Low mutational load and high mutation rate variation in gut commensal bacteria.

Bacteria generally live in species-rich communities, such as the gut microbiota. Yet little is known about bacterial evolution in natural ecosystems. Here, we followed the long-term evolution of commensal Escherichia coli in the mouse gut. We observe the emergence of mutation rate polymorphism, ranging from wild-type levels to 1,000-fold higher. By combining experiments, whole-genome sequencing, and in silico simulations, we identify the molecular causes and explore the evolutionary conditions allowing these hypermutators to emerge and coexist within the microbiota. The hypermutator phenotype is caused by mutations in DNA polymerase III proofreading and catalytic subunits, which increase mutation rate by approximately 1,000-fold and stabilise hypermutator fitness, respectively. Strong mutation rate variation persists for >1,000 generations, with coexistence between lineages carrying 4 to >600 mutations. The in vivo molecular evolution pattern is consistent with fitness effects of deleterious mutations sd ≤ 10-4/generation, assuming a constant effect or exponentially distributed effects with a constant mean. Such effects are lower than typical in vitro estimates, leading to a low mutational load, an inference that is observed in in vivo and in vitro competitions. Despite large numbers of deleterious mutations, we identify multiple beneficial mutations that do not reach fixation over long periods of time. This indicates that the dynamics of beneficial mutations are not shaped by constant positive Darwinian selection but could be explained by other evolutionary mechanisms that maintain genetic diversity. Thus, microbial evolution in the gut is likely characterised by partial sweeps of beneficial mutations combined with hitchhiking of slightly deleterious mutations, which take a long time to be purged because they impose a low mutational load. The combination of these two processes could allow for the long-term maintenance of intraspecies genetic diversity, including mutation rate polymorphism. These results are consistent with the pattern of genetic polymorphism that is emerging from metagenomics studies of the human gut microbiota, suggesting that we have identified key evolutionary processes shaping the genetic composition of this community.

Radial Expansion Facilitates the Maintenance of Double Antibiotic Resistances.

Most microbes live in spatially confined subpopulations. Under spatial structure conditions, the efficacy of natural selection is often reduced (relative to homogeneous conditions) due to the increased importance of genetic drift and local competition. Additionally, under spatial structure conditions, the fittest genotype may not always be the one with better access to the heterogeneous distribution of nutrients. The effect of radial expansion may be particularly relevant for the elimination of antibiotic resistance mutations, as their dynamics within bacterial populations are strongly dependent on their growth rate. Here, we use Escherichia coli to systematically compare the allele frequency of streptomycin, rifampin, and fluoroquinolone single and double resistance mutants after 24 h of coexistence with a susceptible strain under radial expansion (local competition) and homogeneous (global competition) conditions. We show that there is a significant effect of structure on the maintenance of double resistances which is not observed for single resistances. Radial expansion also facilitates the persistence of double resistances when competing against their single counterparts. Importantly, we found that spatial structure reduces the rate of compensation of the double mutant RpsLK43T RpoBH526Y and that a strongly compensatory mutation in homogeneous conditions becomes deleterious under spatial structure conditions. Overall, our results unravel the importance of spatial structure for facilitating the maintenance and accumulation of multiple resistances over time and for determining the identity of compensatory mutations.

A Mutational Hotspot and Strong Selection Contribute to the Order of Mutations Selected for during Escherichia coli Adaptation to the Gut.

The relative role of drift versus selection underlying the evolution of bacterial species within the gut microbiota remains poorly understood. The large sizes of bacterial populations in this environment suggest that even adaptive mutations with weak effects, thought to be the most frequently occurring, could substantially contribute to a rapid pace of evolutionary change in the gut. We followed the emergence of intra-species diversity in a commensal Escherichia coli strain that previously acquired an adaptive mutation with strong effect during one week of colonization of the mouse gut. Following this first step, which consisted of inactivating a metabolic operon, one third of the subsequent adaptive mutations were found to have a selective effect as high as the first. Nevertheless, the order of the adaptive steps was strongly affected by a mutational hotspot with an exceptionally high mutation rate of 10-5. The pattern of polymorphism emerging in the populations evolving within different hosts was characterized by periodic selection, which reduced diversity, but also frequency-dependent selection, actively maintaining genetic diversity. Furthermore, the continuous emergence of similar phenotypes due to distinct mutations, known as clonal interference, was pervasive. Evolutionary change within the gut is therefore highly repeatable within and across hosts, with adaptive mutations of selection coefficients as strong as 12% accumulating without strong constraints on genetic background. In vivo competitive assays showed that one of the second steps (focA) exhibited positive epistasis with the first, while another (dcuB) exhibited negative epistasis. The data shows that strong effect adaptive mutations continuously recur in gut commensal bacterial species.

