In Vivo Microbial Coevolution Favors Host Protection and Plastic Downregulation of Immunity

Microbiota can protect their hosts from infection. The short timescales in which microbes can evolve presents the possibility that “protective microbes” can take-over from the immune system of longer-lived hosts in the coevolutionary race against pathogens. Here, we found that coevolution between a protective bacterium (Enterococcus faecalis) and a virulent pathogen (Staphylococcus aureus) within an animal population (Caenorhabditis elegans) resulted in more disease suppression than when the protective bacterium adapted to uninfected hosts. At the same time, more protective E. faecalis populations became costlier to harbor and altered the expression of 134 host genes. Many of these genes appear to be related to the mechanism of protection, reactive oxygen species production. Crucially, more protective E. faecalis populations downregulated a key immune gene, , known to be effective against S. aureus infection. These results suggest that a microbial line of defense is favored by microbial coevolution and may cause hosts to plastically divest of their own immunity.     

Evolution and maintenance of microbe‐mediated protection under occasional pathogen infection

Every host is colonized by a variety of microbes, some of which can protect their hosts from pathogen infection. However, pathogen presence naturally varies over time in nature, such as in the case of seasonal epidemics. We experimentally coevolved populations of Caenorhabditis elegans worm hosts with bacteria possessing protective traits (Enterococcus faecalis), in treatments varying the infection frequency with pathogenic Staphylococcus aureus every host generation, alternating host generations, every fifth host generation, or never. We additionally investigated the effect of initial pathogen presence at the formation of the defensive symbiosis. Our results show that enhanced microbe‐mediated protection evolved during host‐protective microbe coevolution when faced with rare infections by a pathogen. Initial pathogen presence had no effect on the evolutionary outcome of microbe‐mediated protection. We also found that protection was only effective at preventing mortality during the time of pathogen infection. Overall, our results suggest that resident microbes can be a form of transgenerational immunity against rare pathogen infection.     

The Lysozyme-Induced Peptidoglycan N-Acetylglucosamine Deacetylase PgdA (EF1843) Is Required for Enterococcus faecalis Virulence

Lysozyme is a key component of the innate immune response in humans that provides a first line of defense against microbes. The bactericidal effect of lysozyme relies both on the cell wall lytic activity of this enzyme and on a cationic antimicrobial peptide activity that leads to membrane permeabilization. Among Gram-positive bacteria, the opportunistic pathogen Enterococcus faecalis has been shown to be extremely resistant to lysozyme. This unusual resistance is explained partly by peptidoglycan O-acetylation, which inhibits the enzymatic activity of lysozyme, and partly by d-alanylation of teichoic acids, which is likely to inhibit binding of lysozyme to the bacterial cell wall. Surprisingly, combined mutations abolishing both peptidoglycan O-acetylation and teichoic acid alanylation are not sufficient to confer lysozyme susceptibility. In this work, we identify another mechanism involved in E. faecalis lysozyme resistance. We show that exposure to lysozyme triggers the expression of EF1843, a protein that is not detected under normal growth conditions. Analysis of peptidoglycan structure from strains with EF1843 loss- and gain-of-function mutations, together with in vitro assays using recombinant protein, showed that EF1843 is a peptidoglycan N-acetylglucosamine deacetylase. EF1843-mediated peptidoglycan deacetylation was shown to contribute to lysozyme resistance by inhibiting both lysozyme enzymatic activity and, to a lesser extent, lysozyme cationic antimicrobial activity. Finally, EF1843 mutation was shown to reduce the ability of E. faecalis to cause lethality in the Galleria mellonella infection model. Taken together, our results reveal that peptidoglycan deacetylation is a component of the arsenal that enables E. faecalis to thrive inside mammalian hosts, as both a commensal and a pathogen.     

