Virulence phenotypes result from interactions between pathogen ploidy and genetic background

Studying fungal virulence is often challenging and frequently depends on many contexts, including host immune status and pathogen genetic background. However, the role of ploidy has often been overlooked when studying virulence in eukaryotic pathogens. Since fungal pathogens, including the human opportunistic pathogen Candida albicans, can display extensive ploidy variation, assessing how ploidy impacts virulence has important clinical relevance. As an opportunistic pathogen, C. albicans causes nonlethal, superficial infections in healthy individuals, but life‐threatening bloodstream infections in individuals with compromised immune function. Here, we determined how both ploidy and genetic background of C. albicans impacts virulence phenotypes in healthy and immunocompromised nematode hosts by characterizing virulence phenotypes in four near‐isogenic diploid and tetraploid pairs of strains, which included both laboratory and clinical genetic backgrounds. We found that C. albicans infections decreased host survival and negatively impacted host reproduction, and we leveraged these two measures to survey both lethal and nonlethal virulence phenotypes across the multiple C. albicans strains. In this study, we found that regardless of pathogen ploidy or genetic background, immunocompromised hosts were susceptible to fungal infection compared to healthy hosts. Furthermore, for each host context, we found a significant interaction between C. albicans genetic background and ploidy on virulence phenotypes, but no global differences between diploid and tetraploid pathogens were observed.     

Dynamic genome plasticity during unisexual reproduction in the human fungal pathogen Cryptococcus deneoformans

Genome copy number variation occurs during each mitotic and meiotic cycle and it is crucial for organisms to maintain their natural ploidy. Defects in ploidy transitions can lead to chromosome instability, which is a hallmark of cancer. Ploidy in the haploid human fungal pathogen Cryptococcus neoformans is exquisitely orchestrated and ranges from haploid to polyploid during sexual development and under various environmental and host conditions. However, the mechanisms controlling these ploidy transitions are largely unknown. During C. deneoformans (formerly C. neoformans var. neoformans, serotype D) unisexual reproduction, ploidy increases prior to the onset of meiosis, can be independent from cell-cell fusion and nuclear fusion, and likely occurs through an endoreplication pathway. To elucidate the molecular mechanisms underlying this ploidy transition, we identified twenty cell cycle-regulating genes encoding cyclins, cyclin-dependent kinases (CDK), and CDK regulators. We characterized four cyclin genes and two CDK regulator genes that were differentially expressed during unisexual reproduction and contributed to diploidization. To detect ploidy transition events, we generated a ploidy reporter, called NURAT, which can detect copy number increases via double selection for nourseothricin-resistant, uracil-prototrophic cells. Utilizing this ploidy reporter, we showed that ploidy transition from haploid to diploid can be detected during the early phases of unisexual reproduction. Interestingly, selection for the NURAT reporter revealed several instances of segmental aneuploidy of multiple chromosomes, which conferred azole resistance in some isolates. These findings provide further evidence of ploidy plasticity in fungi with significant biological and public health implications.     

Parasexuality and Ploidy Change in Candida tropicalis

Candida species exhibit a variety of ploidy states and modes of sexual reproduction. Most species possess the requisite genes for sexual reproduction, recombination, and meiosis, yet only a few have been reported to undergo a complete sexual cycle including mating and sporulation. Candida albicans, the most studied Candida species and a prevalent human fungal pathogen, completes its sexual cycle via a parasexual process of concerted chromosome loss rather than a conventional meiosis. In this study, we examine ploidy changes in Candida tropicalis, a closely related species to C. albicans that was recently revealed to undergo sexual mating. C. tropicalis diploid cells mate to form tetraploid cells, and we show that these can be induced to undergo chromosome loss to regenerate diploid forms by growth on sorbose medium. The diploid products are themselves mating competent, thereby establishing a parasexual cycle in this species for the first time. Extended incubation (>120 generations) of C. tropicalis tetraploid cells under rich culture conditions also resulted in instability of the tetraploid form and a gradual reduction in ploidy back to the diploid state. The fitness levels of C. tropicalis diploid and tetraploid cells were compared, and diploid cells exhibited increased fitness relative to tetraploid cells in vitro, despite diploid and tetraploid cells having similar doubling times. Collectively, these experiments demonstrate distinct pathways by which a parasexual cycle can occur in C. tropicalis and indicate that nonmeiotic mechanisms drive ploidy changes in this prevalent human pathogen.     

How Did Host Domestication Modify Life History Traits of Its Pathogens?