Recurrent Reverse Evolution Maintains Polymorphism after Strong Bottlenecks in Commensal Gut Bacteria.

The evolution of new strains within the gut ecosystem is poorly understood. We used a natural but controlled system to follow the emergence of intraspecies diversity of commensal Escherichia coli, during three rounds of adaptation to the mouse gut (∼1,300 generations). We previously showed that, in the first round, a strongly beneficial phenotype (loss-of-function for galactitol consumption; gat-negative) spread to >90% frequency in all colonized mice. Here, we show that this loss-of-function is repeatedly reversed when a gat-negative clone colonizes new mice. The regain of function occurs via compensatory mutation and reversion, the latter leaving no trace of past adaptation. We further show that loss-of-function adaptive mutants reevolve, after colonization with an evolved gat-positive clone. Thus, even under strong bottlenecks a regime of strong-mutation-strong-selection dominates adaptation. Coupling experiments and modeling, we establish that reverse evolution recurrently generates two coexisting phenotypes within the microbiota that can or not consume galactitol (gat-positive and gat-negative, respectively). Although the abundance of the dominant strain, the gat-negative, depends on the microbiota composition, gat-positive abundance is independent of the microbiota composition and can be precisely manipulated by supplementing the diet with galactitol. These results show that a specific diet is able to change the abundance of specific strains. Importantly, we find polymorphism for these phenotypes in indigenous Enterobacteria of mice and man. Our results demonstrate that natural selection can greatly overwhelm genetic drift at structuring the strain diversity of gut commensals and that competition for limiting resources may be a key mechanism for maintaining polymorphism in the gut.

Facilitation through altered resource availability in a mixed-species rodent malaria infection.

A major challenge in disease ecology is to understand how co-infecting parasite species interact. We manipulate in vivo resources and immunity to explain interactions between two rodent malaria parasites, Plasmodium chabaudi and P. yoelii. These species have analogous resource-use strategies to the human parasites Plasmodium falciparum and P. vivax: P. chabaudi and P. falciparum infect red blood cells (RBC) of all ages (RBC generalist); P. yoelii and P. vivax preferentially infect young RBCs (RBC specialist). We find that: (1) recent infection with the RBC generalist facilitates the RBC specialist (P. yoelii density is enhanced ~10 fold). This occurs because the RBC generalist increases availability of the RBC specialist's preferred resource; (2) co-infections with the RBC generalist and RBC specialist are highly virulent; (3) and the presence of an RBC generalist in a host population can increase the prevalence of an RBC specialist. Thus, we show that resources shape how parasite species interact and have epidemiological consequences.

Hybridization and pre-zygotic reproductive barriers in Plasmodium.

Sexual reproduction is an obligate step in the life cycle of many parasites, including the causative agents of malaria (Plasmodium). Mixed-species infections are common in nature and consequently, interactions between heterospecific gametes occur. Given the importance of managing gene flow across parasite populations, remarkably little is understood about how reproductive isolation between species is maintained. We use the rodent malaria parasites P. berghei and P. yoelii to investigate the ecology of mixed-species mating groups, identify proteins involved in pre-zygotic barriers, and examine their evolution. Specifically, we show that (i) hybridization occurs, but at low frequency; (ii) hybridization reaches high levels when female gametes lack the surface proteins P230 or P48/45, demonstrating that these proteins are key for pre-zygotic reproductive isolation; (iii) asymmetric reproductive interference occurs, where the fertility of P. berghei gametes is reduced in the presence of P. yoelii and (iv) as expected for gamete recognition proteins, strong positive selection acts on a region of P230 and P47 (P48/45 paralogue). P230 and P48/45 are leading candidates for interventions to block malaria transmission. Our results suggest that depending on the viability of hybrids, applying such interventions to populations where mixed-species infections occur could either facilitate or hinder malaria control.

The genetic basis of Escherichia coli pathoadaptation to macrophages.

Antagonistic interactions are likely important driving forces of the evolutionary process underlying bacterial genome complexity and diversity. We hypothesized that the ability of evolved bacteria to escape specific components of host innate immunity, such as phagocytosis and killing by macrophages (MΦ), is a critical trait relevant in the acquisition of bacterial virulence. Here, we used a combination of experimental evolution, phenotypic characterization, genome sequencing and mathematical modeling to address how fast, and through how many adaptive steps, a commensal Escherichia coli (E. coli) acquire this virulence trait. We show that when maintained in vitro under the selective pressure of host MΦ commensal E. coli can evolve, in less than 500 generations, virulent clones that escape phagocytosis and MΦ killing in vitro, while increasing their pathogenicity in vivo, as assessed in mice. This pathoadaptive process is driven by a mechanism involving the insertion of a single transposable element into the promoter region of the E. coli yrfF gene. Moreover, transposition of the IS186 element into the promoter of Lon gene, encoding an ATP-dependent serine protease, is likely to accelerate this pathoadaptive process. Competition between clones carrying distinct beneficial mutations dominates the dynamics of the pathoadaptive process, as suggested from a mathematical model, which reproduces the observed experimental dynamics of E. coli evolution towards virulence. In conclusion, we reveal a molecular mechanism explaining how a specific component of host innate immunity can modulate microbial evolution towards pathogenicity.

