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     

A Phylogeny-aware GWAS Framework to Correct for Heritable Pathogen Effects on Infectious Disease Traits

Infectious diseases are particularly challenging for genome-wide association studies (GWAS) because genetic effects from two organisms (pathogen and host) can influence a trait. Traditional GWAS assume individual samples are independent observations. However, pathogen effects on a trait can be heritable from donor to recipient in transmission chains. Thus, residuals in GWAS association tests for host genetic effects may not be independent due to shared pathogen ancestry. We propose a new method to estimate and remove heritable pathogen effects on a trait based on the pathogen phylogeny prior to host GWAS, thus restoring independence of samples. In simulations, we show this additional step can increase GWAS power to detect truly associated host variants when pathogen effects are highly heritable, with strong phylogenetic correlations. We applied our framework to data from two different host–pathogen systems, HIV in humans and X. arboricola in A. thaliana. In both systems, the heritability and thus phylogenetic correlations turn out to be low enough such that qualitative results of GWAS do not change when accounting for the pathogen shared ancestry through a correction step. This means that previous GWAS results applied to these two systems should not be biased due to shared pathogen ancestry. In summary, our framework provides additional information on the evolutionary dynamics of traits in pathogen populations and may improve GWAS if pathogen effects are highly phylogenetically correlated amongst individuals in a cohort.     

Factors affecting host range in a generalist seed pathogen of semi-arid shrublands

Generalist pathogens can exhibit differential success on different hosts, resulting in complex host range patterns. Several factors operate to reduce realized host range relative to potential host range, particularly under field conditions. We explored factors influencing host range of the naturally occurring generalist ascomycete grass seed pathogen Pyrenophora semeniperda. We measured potential host range in laboratory experiments at high inoculum loads with 26 grass species, including the primary host Bromus tectorum, and developed models to predict susceptibility and tolerance based on host traits, including germination speed, seed hardness, seed size, and phylogenetic relations. We also examined pathogen and host density effects on infection and mortality. All species tested were at least somewhat susceptible to the pathogen at high inoculum loads, but both infection and mortality varied widely. Species more closely related to the original host (B. tectorum) were more susceptible to infection, whereas species with slower germination were less tolerant and therefore more likely to suffer mortality. Infection and mortality were sharply reduced as inoculum load was reduced. Intermediate loads had major negative impacts on dormant B. tectorum seeds but generally minimal effects on native species. In addition, field seed bank studies determined that P. semeniperda rarely exploits native grass species as hosts. This marked reduction in realized host range relative to potential host range indicates that laboratory host range studies are potentially a poor predictor of either the current or possible future realized host range for wildland plant pathogens.     

Leptospira interrogans Enolase Is Secreted Extracellularly and Interacts with Plasminogen

Leptospira interrogans is the agent for leptospirosis, an important zoonosis in humans and animals across the globe. Surface proteins of invading pathogens, such as L. interrogans, are thought to be responsible for successful microbial persistence in vivo via interaction with specific host components. In particular, a number of invasive infectious agents exploit host proteolytic pathways, such as one involving plasminogen (Pg), which aid in efficient pathogen dissemination within the host. Here we show that L. interrogans serovar Lai binds host Pg and that the leptospiral gene product LA1951, annotated as enolase, is involved in this interaction. Interestingly, unlike in related pathogenic Spirochetes, such as Borrelia burgdorferi, LA1951 is not readily detectable in the L. interrogans outer membrane. We show that the antigen is indeed secreted extracellularly; however, it can reassociate with the pathogen surface, where it displays Pg-binding and measurable enzymatic activity. Hamsters infected with L. interrogans also develop readily detectable antibody responses against enolase. Taken together, our results suggest that the L. interrogans enolase has evolved to play a role in pathogen interaction with host molecules, which may contribute to the pathogenesis of leptospirosis.     

