Infection in a dish: high-throughput analyses of bacterial pathogenesis

Diverse aspects of host-pathogen interactions have been studied using non-mammalian hosts such as Dictyostelium discoideum, Caenorhabditis elegans, Drosophila melanogaster and Danio rerio for more than 20 years. Over the past two years, the use of these model hosts to dissect bacterial virulence mechanisms has been expanded to include the important human pathogens Vibrio cholerae and Yersinia pestis. Innovative approaches using these alternative hosts have also been developed, enabling the isolation of new antimicrobials through screening large libraries of compounds in a C. elegans Enterococcus faecalis infection model. Host proteins required by Mycobacterium and Listeria during their invasion and intracellular growth have been uncovered using high-throughput dsRNA screens in a Drosophila cell culture system, and immune evasion mechanisms deployed by Pseudomonas aeruginosa during its infection of flies have been identified. Together, these reports further illustrate the potential and relevance of these non-mammalian hosts for modelling many facets of bacterial infection in mammals.     

Environmental predators as models for bacterial pathogenesis

Environmental bacteria are constantly threatened by bacterivorous predators such as free-living protozoa and nematodes. In the course of their coevolution with environmental predators, some bacteria developed sophisticated defence mechanisms, including the secretion of toxins, or the capacity to avoid lysosomal killing and to replicate intracellularly within protozoa. To analyse the interactions with bacterial pathogens on a molecular, cellular or organismic level, protozoa and other non-mammalian hosts are increasingly used. These include amoebae, as well as genetically tractable hosts, such as the social amoeba Dictyostelium discoideum, the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster. Using these hosts, the virulence mechanisms of opportunistic pathogenic bacteria such as Legionella, Mycobacterium, Pseudomonas or Vibrio were found to be not only relevant for the interactions of the bacteria with protozoa, nematodes and insect phagocytes, but also with mammalian hosts including humans. Thus, non-mammalian model hosts provide valuable insight into the pathogenesis of environmental bacteria.     

The pursuit of cryptococcal pathogenesis: heterologous hosts and the study of cryptococcal host-pathogen interactions

Analysis of the molecular mechanisms by which a pathogen interacts with the human host is most commonly performed using a mammalian model of infection. However, several virulence-related genes previously shown to be involved in mammalian infection with Cryptococcus neoformans have also been shown to play a role in the interaction of these pathogens with invertebrates, such as Acanthamoeba castellanii, Caenorhabditis elegans, Dictyostelium discoideum, Drosophila melanogaster and Galleria mellonella. The study of host-pathogen interactions using these model hosts has allowed rapid screening of mutant libraries and can be used for the study of evolutionarily preserved aspects of microbial virulence and host response.     

Cryptococcus neoformans Kin1 protein kinase homologue, identified through a Caenorhabditis elegans screen, promotes virulence in mammals

Cryptococcal infections are a global cause of significant morbidity and mortality. Recent studies support the hypothesis that virulence of Cryptococcus neoformans may have evolved via survival selection in environmental hosts, such as amoebae and free-living nematodes. We used killing of the nematode Caenorhabditis elegans by C. neoformans as an assay to screen a library of random C. neoformans insertion mutants. Of 350 mutants tested, seven were identified with attenuated virulence that persisted after crossing the mutation back into a wild-type strain. Genetic analysis of one strain revealed an insertion in a gene homologous to Saccharomyces cerevisiae KIN1, which encodes a serine/threonine protein kinase. C. neoformans kin1 mutants exhibited significant defects in virulence in murine inhalation and haematogenous infection models and displayed increased binding to alveolar and peritoneal macrophages. The kin1 mutant phenotypes were complemented by the wild-type KIN1 gene. These findings show that the C. neoformans Kin1 kinase homologue is required for full virulence in disparate hosts and that C. elegans can be used as a substitute host to identify novel factors involved in fungal pathogenesis in mammals.     