Understanding evolutionary dynamics of pathogens during domestication of their hosts and rise of agro-ecosystems is essential for durable disease management. Here, we investigated changes in life-history traits of the fungal pathogen Venturia inaequalis during domestication of the apple. Life traits linked to fungal dispersal were compared between 60 strains that were sampled in domestic and wild habitats in Kazakhstan, the center of origin of both host and pathogen. Our two main findings are that transition from wild to agro-ecosystems was associated with an increase of both spore size and sporulation capacity; and that distribution of quantitative traits of the domestic population mostly overlapped with those of the wild population. Our results suggest that apple domestication had a considerable impact on fungal characters linked to its dispersal through selection from standing phenotypic diversity. We showed that pestification of V. inaequalis in orchards led to an enhanced allocation in colonization ability from standing variation in the wild area. This study emphasizes the potential threat that pathogenic fungal populations living in wild environments represent for durability of resistance in agro-ecosystems.     

The diverse roles of autophagy in medically important fungi

Autophagy is a highly conserved eukaryotic mechanism whereby cells recycle cellular elements to survive under adverse conditions. Surprisingly, of the three fungal pathogens of greatest relevance to human health, only Cryptococcus neoformans has been shown to require this process during infection. In contrast, autophagy is dispensable for the virulence of both Candida albicans and Aspergillus fumigatus. The divergent roles for autophagy in these opportunistic species underscore the uniqueness of the host infection niche occupied by each fungus and provide insights into the evolutionary pressures that may have influenced the need for autophagy during infection. Further study of fungal autophagy may reveal the host signals which induce this protective response and determine if these signals differ between host cells or tissues. In addition, a comprehensive understanding of the autophagy machinery in fungal pathogens may provide a rational basis for the design of future therapeutic interventions to improve outcome in patients who are at risk for these infections.     

A Tetraploid Intermediate Precedes Aneuploid Formation in Yeasts Exposed to Fluconazole

Candida albicans, the most prevalent human fungal pathogen, is generally diploid. However, 50% of isolates that are resistant to fluconazole (FLC), the most widely used antifungal, are aneuploid and some aneuploidies can confer FLC resistance. To ask if FLC exposure causes or only selects for aneuploidy, we analyzed diploid strains during exposure to FLC using flow cytometry and epifluorescence microscopy. FLC exposure caused a consistent deviation from normal cell cycle regulation: nuclear and spindle cycles initiated prior to bud emergence, leading to “trimeras,” three connected cells composed of a mother, daughter, and granddaughter bud. Initially binucleate, trimeras underwent coordinated nuclear division yielding four daughter nuclei, two of which underwent mitotic collapse to form a tetraploid cell with extra spindle components. In subsequent cell cycles, the abnormal number of spindles resulted in unequal DNA segregation and viable aneuploid progeny. The process of aneuploid formation in C. albicans is highly reminiscent of early stages in human tumorigenesis in that aneuploidy arises through a tetraploid intermediate and subsequent unequal DNA segregation driven by multiple spindles coupled with a subsequent selective advantage conferred by at least some aneuploidies during growth under stress. Finally, trimera formation was detected in response to other azole antifungals, in related Candida species, and in an in vivo model for Candida infection, suggesting that aneuploids arise due to azole treatment of several pathogenic yeasts and that this can occur during the infection process.     

An ecological framework for understanding the roles of Epichloë endophytes on plant defenses against fungal diseases

Plants harbor a wide diversity of microorganisms in their tissues. Some of them have a long co-evolutionary history with their hosts, likely playing a pivotal role in regulating the plant interaction with other microbes such as pathogens. Some cool-season grasses are symbiotic with Epichloë fungal endophytes that grow symptomless and systemically in aboveground tissues. Among the many benefits that have been ascribed to endophytes, their role in mediating plant interactions with pathogens has been scarcely developed. Here, we explored the effects of Epichloë fungal endophytes on the interaction of host grasses with fungal pathogens. We made a meta-analysis that covered a total of 18 host grass species, 11 fungal endophyte species, and 22 fungal pathogen species. We observed endophyte-mediated negative effects on pathogens in vitro and in planta. Endophyte negative effects on pathogens were apparent not only in laboratory but also in greenhouse and field experiments. Epichloë fungal endophytes had negative effects on pathogen growth and spores' germination. On living plants, endophytes reduced both severity and incidence of the disease as well as colonization and subsequent infection of seeds. Symbiosis with endophytes showed an inhibitory effect on debilitator and killer pathogens, but not on castrators, and this effect did not differ among biotrophic or necrotrophic lifestyles. We found that this protection can be direct through the production of fungistatic compounds, the competition for a common resource, or the induction of plant defenses, and indirect associated with endophyte-generated changes in the abiotic or the biotic environment. Several mechanisms operate simultaneously and contribute differentially to the reduction of disease within grass populations.     