Lack of Host Specialization on Winter Annual Grasses in the Fungal Seed Bank Pathogen Pyrenophora semeniperda

Generalist plant pathogens may have wide host ranges, but many exhibit varying degrees of host specialization, with multiple pathogen races that have narrower host ranges. These races are often genetically distinct, with each race causing highest disease incidence on its host of origin. We examined host specialization in the seed pathogen Pyrenophora semeniperda by reciprocally inoculating pathogen strains from Bromus tectorum and from four other winter annual grass weeds (Bromus diandrus, Bromus rubens, Bromus arvensis and Taeniatherum caput-medusae) onto dormant seeds of B. tectorum and each alternate host. We found that host species varied in resistance and pathogen strains varied in aggressiveness, but there was no evidence for host specialization. Most variation in aggressiveness was among strains within populations and was expressed similarly on both hosts, resulting in a positive correlation between strain-level disease incidence on B. tectorum and on the alternate host. In spite of this lack of host specialization, we detected weak but significant population genetic structure as a function of host species using two neutral marker systems that yielded similar results. This genetic structure is most likely due to founder effects, as the pathogen is known to be dispersed with host seeds. All host species were highly susceptible to their own pathogen races. Tolerance to infection (i.e., the ability to germinate even when infected and thereby avoid seed mortality) increased as a function of seed germination rate, which in turn increased as dormancy was lost. Pyrenophora semeniperda apparently does not require host specialization to fully exploit these winter annual grass species, which share many life history features that make them ideal hosts for this pathogen.     

Dynamic optimization reveals alveolar epithelial cells as key mediators of host defense in invasive aspergillosis

Aspergillus fumigatus is an important human fungal pathogen and its conidia are constantly inhaled by humans. In immunocompromised individuals, conidia can grow out as hyphae that damage lung epithelium. The resulting invasive aspergillosis is associated with devastating mortality rates. Since infection is a race between the innate immune system and the outgrowth of A. fumigatus conidia, we use dynamic optimization to obtain insight into the recruitment and depletion of alveolar macrophages and neutrophils. Using this model, we obtain key insights into major determinants of infection outcome on host and pathogen side. On the pathogen side, we predict in silico and confirm in vitro that germination speed is an important virulence trait of fungal pathogens due to the vulnerability of conidia against host defense. On the host side, we found that epithelial cells, which have been underappreciated, play a role in fungal clearance and are potent mediators of cytokine release. Both predictions were confirmed by in vitro experiments on established cell lines as well as primary lung cells. Further, our model affirms the importance of neutrophils in invasive aspergillosis and underlines that the role of macrophages remains elusive. We expect that our model will contribute to improvement of treatment protocols by focusing on the critical components of immune response to fungi but also fungal virulence traits.     

Characterization of the small RNA transcriptome in plant–microbe (Brassica/Erwinia) interactions by high-throughput sequencing

Non-coding, small RNAs (sRNAs) have been identified in a wide spectrum of organisms ranging from bacteria to humans; however, the role and mechanisms of these sRNA in plant immunity is largely unknown. To determine possible roles of sRNA in plant–pathogen interaction, we carried out a high-throughput sRNA sequencing of Brassica campestris using non-infected plants and plants infected with Erwinia carotovora. Consistent with our hypothesis that distinct classes of host sRNAs alerts their expression levels in response to infection, we found that: (1) host 28-nt sRNAs were strongly increased under pathogen infection; and (2) a group of host sRNAs homologous to the pathogen genome also accumulated at significantly higher level. Our data thus suggest several distinct classes of the host sRNAs may enhance their function by up-regulation of their expression/stability in response to bacterial pathogen challenges.     

Hidden Population Structure and Cross-species Transmission of Whipworms (Trichuris sp.) in Humans and Non-human Primates in Uganda

Background

Whipworms (Trichuris sp.) are a globally distributed genus of parasitic helminths that infect a diversity of mammalian hosts. Molecular methods have successfully resolved porcine whipworm, Trichuris suis, from primate whipworm, T. trichiura. However, it remains unclear whether T. trichiura is a multi-host parasite capable of infecting a wide taxonomic breadth of primate hosts or a complex of host specific parasites that infect one or two closely related hosts.