Host genotype and genetic diversity shape the evolution of a novel bacterial infection

Pathogens continue to emerge from increased contact with novel host species. Whilst these hosts can represent distinct environments for pathogens, the impacts of host genetic background on how a pathogen evolves post-emergence are unclear. In a novel interaction, we experimentally evolved a pathogen (Staphylococcus aureus) in populations of wild nematodes (Caenorhabditis elegans) to test whether host genotype and genetic diversity affect pathogen evolution. After ten rounds of selection, we found that pathogen virulence evolved to vary across host genotypes, with differences in host metal ion acquisition detected as a possible driver of increased host exploitation. Diverse host populations selected for the highest levels of pathogen virulence, but infectivity was constrained, unlike in host monocultures. We hypothesise that population heterogeneity might pool together individuals that contribute disproportionately to the spread of infection or to enhanced virulence. The genomes of evolved populations were sequenced, and it was revealed that pathogens selected in distantly-related host genotypes diverged more than those in closely-related host genotypes. S. aureus nevertheless maintained a broad host range. Our study provides unique empirical insight into the evolutionary dynamics that could occur in other novel infections of wildlife and humans.Subject terms: Molecular evolution, Bacterial evolution, Bacterial genetics     

Enemies make you stronger: Coevolution between fruit fly host and bacterial pathogen increases postinfection survivorship in the host

Multiple laboratory studies have evolved hosts against a nonevolving pathogen to address questions about evolution of immune responses. However, an ecologically more relevant scenario is one where hosts and pathogens can coevolve. Such coevolution between the antagonists, depending on the mutual selection pressure and additive variance in the respective populations, can potentially lead to a different pattern of evolution in the hosts compared to a situation where the host evolves against a nonevolving pathogen. In the present study, we used Drosophila melanogaster as the host and Pseudomonas entomophila as the pathogen. We let the host populations either evolve against a nonevolving pathogen or coevolve with the same pathogen. We found that the coevolving hosts on average evolved higher survivorship against the coevolving pathogen and ancestral (nonevolving) pathogen relative to the hosts evolving against a nonevolving pathogen. The coevolving pathogens evolved greater ability to induce host mortality even in nonlocal (novel) hosts compared to infection by an ancestral (nonevolving) pathogen. Thus, our results clearly show that the evolved traits in the host and the pathogen under coevolution can be different from one‐sided adaptation. In addition, our results also show that the coevolving host–pathogen interactions can involve certain general mechanisms in the pathogen, leading to increased mortality induction in nonlocal or novel hosts.     

Identification of Polyketide Inhibitors Targeting 3-Dehydroquinate Dehydratase in the Shikimate Pathway of Enterococcus faecalis

Due to the emergence of resistance toward current antibiotics, there is a pressing need to develop the next generation of antibiotics as therapeutics against infectious and opportunistic diseases of microbial origins. The shikimate pathway is exclusive to microbes, plants and fungi, and hence is an attractive and logical target for development of antimicrobial therapeutics. The Gram-positive commensal microbe, Enterococcus faecalis, is a major human pathogen associated with nosocomial infections and resistance to vancomycin, the “drug of last resort”. Here, we report the identification of several polyketide-based inhibitors against the E. faecalis shikimate pathway enzyme, 3-dehydroquinate dehydratase (DHQase). In particular, marein, a flavonoid polyketide, both inhibited DHQase and retarded the growth of Enterococcus faecalis. The purification, crystallization and structural resolution of recombinant DHQase from E. faecalis (at 2.2 Å resolution) are also reported. This study provides a route in the development of polyketide-based antimicrobial inhibitors targeting the shikimate pathway of the human pathogen E. faecalis.     

Eep Confers Lysozyme Resistance to Enterococcus faecalis via the Activation of the Extracytoplasmic Function Sigma Factor SigV

Enterococcus faecalis is a commensal bacterium found in the gastrointestinal tract of most mammals, including humans, and is one of the leading causes of nosocomial infections. One of the hallmarks of E. faecalis pathogenesis is its unusual ability to tolerate high concentrations of lysozyme, which is an important innate immune component of the host. Previous studies have shown that the presence of lysozyme leads to the activation of SigV, an extracytoplasmic function (ECF) sigma factor in E. faecalis, and that the deletion of sigV increases the susceptibility of the bacterium toward lysozyme. Here, we describe the contribution of Eep, a membrane-bound zinc metalloprotease, to the activation of SigV under lysozyme stress by its effects on the stability of the anti-sigma factor RsiV. We demonstrate that the Δeep mutant phenocopies the ΔsigV mutant in lysozyme, heat, ethanol, and acid stress susceptibility. We also show, using an immunoblot analysis, that in an eep deletion mutant, the anti-sigma factor RsiV is only partially degraded after lysozyme exposure, suggesting that RsiV is processed by unknown protease(s) prior to the action of Eep. An additional observation is that the deletion of rsiV, which results in constitutive SigV expression, leads to chaining of cells, suggesting that SigV might be involved in regulating cell wall-modifying enzymes important in cell wall turnover. We also demonstrate that, in the absence of eep or sigV, enterococci bind significantly more lysozyme, providing a plausible explanation for the increased sensitivity of these mutants toward lysozyme.     