Overexpression of mannitol dehydrogenase in zonal geranium confers increased resistance to the mannitol secreting fungal pathogen Botrytis cinerea

The sugar alcohol mannitol is a carbohydrate with well-documented roles in both metabolism and osmoprotection in plants and fungi. In addition, however, mannitol is an antioxidant, and current research suggests that pathogenic fungi can secrete mannitol into the plant’s extracellular spaces during infection to suppress reactive oxygen-mediated host defenses. In response to pathogen attack, plants have been shown to secrete the normally symplastic enzyme, mannitol dehydrogenase (MTD). Given that MTD converts mannitol to the sugar mannose, extracellular MTD may be an important defense against mannitol-secreting fungal pathogens. Previous work demonstrated that overexpression of MTD in tobacco did, in fact, provide increased resistance to the mannitol-secreting fungal pathogen Alternaria alternata. In the present work we demonstrate that the fungal pathogen Botrytis cinerea also can secrete mannitol, and that overexpression of MTD in zonal geranium (Pelargonium × hortorum) in turn provides increased resistance to B. cinerea. These results are not only an important validation of previous work, but support the idea that MTD-overexpression might be used to engineer a broad variety of plants for resistance to mannitol-secreting fungal pathogens like B. cinerea for which specific resistance is lacking.     

Rust secreted protein Ps87 is conserved in diverse fungal pathogens and contains a RXLR-like motif sufficient for translocation into plant cells

Background

Effector proteins of biotrophic plant pathogenic fungi and oomycetes are delivered into host cells and play important roles in both disease development and disease resistance response. How obligate fungal pathogen effectors enter host cells is poorly understood. The Ps87 gene of Puccinia striiformis encodes a protein that is conserved in diverse fungal pathogens. Ps87 homologs from a clade containing rust fungi are predicted to be secreted. The aim of this study is to test whether Ps87 may act as an effector during Puccinia striiformis infection.

Methodology/Principal Findings

Yeast signal sequence trap assay showed that the rust protein Ps87 could be secreted from yeast cells, but a homolog from Magnaporthe oryzae that was not predicted to be secreted, could not. Cell re-entry and protein uptake assays showed that a region of Ps87 containing a conserved RXLR-like motif [K/R]RLTG was confirmed to be capable of delivering oomycete effector Avr1b into soybean leaf cells and carrying GFP into soybean root cells. Mutations in the Ps87 motif (KRLTG) abolished the protein translocation ability.

Conclusions/Significance

The results suggest that Ps87 and its secreted homologs could utilize similar protein translocation machinery as those of oomycete and other fungal pathogens. Ps87 did not show direct suppression activity on plant defense responses. These results suggest Ps87 may represent an “emerging effector” that has recently acquired the ability to enter plant cells but has not yet acquired the ability to alter host physiology.     

Ploidy variation in multinucleate cells changes under stress

Ploidy variation is found in contexts as diverse as solid tumors, drug resistance in fungal infection, and normal development. Altering chromosome or genome copy number supports adaptation to fluctuating environments but is also associated with fitness defects attributed to protein imbalances. Both aneuploidy and polyploidy can arise from multinucleate states after failed cytokinesis or cell fusion. The consequences of ploidy variation in syncytia are difficult to predict because protein imbalances are theoretically buffered by a common cytoplasm. We examined ploidy in a naturally multinucleate fungus, Ashbya gossypii. Using integrated lac operator arrays, we found that chromosome number varies substantially among nuclei sharing a common cytoplasm. Populations of nuclei range from 1N to >4N, with different polyploidies in the same cell and low levels of aneuploidy. The degree of ploidy variation increases as cells age. In response to cellular stress, polyploid nuclei diminish and haploid nuclei predominate. These data suggest that mixed ploidy is tolerated in these syncytia; however, there may be costs associated with variation as stress homogenizes the genome content of nuclei. Furthermore, the results suggest that sharing of gene products is limited, and thus there is incomplete buffering of ploidy variation despite a common cytosol.     