Methods and Findings

We examined the phylogenetic structure of whipworms in a multi-species community of non-human primates and humans in Western Uganda, using both traditional microscopy and molecular methods. A newly developed nested polymerase chain reaction (PCR) method applied to non-invasively collected fecal samples detected Trichuris with 100% sensitivity and 97% specificity relative to microscopy. Infection rates varied significantly among host species, from 13.3% in chimpanzees (Pan troglodytes) to 88.9% in olive baboons (Papio anubis). Phylogenetic analyses based on nucleotide sequences of the Trichuris internal transcribed spacer regions 1 and 2 of ribosomal DNA revealed three co-circulating Trichuris groups. Notably, one group was detected only in humans, while another infected all screened host species, indicating that whipworms from this group are transmitted among wild primates and humans.

Conclusions and Significance

Our results suggest that the host range of Trichuris varies by taxonomic group, with some groups showing host specificity, and others showing host generality. In particular, one Trichuris taxon should be considered a multi-host pathogen that is capable of infecting wild primates and humans. This challenges past assumptions about the host specificity of this and similar helminth parasites and raises concerns about animal and human health.     

Predation on Multiple Trophic Levels Shapes the Evolution of Pathogen Virulence

The pathogen virulence is traditionally thought to co-evolve as a result of reciprocal selection with its host organism. In natural communities, pathogens and hosts are typically embedded within a web of interactions with other species, which could affect indirectly the pathogen virulence and host immunity through trade-offs. Here we show that selection by predation can affect both pathogen virulence and host immune defence. Exposing opportunistic bacterial pathogen Serratia marcescens to predation by protozoan Tetrahymena thermophila decreased its virulence when measured as host moth Parasemia plantaginis survival. This was probably because the bacterial anti-predatory traits were traded off with bacterial virulence factors, such as motility or resource use efficiency. However, the host survival depended also on its allocation to warning signal that is used against avian predation. When infected with most virulent ancestral bacterial strain, host larvae with a small warning signal survived better than those with an effective large signal. This suggests that larval immune defence could be traded off with effective defence against bird predators. However, the signal size had no effect on larval survival when less virulent control or evolved strains were used for infection suggesting that anti-predatory defence against avian predators, might be less constrained when the invading pathogen is rather low in virulence. Our results demonstrate that predation can be important indirect driver of the evolution of both pathogen virulence and host immunity in communities with multiple species interactions. Thus, the pathogen virulence should be viewed as a result of both past evolutionary history, and current ecological interactions.     

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.     

Transmission of Hypervirulence Traits via Sexual Reproduction within and between Lineages of the Human Fungal Pathogen Cryptococcus gattii

Since 1999 a lineage of the pathogen Cryptococcus gattii has been infecting humans and other animals in Canada and the Pacific Northwest of the USA. It is now the largest outbreak of a life-threatening fungal infection in a healthy population in recorded history. The high virulence of outbreak strains is closely linked to the ability of the pathogen to undergo rapid mitochondrial tubularisation and proliferation following engulfment by host phagocytes. Most outbreaks spread by geographic expansion across suitable niches, but it is known that genetic re-assortment and hybridisation can also lead to rapid range and host expansion. In the context of C. gattii, however, the likelihood of virulence traits associated with the outbreak lineages spreading to other lineages via genetic exchange is currently unknown. Here we address this question by conducting outgroup crosses between distantly related C. gattii lineages (VGII and VGIII) and ingroup crosses between isolates from the same molecular type (VGII). Systematic phenotypic characterisation shows that virulence traits are transmitted to outgroups infrequently, but readily inherited during ingroup crosses. In addition, we observed higher levels of biparental (as opposed to uniparental) mitochondrial inheritance during VGII ingroup sexual mating in this species and provide evidence for mitochondrial recombination following mating. Taken together, our data suggest that hypervirulence can spread among the C. gattii lineages VGII and VGIII, potentially creating novel hypervirulent genotypes, and that current models of uniparental mitochondrial inheritance in the Cryptococcus genus may not be universal.     