Sex and death: the effects of innate immune factors on the sexual reproduction of malaria parasites.

Malaria parasites must undergo a round of sexual reproduction in the blood meal of a mosquito vector to be transmitted between hosts. Developing a transmission-blocking intervention to prevent parasites from mating is a major goal of biomedicine, but its effectiveness could be compromised if parasites can compensate by simply adjusting their sex allocation strategies. Recently, the application of evolutionary theory for sex allocation has been supported by experiments demonstrating that malaria parasites adjust their sex ratios in response to infection genetic diversity, precisely as predicted. Theory also predicts that parasites should adjust sex allocation in response to host immunity. Whilst data are supportive, the assumptions underlying this prediction - that host immune responses have differential effects on the mating ability of males and females - have not yet been tested. Here, we combine experimental work with theoretical models in order to investigate whether the development and fertility of male and female parasites is affected by innate immune factors and develop new theory to predict how parasites' sex allocation strategies should evolve in response to the observed effects. Specifically, we demonstrate that reactive nitrogen species impair gametogenesis of males only, but reduce the fertility of both male and female gametes. In contrast, tumour necrosis factor-α does not influence gametogenesis in either sex but impairs zygote development. Therefore, our experiments demonstrate that immune factors have complex effects on each sex, ranging from reducing the ability of gametocytes to develop into gametes, to affecting the viability of offspring. We incorporate these results into theory to predict how the evolutionary trajectories of parasite sex ratio strategies are shaped by sex differences in gamete production, fertility and offspring development. We show that medical interventions targeting offspring development are more likely to be 'evolution-proof' than interventions directed at killing males or females. Given the drive to develop medical interventions that interfere with parasite mating, our data and theoretical models have important implications.

Molecular evolution and phylogenetics of rodent malaria parasites.

BACKGROUND: Over the last 6 decades, rodent Plasmodium species have become key model systems for understanding the basic biology of malaria parasites. Cell and molecular parasitology have made much progress in identifying genes underpinning interactions between malaria parasites, hosts, and vectors. However, little attention has been paid to the evolutionary genetics of parasites, which provides context for identifying potential therapeutic targets and for understanding the selective forces shaping parasites in natural populations. Additionally, understanding the relationships between species, subspecies, and strains, is necessary to maximize the utility of rodent malaria parasites as medically important infectious disease models, and for investigating the evolution of host-parasite interactions. RESULTS: Here, we collected multi-locus sequence data from 58 rodent malaria genotypes distributed throughout 13 subspecies belonging to P. berghei, P. chabaudi, P. vinckei, and P. yoelii. We employ multi-locus methods to infer the subspecies phylogeny, and use population-genetic approaches to elucidate the selective patterns shaping the evolution of these organisms. Our results reveal a time-line for the evolution of rodent Plasmodium and suggest that all the subspecies are independently evolving lineages (i.e. species). We show that estimates of species-level polymorphism are inflated if subspecies are not explicitly recognized, and detect purifying selection at most loci. CONCLUSIONS: Our work resolves previous inconsistencies in the phylogeny of rodent malaria parasites, provides estimates of important parameters that relate to the parasite's natural history and provides a much-needed evolutionary context for understanding diverse biological aspects from the cross-reactivity of immune responses to parasite mating patterns.

Host lung microbiota promotes malaria-associated acute respiratory distress syndrome.

Severe malaria can manifest itself with a variety of well-recognized clinical phenotypes that are highly predictive of death - severe anaemia, coma (cerebral malaria), multiple organ failure, and respiratory distress. The reasons why an infected individual develops one pathology rather than another remain poorly understood. Here we use distinct rodent models of infection to show that the host microbiota is a contributing factor for the development of respiratory distress syndrome and host mortality in the context of malaria infections (malaria-associated acute respiratory distress syndrome, MA-ARDS). We show that parasite sequestration in the lung results in sustained immune activation. Subsequent production of the anti-inflammatory cytokine IL-10 by T cells compromises microbial control, leading to severe lung disease. Notably, bacterial clearance with linezolid, an antibiotic commonly used in the clinical setting to control lung-associated bacterial infections, prevents MA-ARDS-associated lethality. Thus, we propose that the host's anti-inflammatory response to limit tissue damage can result in loss of microbial control, which promotes MA-ARDS. This must be considered when intervening against life-threatening respiratory complications.

