TA systems this could have important consequences for the role of CRISPR-Cas systems in limiting the spread of antibiotic resistance.

CRISPR-Cas is an adaptive prokaryotic immune system that prevents phage infection. By incorporating phage-derived 'spacer' sequences into CRISPR loci on the host genome, future infections from the same phage genotype can be recognized and the phage genome cleaved. However, the phage can escape CRISPR degradation by mutating the sequence targeted by the spacer, allowing them to re-infect previously CRISPR-immune hosts, and theoretically leading to coevolution. Previous studies have shown that phage can persist over long periods in populations of Streptococcus thermophilus that can acquire CRISPR-Cas immunity, but it has remained less clear whether this coexistence was owing to coevolution, and if so, what type of coevolutionary dynamics were involved. In this study, we performed highly replicated serial transfer experiments over 30 days with S. thermophilus and a lytic phage. Using a combination of phenotypic and genotypic data, we show that CRISPR-mediated resistance and phage infectivity coevolved over time following an arms race dynamic, and that asymmetry between phage infectivity and host resistance within this system eventually causes phage extinction. This work provides further insight into the way CRISPR-Cas systems shape the population and coevolutionary dynamics of bacteria-phage interactions. This article is part of a discussion meeting issue 'The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems'.

Bacteriophage (phage), viruses of bacteria, are the most abundant and diverse biological entities on Earth. There are an estimated 10³⁰ phages in the biosphere; collectively they outnumber their prokaryotic hosts tenfold, and they are thought to destroy up to half of the World’s bacteria every 48 hours. Yet, despite the ubiquity of phage and the influence they exert over bacterial populations, we lack a detailed understanding of the complex bacteria-phage interactions that govern the coexistence of both species. Lytic phages – which require cell death to complete their life cycle – impose continuous selection pressures on bacteria to evolve resistance or face extinction. Likewise, increasing bacterial resistance creates strong selection pressures on phage to evolve greater infectivity. These reciprocal adaptations can lead to coevolution, which has profound consequences for the genetic diversity and evolutionary trajectories of both species.

One such phage-resistance mechanism in bacteria is CRISPR-Cas (clustered regularly interspaced short palindromic repeats - and associated Cas proteins); a heritable, adaptive immune system that is found in approximately half of all bacteria. CRISPR provides immunity against phages by incorporating phage-derived ‘spacer’ sequences into CRISPR loci on the bacterial genome. Transcriptions of these spacers can recognise and cleave complementary DNA from invading phage genotypes, but phage can ‘escape’ CRISPR by mutating the regions targeted by the spacers, enabling them to re-infect previously-resistant bacteria and potentially leading to coevolution. To date, very few CRISPR-phage systems have been studied in detail, but two of the best-known systems; Pseudomonas aeruginosa and Streptococcus thermophilus, show great variation in their efficacy at preventing phage infections. Whilst phage are rapidly driven extinct in P. aeruginosa, they can coexist over long periods in S. thermophilus; but it is unclear whether this phage persistence is due to coevolution, and if so, what kind of coevolutionary dynamics are associated with this system.

In this thesis, I examine P. aeruginosa and S. thermophilus together with their lytic phages in a coevolutionary context to better understand the role of coevolution in CRISPR-phage interactions. I first explain how the diversity of CRISPR spacers generated in P. aeruginosa rapidly drives phage extinct, but that phage can become locally adapted when CRISPR spacer diversity is low. I then present the first empirical evidence of CRISPR-phage coevolution in S. thermophilus and show that coevolution follows an arms race dynamic in this system. I conclude by discussing the importance of CRISPR spacer diversity in determining the outcome of CRISPR-phage interactions and how low spacer diversity can lead to coevolution and local adaptation.

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'.

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.

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.

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.

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.

Specificity in the interactions between hosts and their parasites can lead to local adaptation. However, the degree of local adaptation is predicted to depend upon the diversity of resistance alleles within the host population; increasing host diversity should decrease mean parasite infectivity and hence reduce local adaptation. In this study, we empirically test this prediction using the highly specific interactions between bacteria with clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) immunity and their bacteriophage. Bacteria acquire immunity to phage by incorporating a phage-derived spacer sequence into CRISPR loci on the host genome, and phage can escape the CRISPR-mediated immunity of a specific clone by mutating the targeted sequence. We found that high levels of CRISPR allele diversity that naturally evolve in host populations exposed to phage (because each bacterial clone captures a unique phage-derived sequence) prevents phage from becoming locally adapted. By manipulating the number of CRISPR alleles in the host population, we show that phage can become locally adapted to their bacterial hosts but only when CRISPR allele diversity is low.

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.

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.

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.

Prokaryotic CRISPR-Cas adaptive immune systems insert spacers derived from viruses and other parasitic DNA elements into CRISPR loci to provide sequence-specific immunity. This frequently results in high within-population spacer diversity, 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 mechanisms.

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.

Summary in 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.

Many parasites enhance their dispersal and transmission by manipulating host behaviour. One intriguing example concerns baculoviruses that induce hyperactivity and tree-top disease (i.e. climbing to elevated positions prior to death) in their caterpillar hosts. Little is known about the underlying mechanisms of such parasite-induced behavioural changes. Here, we studied the role of the ecdysteroid UDP-glucosyltransferase (egt) gene of Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) in tree-top disease in S. exigua larvae. Larvae infected with a mutant virus lacking the egt gene exhibited a shorter time to death and died before the induction of tree-top disease. Moreover, deletion of either the open reading frame or the ATG start codon of the egt gene prevented tree-top disease, indicating that the EGT protein is involved in this process. We hypothesize that SeMNPV EGT facilitates tree-top disease in S. exigua larvae by prolonging the larval time to death. Additionally, we discuss the role of egt in baculovirus-induced tree-top disease.

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.

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.

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.

Many parasites manipulate host behavior to increase the probability of transmission. To date, direct evidence for parasitic genes underlying such behavioral manipulations is scarce. Here we show that the baculovirus Autographa californica nuclear polyhedrovirus (AcMNPV) induces hyperactive behavior in Spodoptera exigua larvae at three days after infection. Furthermore, we identify the viral protein tyrosine phosphatase (ptp) gene as a key player in the induction of hyperactivity in larvae, and show that mutating the catalytic site of the encoded phosphatase enzyme prevents this induced behavior. Phylogenetic inference points at a lepidopteran origin of the ptp gene and shows that this gene is well-conserved in a group of related baculoviruses. Our study suggests that ptp-induced behavioral manipulation is an evolutionarily conserved strategy of this group of baculoviruses to enhance virus transmission, and represents an example of the extended phenotype concept. Overall, these data provide a firm base for a deeper understanding of the mechanisms behind baculovirus-induced insect behavior.