Development and Validation of a Real-Time PCR for Chlamydia suis Diagnosis in Swine and Humans

Pigs are the natural host for Chlamydia suis, a pathogen which is phylogenetically highly related to the human pathogen C. trachomatis. Chlamydia suis infections are generally treated with tetracyclines. In 1998, tetracyline resistant C. suis strains emerged on U.S. pig farms and they are currently present in the Belgian, Cypriote, German, Israeli, Italian and Swiss pig industry. Infections with tetracycline resistant C. suis strains are mainly associated with severe reproductive failure leading to marked economical loss. We developed a sensitive and specific TaqMan probe-based C. suis real-time PCR for examining clinical samples of both pigs and humans. The analytical sensitivity of the real-time PCR is 10 rDNA copies/reaction without cross-amplifying DNA of other Chlamydia species. The PCR was successfully validated using conjunctival, pharyngeal and stool samples of slaughterhouse employees, as well as porcine samples from two farms with evidence of reproductive failure and one farm without clinical disease. Chlamydia suis was only detected in diseased pigs and in the eyes of humans. Positive humans had no clinical complaints. PCR results were confirmed by culture in McCoy cells. In addition, Chlamydia suis isolates were also examined by the tet(C) PCR, designed for demonstrating the tetracycline resistance gene tet(C). The tet(C) gene was only present in porcine C. suis isolates.     

In vitro, ex vivo and in vivo models: A comparative analysis of Paracoccidioides spp. proteomic studies

Members of the Paracoccidioides complex are human pathogens that infect different anatomic sites in the host. The ability of Paracoccidioides spp. to infect host niches is putatively supported by a wide range of virulence factors, as well as fitness attributes that may comprise the transition from mycelia/conidia to yeast cells, response to deprivation of micronutrients in the host, expression of adhesins on the cell surface, response to oxidative and nitrosative stresses, as well as the secretion of hydrolytic enzymes in the host tissue. Our understanding of how those molecules can contribute to the infection establishment has been increasing significantly, through the utilization of several models, including in vitro, ex vivo and in vivo infection in animal models. In this review we present an update of our understanding on the strategies used by the pathogen to establish infection. Our results were obtained through a comparative proteomic analysis of Paracoccidioides spp. in models of infection.     

Hyphal Growth of Phagocytosed Fusarium oxysporum Causes Cell Lysis and Death of Murine Macrophages

Fusarium oxysporum is an important plant pathogen and an opportunistic pathogen of humans. Here we investigated phagocytosis of F. oxysporum by J774.1 murine cell line macrophages using live cell video microscopy. Macrophages avidly migrated towards F. oxysporum germlings and were rapidly engulfed after cell-cell contact was established. F. oxysporum germlings continued hyphal growth after engulfment by macrophages, leading to associated macrophage lysis and escape. Macrophage killing depended on the multiplicity of infection. After engulfment, F. oxysporum inhibited macrophages from completing mitosis, resulting in large daughter cells fused together by means of a F. oxysporum hypha. These results shed new light on the initial stages of Fusarium infection and the innate immune response of the mammalian host.     

A field test of the dilution effect hypothesis in four avian multi-host pathogens