Host miRNA-21 promotes liver dysfunction by targeting small intestinal Lactobacillus in mice.

New evidence shows that host-microbiota crosstalk can be modulated via endogenous miRNAs. We have previously reported that miR-21 ablation protects against liver injury in cholestasis. In this study, we investigated the role of miR-21 in modulating the gut microbiota during cholestasis and its effects in liver dysfunction. Mice lacking miR-21 had reduced liver damage and were protected against small intestinal injury as well as from gut microbiota dysbiosis when subjected to bile duct ligation surgery. The unique microbiota profile of miR-21KO mice was characterized by an increase in Lactobacillus, a key microbiome genus for gut homeostasis. Interestingly, in vitro incubation of synthetic miR-21 directly reduced Lactobacillus load. Moreover, supplementation with Lactobacillus reuteri revealed reduced liver fibrosis in acute bile duct-ligated mice, mimicking the protective effects in miR-21 knockout mice. D-lactate, a main product of Lactobacillus, regulates gut homeostasis that may link with reduced liver fibrosis. Altogether, our results demonstrate that miR-21 promotes liver dysfunction through direct modulation of the gut microbiota and highlight the potential therapeutic effects of Lactobacillus supplementation in gut and liver homeostasis.

Evolution of commensal bacteria in the intestinal tract of mice.

Hundreds of different bacterial species inhabit our intestines and contribute to our health status, with significant loss of species diversity typically observed in disease conditions. Within each microbial species a great deal of diversity is hidden and such intra-specific variation is also key to the proper homeostasis between the host and its microbial inhabitants. Indeed, it is at this level that new mechanisms of antibiotic resistance emerge and pathogenic characteristics evolve. Yet, our knowledge on intra-species variation in the gut is still limited and an understanding of the evolutionary mechanisms acting on it is extremely reduced. Here we review recent work that has begun to reveal that adaptation of commensal bacteria to the mammalian intestine may be fast and highly repeatable, and that the time scales of evolutionary and ecological change can be very similar in these ecosystems.

Macrophage adaptation leads to parallel evolution of genetically diverse Escherichia coli small-colony variants with increased fitness in vivo and antibiotic collateral sensitivity.

Small-colony variants (SCVs) are commonly observed in evolution experiments and clinical isolates, being associated with antibiotic resistance and persistent infections. We recently observed the repeated emergence of Escherichia coli SCVs during adaptation to the interaction with macrophages. To identify the genetic targets underlying the emergence of this clinically relevant morphotype, we performed whole-genome sequencing of independently evolved SCV clones. We uncovered novel mutational targets, not previously associated with SCVs (e.g. cydA, pepP) and observed widespread functional parallelism. All SCV clones had mutations in genes related to the electron-transport chain. As SCVs emerged during adaptation to macrophages, and often show increased antibiotic resistance, we measured SCV fitness inside macrophages and measured their antibiotic resistance profiles. SCVs had a fitness advantage inside macrophages and showed increased aminoglycoside resistance in vitro, but had collateral sensitivity to other antibiotics (e.g. tetracycline). Importantly, we observed similar results in vivo. SCVs had a fitness advantage upon colonization of the mouse gut, which could be tuned by antibiotic treatment: kanamycin (aminoglycoside) increased SCV fitness, but tetracycline strongly reduced it. Our results highlight the power of using experimental evolution as the basis for identifying the causes and consequences of adaptation during host-microbe interactions.

Plastic parasites: sophisticated strategies for survival and reproduction?

Adaptive phenotypic plasticity in life history traits, behaviours, and strategies is ubiquitous in biological systems. It is driven by variation in selection pressures across environmental gradients and operates under constraints imposed by trade-offs. Phenotypic plasticity has been thoroughly documented for multicellular taxa, such as insects, birds and mammals, and in many cases the underlying selective pressures are well understood. Whilst unicellular parasites face many of the same selective pressures and trade-offs, plasticity in their phenotypic traits has been largely overlooked and remains poorly understood. Here, we demonstrate that evolutionary theory, developed to explain variation observed in the life-history traits of multicellular organisms, can be applied to parasites. Though our message is general - we can expect the life-histories of all parasites to have evolved phenotypic plasticity - we focus our discussion on malaria parasites. We use an evolutionary framework to explain the trade-offs that parasites face and how plasticity in their life history traits will be expressed according to changes in their in-host environment. Testing whether variation in parasites traits is adaptive will provide new and fundamental insights into the basic biology of parasites, their epidemiology and the processes of disease during individual infections.