Diversity and bioprospecting of cultivable fungal assemblages in sediments of lakes in the Antarctic Peninsula

We recovered 195 fungal isolates from the sediments of different lakes in the Antarctic Peninsula, which were screened to detect bioactive compounds. Forty-two taxa belonging to the phyla Ascomycota, Basidiomycota, and Mortierellomycota were identified. Thelebolus globosus, Antarctomyces psychrotrophicus, Pseudogymnoascus verrucosus, Vishniacozyma victoriae, and Phenoliferia sp. were found to be the most prevalent. The fungal assemblages showed high diversity and richness, but low dominance values. However, the diversity indices and fungal distribution ranged according to the different lake sediments. Sixty fungal extracts displayed at least one biological activity against the evaluated targets. Among them, Pseudogymnoascus destructans showed selective trypanocidal activity, Cladosporium sp. 1 and Trichoderma polysporum showed antifungal activity, and Pseudogymnoascus appendiculatus and Helotiales sp. showed high herbicidal activity. We detected a rich and diverse fungal community composed of cold cosmopolitan and psychrophilic endemic taxa recognized as decomposers, symbiotics, pathogens, and potential new species, in the sediments of Antarctic lakes. The dynamics and balance of this fungal community represents an interesting aquatic web model for further ecological and evolutionary studies under extreme conditions and potential climate changes in the regions. In addition, we detected fungal taxa and isolates able to produce bioactive compounds that may represent the source of prototype molecules for applications in medicine and agriculture.     

Z-3-Hexenylacetate emissions induced by the endophyte Epichloë occultans at different levels of defoliation during the host plant's life cycle

Vertically transmitted fungal endophytes are common defensive symbionts of cool-season grasses. Protection against herbivores has been generally associated with alkaloids produced in the grass-endophyte symbiosis. However, many other changes occur in host metabolism like the release of VOCs. We aimed at characterizing the profile of volatile organic compounds (VOCs) induced by simultaneous fungal endophyte symbiosis and defoliation during the entire life cycle of the annual host grass and the asexual symbiont. We designed an outdoor factorial experiment with plots dominated by intact and damaged Lolium multiflorum plants with high and low infection levels with Epichloë occultans. After exploring the entire VOC profile, the green leaf volatile (Z)-3-hexenyl acetate (Z3-HAC) was found as the main compound emitted by the plants under field conditions. While in low-infected patches there were no differences in volatile emission during the plant life cycle, highly infected patches emitted more Z3-HAC in seedling and vegetative phases than in the reproductive phase. The role of Z3-HAC provided by symbiotic to neighbouring non-symbiotic plants in the associational protection against herbivores and fungal pathogens is discussed.     

Adaptation to iron deficiency in human pathogenic fungi

Iron is an essential micronutrient for virtually all eukaryotic organisms and plays a central role during microbial infections. Invasive fungal diseases are associated with strikingly high rates of mortality, but their impact on human health is usually underestimated. Upon a fungal infection, hosts restrict iron availability in order to limit the growth and virulence of the pathogen. Here, we use two model yeasts, Saccharomyces cerevisiae and Schizosaccharomyces pombe, to delve into the response to iron deficiency of human fungal pathogens, such as Candida glabrata, Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans. Fungi possess common and species-specific mechanisms to acquire iron and to control the response to iron limitation. Upon iron scarcity, fungi activate a wide range of elegant strategies to capture and import exogenous iron, mobilize iron from intracellular stores, and modulate their metabolism to economize and prioritize iron utilization. Hence, iron homeostasis genes represent remarkable virulence factors that can be used as targets for the development of novel antifungal treatments.     

Skin fungal assemblages of bats vary based on susceptibility to white-nose syndrome

Microbial skin assemblages, including fungal communities, can influence host resistance to infectious diseases. The diversity-invasibility hypothesis predicts that high-diversity communities are less easily invaded than species-poor communities, and thus diverse microbial communities may prevent pathogens from colonizing a host. To explore the hypothesis that host fungal communities mediate resistance to infection by fungal pathogens, we investigated characteristics of bat skin fungal communities as they relate to susceptibility to the emerging disease white-nose syndrome (WNS). Using a culture-based approach, we compared skin fungal assemblage characteristics of 10 bat species that differ in susceptibility to WNS across 10 eastern U.S. states. The fungal assemblages on WNS-susceptible bat species had significantly lower alpha diversity and abundance compared to WNS-resistant species. Overall fungal assemblage structure did not vary based on WNS-susceptibility, but several yeast species were differentially abundant on WNS-resistant bat species. One yeast species inhibited Pseudogymnoascus destructans (Pd), the causative agent on WNS, in vitro under certain conditions, suggesting a possible role in host protection. Further exploration of interactions between Pd and constituents of skin fungal assemblages may prove useful for predicting susceptibility of bat populations to WNS and for developing effective mitigation strategies.Subject terms: Microbiome, Fungal ecology     