Special Issue Oceans and Humans Health: The Ecology of Marine Opportunists

Opportunistic marine pathogens, like opportunistic terrestrial pathogens, are ubiquitous in the environment (waters, sediments, and organisms) and only cause disease in immune-compromised or stressed hosts. In this review, we discuss four host–pathogen interactions within the marine environment that are typically considered opportunistic: sea fan coral–fungus, eelgrass–Labyrinthula zosterae, sea fan–Labyrinthulomycetes, and hard clam–Quahog Parasite Unknown with particular focus on disease ecology, parasite pathology, host response, and known associated environmental conditions. Disease is a natural part of all ecosystems; however, in some cases, a shift in the balance between the host, pathogen, and the environment may lead to epizootics in natural or cultured populations. In marine systems, host–microbe interactions are less understood than their terrestrial counterparts. The biological and physical changes to the world’s oceans, coupled with other anthropogenic influences, will likely lead to more opportunistic diseases in the marine environment.     

Virus Satellites Drive Viral Evolution and Ecology

Virus satellites are widespread subcellular entities, present both in eukaryotic and in prokaryotic cells. Their modus vivendi involves parasitism of the life cycle of their inducing helper viruses, which assures their transmission to a new host. However, the evolutionary and ecological implications of satellites on helper viruses remain unclear. Here, using staphylococcal pathogenicity islands (SaPIs) as a model of virus satellites, we experimentally show that helper viruses rapidly evolve resistance to their virus satellites, preventing SaPI proliferation, and SaPIs in turn can readily evolve to overcome phage resistance. Genomic analyses of both these experimentally evolved strains as well as naturally occurring bacteriophages suggest that the SaPIs drive the coexistence of multiple alleles of the phage-coded SaPI inducing genes, as well as sometimes selecting for the absence of the SaPI depressing genes. We report similar (accidental) evolution of resistance to SaPIs in laboratory phages used for Staphylococcus aureus typing and also obtain the same qualitative results in both experimental evolution and phylogenetic studies of Enterococcus faecalis phages and their satellites viruses. In summary, our results suggest that helper and satellite viruses undergo rapid coevolution, which is likely to play a key role in the evolution and ecology of the viruses as well as their prokaryotic hosts.     

How Many Parameters Does It Take to Describe Disease Tolerance?

The study of infectious disease has been aided by model organisms, which have helped to elucidate molecular mechanisms and contributed to the development of new treatments; however, the lack of a conceptual framework for unifying findings across models, combined with host variability, has impeded progress and translation. Here, we fill this gap with a simple graphical and mathematical framework to study disease tolerance, the dose response curve relating health to microbe load; this approach helped uncover parameters that were previously overlooked. Using a model experimental system in which we challenged Drosophila melanogaster with the pathogen Listeria monocytogenes, we tested this framework, finding that microbe growth, the immune response, and disease tolerance were all well represented by sigmoid models. As we altered the system by varying host or pathogen genetics, disease tolerance varied, as we would expect if it was indeed governed by parameters controlling the sensitivity of the system (the number of bacteria required to trigger a response) and maximal effect size according to a logistic equation. Though either the pathogen or host immune response or both together could theoretically be the proximal cause of pathology that killed the flies, we found that the pathogen, but not the immune response, drove damage in this model. With this new understanding of the circuitry controlling disease tolerance, we can now propose better ways of choosing, combining, and developing treatments.     