The Dilution Effect Hypothesis (DEH) argues that greater biodiversity lowers the risk of disease and reduces the rates of pathogen transmission since more diverse communities harbour fewer competent hosts for any given pathogen, thereby reducing host exposure to the pathogen. DEH is expected to operate most intensely in vector-borne pathogens and when species-rich communities are not associated with increased host density. Overall, dilution will occur if greater species diversity leads to a lower contact rate between infected vectors and susceptible hosts, and between infected hosts and susceptible vectors. Field-based tests simultaneously analysing the prevalence of several multi-host pathogens in relation to host and vector diversity are required to validate DEH. We tested the relationship between the prevalence in house sparrows (Passer domesticus) of four vector-borne pathogens–three avian haemosporidians (including the avian malaria parasite Plasmodium and the malaria-like parasites Haemoproteus and Leucocytozoon) and West Nile virus (WNV)–and vertebrate diversity. Birds were sampled at 45 localities in SW Spain for which extensive data on vector (mosquitoes) and vertebrate communities exist. Vertebrate censuses were conducted to quantify avian and mammal density, species richness and evenness. Contrary to the predictions of DEH, WNV seroprevalence and haemosporidian prevalence were not negatively associated with either vertebrate species richness or evenness. Indeed, the opposite pattern was found, with positive relationships between avian species richness and WNV seroprevalence, and Leucocytozoon prevalence being detected. When vector (mosquito) richness and evenness were incorporated into the models, all the previous associations between WNV prevalence and the vertebrate community variables remained unchanged. No significant association was found for Plasmodium prevalence and vertebrate community variables in any of the models tested. Despite the studied system having several characteristics that should favour the dilution effect (i.e., vector-borne pathogens, an area where vector and host densities are unrelated, and where host richness is not associated with an increase in host density), none of the relationships between host species diversity and species richness, and pathogen prevalence supported DEH and, in fact, amplification was found for three of the four pathogens tested. Consequently, the range of pathogens and communities studied needs to be broadened if we are to understand the ecological factors that favour dilution and how often these conditions occur in nature.     

Climate change accelerates local disease extinction rates in a long‐term wild host–pathogen association

Pathogens are a significant component of all plant communities. In recent years, the potential for existing and emerging pathogens of agricultural crops to cause increased yield losses as a consequence of changing climatic patterns has raised considerable concern. In contrast, the response of naturally occurring, endemic pathogens to a warming climate has received little attention. Here, we report on the impact of a signature variable of global climate change – increasing temperature – on the long‐term epidemiology of a natural host–pathogen association involving the rust pathogen Triphragmium ulmariae and its host plant Filipendula ulmaria. In a host–pathogen metapopulation involving approximately 230 host populations growing on an archipelago of islands in the Gulf of Bothnia we assessed changes in host population size and pathogen epidemiological measures over a 25‐year period. We show how the incidence of disease and its severity declines over that period and most importantly demonstrate a positive association between a long‐term trend of increasing extinction rates in individual pathogen populations of the metapopulation and increasing temperature. Our results are highly suggestive that changing climatic patterns, particularly mean monthly growing season (April‐November) temperature, are markedly influencing the epidemiology of plant disease in this host–pathogen association. Given the important role plant pathogens have in shaping the structure of communities, changes in the epidemiology of pathogens have potentially far‐reaching impacts on ecological and evolutionary processes. For these reasons, it is essential to increase understanding of pathogen epidemiology, its response to warming, and to invoke these responses in forecasts for the future.     

Experimental Evolution of Legionella pneumophila in Mouse Macrophages Leads to Strains with Altered Determinants of Environmental Survival

The Gram-negative bacterium, Legionella pneumophila, is a protozoan parasite and accidental intracellular pathogen of humans. We propose a model in which cycling through multiple protozoan hosts in the environment holds L. pneumophila in a state of evolutionary stasis as a broad host-range pathogen. Using an experimental evolution approach, we tested this hypothesis by restricting L. pneumophila to growth within mouse macrophages for hundreds of generations. Whole-genome resequencing and high-throughput genotyping identified several parallel adaptive mutations and population dynamics that led to improved replication within macrophages. Based on these results, we provide a detailed view of the population dynamics of an experimentally evolving bacterial population, punctuated by frequent instances of transient clonal interference and selective sweeps. Non-synonymous point mutations in the flagellar regulator, fleN, resulted in increased uptake and broadly increased replication in both macrophages and amoebae. Mutations in multiple steps of the lysine biosynthesis pathway were also independently isolated, resulting in lysine auxotrophy and reduced replication in amoebae. These results demonstrate that under laboratory conditions, host restriction is sufficient to rapidly modify L. pneumophila fitness and host range. We hypothesize that, in the environment, host cycling prevents L. pneumophila host-specialization by maintaining pathways that are deleterious for growth in macrophages and other hosts.