The role of apoptosis in non-mammalian host-parasite relationships

It is clear that the roles of apoptosis in the interactions between the parasite and their non-mammalian hosts are multifaceted and highly dependent on individual associations between the two organisms involved. Whilst there are instances where both organisms appear to gain from the apoptotic mechanism induced, in the majority of cases apoptosis appears to favour only one of the parties. In the instances when the parasite benefits, the apoptosis has been related to infectivity and virulence, an interruption of the killing mechanism of the host, and liberation of the pathogen. However, there are occasions where the apoptotic process benefits the host, as controlled cell death has been associated with limiting the pathogen population, parasite migration within the host and, in some instances, actually killing the invading organism. Apoptosis thus appears to play several fundamental roles within the host-parasite relationship which is ultimately reflected in an effect on the host population either mediated through an alteration in host fecundity or reduction in host numbers. The next decade promises to be both exciting and productive with respect to our knowledge of the relationship between apoptosis in non-mammalian animals and infection. Over the last few years the information obtained from studies on the apoptotic process in mammals and invertebrates (i.e. C. elegans and Drosophila) have been effectively used to increase our understanding of the apoptotic process in other animals such as insects, fish and amphibians. Such knowledge has paved the way for extensive studies on the effect of infections to be carried out.     

Elucidating the molecular mechanisms of bacterial virulence using non-mammalian hosts

Several strains of the human opportunistic pathogen Pseudomonas aeruginosa infect plants, nematodes and insects. Our laboratory has developed a multihost pathogenesis system based on the P. aeruginosa clinical isolate PA14, in which non-mammalian hosts are used to screen directly for virulence-attenuated mutants. The majority of PA14 mutants isolated using non-mammalian hosts also displayed reduced virulence in a burned mouse model. Surprisingly, only a few host-specific virulence factors were identified, and many of the P. aeruginosa mutants were attenuated in virulence in all the hosts. These studies illustrate the extensive conservation in the virulence mechanisms used by P. aeruginosa to infect evolutionarily diverged hosts, and validate the multihost method of screening for virulence factors relevant to mammalian pathogenesis. Through the use of genetically tractable hosts, the multihost pathogenesis model also provides tools for elucidating host responses and dissecting the fundamental molecular interactions that underlie bacterial pathogenesis.     

Exploiting amoeboid and non-vertebrate animal model systems to study the virulence of human pathogenic fungi

Experiments with insects, protozoa, nematodes, and slime molds have recently come to the forefront in the study of host-fungal interactions. Many of the virulence factors required for pathogenicity in mammals are also important for fungal survival during interactions with non-vertebrate hosts, suggesting that fungal virulence may have evolved, and been maintained, as a countermeasure to environmental predation by amoebae and nematodes and other small non-vertebrates that feed on microorganisms. Host innate immune responses are also broadly conserved across many phyla. The study of the interaction between invertebrate model hosts and pathogenic fungi therefore provides insights into the mechanisms underlying pathogen virulence and host immunity, and complements the use of mammalian models by enabling whole-animal high throughput infection assays. This review aims to assist researchers in identifying appropriate invertebrate systems for the study of particular aspects of fungal pathogenesis.     

The nematode Caenorhabditis elegans as a model for the study of host-pathogen interactions

For certain pathogens capable of infecting a broad range of organisms, there exist universal virulence factors, necessary for full pathogenicity regardless of the host. This has been most clearly demonstrated by Ausubel and colleagues for the human opportunistic pathogen Pseudomonas aeruginosa. As a consequence, one can use non-mammalian model systems, including the nematode worm Caenorhabditis elegans, to assay for such virulence factors. A significant number of pathogens of C. elegans, that provoke a range of diseases, are now known, including the opportunistic human pathogen Serratia marcescens. After explaining the practical advantages associated with the use of C. elegans, and briefly reviewing previous studies, the results of a screen for S. marcescens virulence factors will be presented.     

Fungal virulence, vertebrate endothermy, and dinosaur extinction: is there a connection?

Fungi are relatively rare causes of life-threatening systemic disease in immunologically intact mammals despite being frequent pathogens in insects, amphibians, and plants. Given that virulence is a complex trait, the capacity of certain soil fungi to infect, persist, and cause disease in animals despite no apparent requirement for animal hosts in replication or survival presents a paradox. In recent years studies with amoeba, slime molds, and worms have led to the proposal that interactions between fungi and other environmental microbes, including predators, select for characteristics that are also suitable for survival in animal hosts. Given that most fungal species grow best at ambient temperatures, the high body temperature of endothermic animals must provide a thermal barrier for protection against infection with a large number of fungi. Fungal disease is relatively common in birds but most are caused by only a few thermotolerant species. The relative resistance of endothermic vertebrates to fungal diseases is likely a result of higher body temperatures combined with immune defenses. Protection against fungal diseases could have been a powerful selective mechanism for endothermy in certain vertebrates. Deforestation and proliferation of fungal spores at cretaceous-tertiary boundary suggests that fungal diseases could have contributed to the demise of dinosaurs and the flourishing of mammalian species.     