Immunosuppressive effect of cyclosporin A on insect humoral immune response

Cyclosporin A suppressed humoral immune response of Galleria mellonella larvae. Insects were immunized with LPS Pseudomonas aeruginosa and then injected with cyclosporin A. Immunosuppressive effects were expressed both, in larvae treated with cyclosporin A at the initial phase of immune response and at the effector phase of antibacterial immunity. Cyclosporin A moderately decreased lysozyme activity and significantly decreased antibacterial activity peptides against Escherichia coli. Immunosuppressive effects of cyclosporin A were observed after immunoblotting with antibodies anti-G. mellonella lysozyme. Tricine SDS/PAGE shown that synthesis of antibacterial peptides of larvae treated with cyclosporin A was considerably inhibited. Insects of impaired immune response by cyclosporin A action lost protective immunity to insect bacterial pathogen P. aeruginosa.     

Unrestricted migration favours virulent pathogens in experimental metapopulations: evolutionary genetics of a rapacious life history

Understanding pathogen infectivity and virulence requires combining insights from epidemiology, ecology, evolution and genetics. Although theoretical work in these fields has identified population structure as important for pathogen life-history evolution, experimental tests are scarce. Here, we explore the impact of population structure on life-history evolution in phage T4, a viral pathogen of Escherichia coli. The host–pathogen system is propagated as a metapopulation in which migration between subpopulations is either spatially restricted or unrestricted. Restricted migration favours pathogens with low infectivity and low virulence. Unrestricted migration favours pathogens that enter and exit their hosts quickly, although they are less productive owing to rapid extirpation of the host population. The rise of such ‘rapacious’ phage produces a ‘tragedy of the commons’, in which better competitors lower productivity. We have now identified a genetic basis for a rapacious life history. Mutations at a single locus (rI) cause increased virulence and are sufficient to account for a negative relationship between phage competitive ability and productivity. A higher frequency of rI mutants under unrestricted migration signifies the evolution of rapaciousness in this treatment. Conversely, spatially restricted migration favours a more ‘prudent’ pathogen strategy, in which the tragedy of the commons is averted. As our results illustrate, profound epidemiological and ecological consequences of life-history evolution in a pathogen can have a simple genetic cause.     

A new role for an old antimicrobial: lysozyme c-1 can function to protect malaria parasites in Anopheles mosquitoes

Background

Plasmodium requires an obligatory life stage in its mosquito host. The parasites encounter a number of insults while journeying through this host and have developed mechanisms to avoid host defenses. Lysozymes are a family of important antimicrobial immune effectors produced by mosquitoes in response to microbial challenge.

Methodology/Principal Findings

A mosquito lysozyme was identified as a protective agonist for Plasmodium. Immunohistochemical analyses demonstrated that Anopheles gambiae lysozyme c-1 binds to oocysts of Plasmodium berghei and Plasmodium falciparum at 2 and 5 days after infection. Similar results were observed with Anopheles stephensi and P. falciparum, suggesting wide occurrence of this phenomenon across parasite and vector species. Lysozyme c-1 did not bind to cultured ookinetes nor did recombinant lysozyme c-1 affect ookinete viability. dsRNA-mediated silencing of LYSC-1 in Anopheles gambiae significantly reduced the intensity and the prevalence of Plasmodium berghei infection. We conclude that this host antibacterial protein directly interacts with and facilitates development of Plasmodium oocysts within the mosquito.

Conclusions/Significance

This work identifies mosquito lysozyme c-1 as a positive mediator of Plasmodium development as its reduction reduces parasite load in the mosquito host. These findings improve our understanding of parasite development and provide a novel target to interrupt parasite transmission to human hosts.     

Borrelia burgdorferi protein interactions critical for microbial persistence in mammals

Borrelia burgdorferi is the causative agent of Lyme disease that persists in a complex enzootic life cycle, involving Ixodes ticks and vertebrate hosts. The microbe invades ticks and vertebrate hosts in spite of active immune surveillance and potent microbicidal responses, and establishes long‐term infection utilising mechanisms that are yet to be unravelled. The pathogen can cause multi‐system disorders when transmitted to susceptible mammalian hosts, including in humans. In the past decades, several studies identified a limited number of B. burgdorferi gene‐products critical for pathogen persistence, transmission between the vectors and the host, and host–pathogen interactions. This review will focus on the interactions between B. burgdorferi proteins, as well as between microbial proteins and host components, protein and non‐protein components, highlighting their roles in pathogen persistence in the mammalian host. A better understanding of the contributions of protein interactions in the microbial virulence and persistence of B. burgdorferi would support development of novel therapeutics against the infection.