Family issues: fungal endophyte protects host grass from the closely related pathogen Claviceps purpurea

Certain cool season grasses establish systemic and asymptomatic symbioses with clavicipitaceous fungi of the genus Neotyphodium, which affect multiple biotic interactions within host neighborhood. The presumed symbiont-mediated plant resistance to pathogens is mostly based on studies performed under laboratory and greenhouse conditions. Here we investigated, in two outdoor experiments, the relation between two fungi of the same family with opposite effects on Lolium multiflorum plants: the mutualist endophyte Neotyphodium occultans, and the pathogen Claviceps purpurea. Natural infection and its consequences on symbiotic and non-symbiotic plants were studied under varying conditions of stress by herbicide. In both experiments, N. occultans reduced significantly the infection by C. purpurea at population levels (70 % less). The percentage of spikes infected by C. purpurea was almost three times lower in endophyte-symbiotic plants than in non-symbiotic ones. However, the protective effect was not maintained under stress condition. Our results show that constitutive symbionts such as the systemic fungal endophytes mediate the interaction between host grasses and pathogens, although the effect may depend on the level of stress in the environment.     

Variability of gene expression profiles in human blood and lymphoblastoid cell lines

Background

Infection of plants by pathogens and the subsequent disease development involves substantial changes in the biochemistry and physiology of both partners. Analysis of genes that are expressed during these interactions represents a powerful strategy to obtain insights into the molecular events underlying these changes. We have employed expressed sequence tag (EST) analysis to identify rice genes involved in defense responses against infection by the blast fungus Magnaporthe oryzae and fungal genes involved in infectious growth within the host during a compatible interaction.

Results

A cDNA library was constructed with RNA from rice leaves (Oryza sativa cv. Hwacheong) infected with M. oryzae strain KJ201. To enrich for fungal genes, subtraction library using PCR-based suppression subtractive hybridization was constructed with RNA from infected rice leaves as a tester and that from uninfected rice leaves as the driver. A total of 4,148 clones from two libraries were sequenced to generate 2,302 non-redundant ESTs. Of these, 712 and 1,562 ESTs could be identified to encode fungal and rice genes, respectively. To predict gene function, Gene Ontology (GO) analysis was applied, with 31% and 32% of rice and fungal ESTs being assigned to GO terms, respectively. One hundred uniESTs were found to be specific to fungal infection EST. More than 80 full-length fungal cDNA sequences were used to validate ab initio annotated gene model of M. oryzae genome sequence.

Conclusion

This study shows the power of ESTs to refine genome annotation and functional characterization. Results of this work have advanced our understanding of the molecular mechanisms underpinning fungal-plant interactions and formed the basis for new hypothesis.     

Secretome analysis of the phytopathogen Macrophomina phaseolina cultivated in liquid medium supplemented with and without soybean leaf infusion

Macrophomina phaseolina (Tassi) Goid. is a fungal pathogen that causes root and stem rot in several economically important crops. However, most of disease control strategies have shown limited effectiveness. Despite its impact on agriculture, molecular mechanisms involved in the interaction with host plant remains poorly understood. Nevertheless, it has been proven that fungal pathogens secrete a variety of proteins and metabolites to successfully infect their host plants. In this study, a proteomic analysis of proteins secreted by M. phaseolina in culture media supplemented with soybean leaf infusion was performed. A total of 250 proteins were identified with a predominance of hydrolytic enzymes. Plant cell wall degrading enzymes together peptidases were found, probably involved in the infection process. Predicted effector proteins were also found that could induce plant cell death or suppress plant immune response. Some of the putative effectors presented similarities to known fungal virulence factors. Expression analysis of ten selected protein-coding genes showed that these genes are induced during host tissue infection and suggested their participation in the infection process. The identification of secreted proteins of M. phaseolina could be used to improve the understanding of the biology and pathogenesis of this fungus. Although leaf infusion was able to induce changes at the proteome level, it is necessary to study the changes induced under conditions that mimic the natural infection process of the soil-borne pathogen M. phaseolina to identify virulence factors.