Pseudomonas aeruginosa virulence genes identified in a Dictyostelium host model

The human pathogen Pseudomonas aeruginosa has been shown previously to use similar virulence factors when infecting mammalian hosts or Dictyostelium amoebae. Here we randomly mutagenized a clinical isolate of P. aeruginosa , and identified mutants with attenuated virulence towards Dictyostelium . These mutant strains also exhibited a strong decrease in virulence when infecting Drosophila and mice, confirming that P. aeruginosa makes use of similar virulence traits to confront these very different hosts. Further characterization of these bacterial mutants showed that TrpD is important for the induction of the quorum-sensing circuit, while PchH and PchI are involved in the induction of the type III secretion system. These results demonstrate the usefulness and the relevance of the Dictyostelium host model to identify and analyse new virulence genes in P. aeruginosa .     

Variation in tolerance and virulence in the chestnut blight fungus-hypovirus interaction

Chestnut blight, caused by the fungus Cryphonectria parasitica, has been effectively controlled with double-stranded RNA hypoviruses in Europe for over 40 years. The marked reduction in the virulence of C. parasitica by hypoviruses is a phenomenon known as hypovirulence. This virus-fungus pathosystem has become a model system for the study of biological control of fungi with viruses. We studied variation in tolerance to hypoviruses in fungal hosts and variation in virulence among virus isolates from a local population in Italy. Tolerance is defined as the relative fitness of a fungal individual when infected with hypoviruses (compared to being uninfected); virulence is defined for each hypovirus as the reduction in fitness of fungal hosts relative to virus-free hosts. Six hypovirus-infected isolates of C. parasitica were sampled from the population, and each hypovirus was transferred into six hypovirus-free recipient isolates. The resulting 36 hypovirus-fungus combinations were used to estimate genetic variation in tolerance to hypoviruses, in hypovirus virulence, and in virus-fungus interactions. Four phenotypes were evaluated for each virus-fungus combination to estimate relative fitness: (i) sporulation, i.e., the number of asexual spores (conidia) produced; (ii) canker area on field-inoculated chestnut trees, (iii) vertical transmission of hypoviruses into conidia, and (iv) conidial germination. Two-way analysis of variance (ANOVA) revealed significant interactions (P < 0.001) between viruses and fungal isolates for sporulation and canker area but not for conidial germination or transmission. One-way ANOVA among hypoviruses (within each fungal isolate) and among fungal isolates (within each hypovirus) revealed significant genetic variation (P < 0.01) in hypovirus virulence and fungal tolerance within several fungal isolates, and hypoviruses, respectively. These interactions and the significant genetic variation in several fitness characters indicate the potential for future evolution of these characters. However, biological control is unlikely to break down due to evolution of tolerance to hypoviruses in the fungus because the magnitudes of tolerance and interactions were relatively small.     

Identification of fungal metabolites of anticonvulsant drug carbamazepine

Carbamazepine, which has been used in the treatments of epilepsy, is often found in the environment. Although metabolism of carbamazepine by humans and rats has been characterized, the environmental fate of carbamazepine has not been studied. In this study, two model fungi Cunninghamella elegans ATCC 9245 and Umbelopsis ramanniana R-56, which have previously shown diverse metabolic activities, were tested for metabolism of carbamazepine. Both fungi produced three metabolites each (C1-C3 and M1-M3). All six metabolites showed [M + H](+) at m/z 253, suggesting addition of one oxygen to the parent compound. High-performance liquid chromatography and liquid chromatography-mass spectrometric analysis detected 10, 11-dihydro-10, 11-epoxycarbamazepine as a major product (C3 (47%) and M3 (85%)) and 3-hydroxycarbamazepine (C2 (15%) and M2 (7%)) from carbamazepine through mixed mono-oxidation reactions in both fungal strains. C. elegans was confirmed to produce 2-hydroxycarbamazepine (C1 (38%)) while U. ramanniana produced a yet unidentified ring-hydroxylated metabolite (M1 (8%)). The current study suggests that carbamazepine is likely to be subjected to initially diverse mono-oxygenation reactions by fungal metabolisms, resulting in the formation of the corresponding metabolites, which were similarly found in mammalian metabolisms.     

The C. elegans touch response facilitates escape from predacious fungi

Predator-prey interactions are vital determinants in the natural selection of behavioral traits. Gentle touch to the anterior half of the body of Caenorhabditis elegans elicits an escape response in which the animal quickly reverses and suppresses exploratory head movements [1, 2]. Here, we investigate the ecological significance of the touch response in predator-prey interactions between C. elegans and predacious fungi that catch nematodes using constricting hyphal rings. We show that the constricting rings of Drechslerella doedycoides catch early larval stages with a diameter similar to the trap opening. There is a delay between the ring entry and ring closure, which allows the animal to withdraw from the trap before being caught. Mutants that fail to suppress head movements in response to touch are caught more efficiently than the wild-type. This demonstrates that the coordination of motor programs allows C. elegans to smoothly retract from a fungal noose and evade capture. Our results suggest that selective pressures imposed by predacious fungi have shaped the evolution of C. elegans escape behavior.     

The origin and maintenance of virulence for the human pathogenic fungus Cryptococcus neoformans

The origin of virulence in environmental fungi that have no requirement for animal hosts in their life cycle is enigmatic. Cryptococcus neoformans is a human pathogenic fungus with virulence factors for mammalian pathogenesis that also contribute to environmental survival. C. neoformans virulence may originate from selection pressures imposed by environmental predators.     

The interactions between plant life form and fungal traits of arbuscular mycorrhizal fungi determine the symbiotic community

Arbuscular mycorrhizal (AM) fungi have traditionally been considered generalist symbionts. However, an increasing number of studies are pointing out the selectivity potential of plant hosts. Plant life form, determined by plant life history traits, seems to drive the AM fungal community composition. The AM fungi also exhibit a wide diversity of functional traits known to be responsible for their distribution in natural ecosystems. However, little is known about the role of plant and fungal traits driving the resultant symbiotic assemblages. With the aim of testing the feedback relationship between plant and fungal traits on the resulting AM fungal community, we inoculated three different plant life forms, i.e. annual herbs, perennial herbs and perennial semi-woody plants, with AM fungal communities sampled in different seasons. We hypothesized that the annual climate variation will induce changes in the mean traits of the AM fungal communities present in the soil throughout the year. Furthermore, the association of plants with different life forms with AM fungi with contrasting life history traits will show certain preferences according to reciprocal traits of the plants and fungi. We found changes in the AM fungal community throughout the year, which were differentially disrupted by disturbance and altered by plant growth form and plant biomass. Both plant and fungal traits clearly contributed to the resultant AM fungal communities. The revealed process can have implications for the functioning of ecosystems since changes in dominant plant life forms or climatic variables could influence the traits of AM fungal communities in soil and hence ecosystem processes.     

Variation in Tolerance and Virulence in the Chestnut Blight Fungus-Hypovirus Interaction

Chestnut blight, caused by the fungus Cryphonectria parasitica, has been effectively controlled with double-stranded RNA hypoviruses in Europe for over 40 years. The marked reduction in the virulence of C. parasitica by hypoviruses is a phenomenon known as hypovirulence. This virus-fungus pathosystem has become a model system for the study of biological control of fungi with viruses. We studied variation in tolerance to hypoviruses in fungal hosts and variation in virulence among virus isolates from a local population in Italy. Tolerance is defined as the relative fitness of a fungal individual when infected with hypoviruses (compared to being uninfected); virulence is defined for each hypovirus as the reduction in fitness of fungal hosts relative to virus-free hosts. Six hypovirus-infected isolates of C. parasitica were sampled from the population, and each hypovirus was transferred into six hypovirus-free recipient isolates. The resulting 36 hypovirus-fungus combinations were used to estimate genetic variation in tolerance to hypoviruses, in hypovirus virulence, and in virus-fungus interactions. Four phenotypes were evaluated for each virus-fungus combination to estimate relative fitness: (i) sporulation, i.e., the number of asexual spores (conidia) produced; (ii) canker area on field-inoculated chestnut trees, (iii) vertical transmission of hypoviruses into conidia, and (iv) conidial germination. Two-way analysis of variance (ANOVA) revealed significant interactions (P < 0.001) between viruses and fungal isolates for sporulation and canker area but not for conidial germination or transmission. One-way ANOVA among hypoviruses (within each fungal isolate) and among fungal isolates (within each hypovirus) revealed significant genetic variation (P < 0.01) in hypovirus virulence and fungal tolerance within several fungal isolates, and hypoviruses, respectively. These interactions and the significant genetic variation in several fitness characters indicate the potential for future evolution of these characters. However, biological control is unlikely to break down due to evolution of tolerance to hypoviruses in the fungus because the magnitudes of tolerance and interactions were relatively small.     

Virulence not only costs but also benefits the transmission of a fungal virus

Current theory suggests that cost-benefit relationships govern the evolution of parasite virulence. The cost of virulence is expected to be high for fungal viruses, which are obligate parasites and completely dependent on their hosts. The majority of fungal viruses infect their hosts without any apparent symptoms. Cryphonectria hypovirus 1 (CHV-1), in contrast, is virulent and debilitates its host, Cryphonectria parasitica. However, the virulence of CHV-1 is associated with high costs for virus transmission, such as an attenuated fungal growth and reduced production of the fungal spores spreading the virus. In this study, we tested the hypothesis that virulence may not only have costs but also benefits for transmitting CHV-1 across vegetative incompatibility barriers between fungi. We investigated viruses with low, medium, and high virulence, and determined their transmission rate per host-to-host contact (transmissibility). The average transmission rate across all combinations tested was 53% for the most virulent virus, 37% for the virus with intermediate virulence, and 20% for the virus with lowest virulence. These results showed that increased virulence was strongly correlated with increased transmissibility, potentially counterbalancing virulence costs. This association of virulence and transmissibility may explain why CHV-1 spread widely and evolved higher virulence than most other fungal viruses.     

The worm has turned--microbial virulence modeled in Caenorhabditis elegans

The nematode Caenorhabditis elegans is emerging as a facile and economical model host for the study of evolutionarily conserved mechanisms of microbial pathogenesis and innate immunity. A rapidly growing number of human and animal microbial pathogens have been shown to injure and kill nematodes. In many cases, microbial genes known to be important for full virulence in mammalian models have been shown to be similarly required for maximum pathogenicity in nematodes. C. elegans has been used in mutation-based screening systems to identify novel virulence-related microbial genes and immune-related host genes, many of which have been validated in mammalian models of disease. C. elegans-based pathogenesis systems hold the potential to simultaneously explore the molecular genetic determinants of both pathogen virulence and host defense.     

Cystic fibrosis-niche adaptation of Pseudomonas aeruginosa reduces virulence in multiple infection hosts

The opportunistic pathogen Pseudomonas aeruginosa is able to thrive in diverse ecological niches and to cause serious human infection. P. aeruginosa environmental strains are producing various virulence factors that are required for establishing acute infections in several host organisms; however, the P. aeruginosa phenotypic variants favour long-term persistence in the cystic fibrosis (CF) airways. Whether P. aeruginosa strains, which have adapted to the CF-niche, have lost their competitive fitness in the other environment remains to be investigated. In this paper, three P. aeruginosa clonal lineages, including early strains isolated at the onset of infection, and late strains, isolated after several years of chronic lung infection from patients with CF, were analysed in multi-host model systems of acute infection. P. aeruginosa early isolates caused lethality in the three non-mammalian hosts, namely Caenorhabditis elegans, Galleria mellonella, and Drosophila melanogaster, while late adapted clonal isolates were attenuated in acute virulence. When two different mouse genetic background strains, namely C57Bl/6NCrl and Balb/cAnNCrl, were used as acute infection models, early P. aeruginosa CF isolates were lethal, while late isolates exhibited reduced or abolished acute virulence. Severe histopathological lesions, including high leukocytes recruitment and bacterial load, were detected in the lungs of mice infected with P. aeruginosa CF early isolates, while late isolates were progressively cleared. In addition, systemic bacterial spread and invasion of epithelial cells, which were detected for P. aeruginosa CF early strains, were not observed with late strains. Our findings indicate that niche-specific selection in P. aeruginosa reduced its ability to cause acute infections across a broad range of hosts while maintaining the capacity for chronic infection in the CF host.