Fungal Pathogens Of Animals Research Articles

Warming and rainfall reduction alter soil microbial diversity and co-occurrence networks and enhance pathogenic fungi in dryland soils

In this 9-year manipulative field experiment, we examined the impacts of experimental warming (2 °C, W), rainfall reduction (30 % decrease in annual rainfall, RR), and their combination (W + RR) on soil microbial communities and native vegetation in a semi-arid shrubland in south-eastern Spain. Warming had strong negative effects on plant performance across five coexisting native shrub species, consistently reducing their aboveground biomass growth and long-term survival. The impacts of rainfall reduction on plant growth and survival were species-specific and more variable. Warming strongly altered the soil microbial community alpha-diversity and changed the co-occurrence network structure. The relative abundance of symbiotic arbuscular mycorrhizal fungi (AMF) increased under W and W + RR, which could help buffer the direct negative impacts of climate change on their host plants nutrition and enhance their resistance to heat and drought stress. Indicator microbial taxa analyses evidenced that the marked sequence abundance of many plant pathogenic fungi, such as Phaeoacremonium, Cyberlindnera, Acremonium, Occultifur, Neodevriesia and Stagonosporopsis, increased significantly in the W and W + RR treatments. Moreover, the relative abundance of fungal animal pathogens and mycoparasites in soil also increased significantly under climate warming. Our findings indicate that warmer and drier conditions sustained over several years can alter the soil microbial community structure, composition, and network topology. The projected warmer and drier climate favours pathogenic fungi, which could offset the benefits of increased AMF abundance under warming and further aggravate the severe detrimental impacts of increased abiotic stress on native vegetation performance and ecosystem services in drylands.

Two-speed genome evolution drives pathogenicity in fungal pathogens of animals

The origins and evolution of virulence in amphibian-infecting chytrids Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal) are largely unknown. Here, we use deep nanopore sequencing of Bsal and comparative genomics against 21 high-quality genome assemblies that span the fungal Chytridiomycota. We discover that Bsal has the most repeat-rich genome of the Chytridiomycota, comprising 40.9% repetitive elements; this genome has expanded to more than 3× the length of its conspecific Bd, with autonomous and fully functional LTR/Gypsy elements contributing significantly to the expansion. The M36 metalloprotease virulence factors are highly expanded (n = 177) in Bsal, most of which (53%) are flanked by transposable elements, suggesting they have a repeat-associated expansion. We find enrichment upstream of M36 metalloprotease genes of three novel repeat families belonging to the repeat superfamily of LINEs that are implicated with gene copy number variations. Additionally, Bsal has a highly compartmentalized genome architecture, with virulence factors enriched in gene-sparse/repeat-rich compartments, while core conserved genes are enriched in gene-rich/repeat-poor compartments. Genes upregulated during infection are primarily found in the gene-sparse/repeat-rich compartment in both Bd and Bsal. Furthermore, genes with signatures of positive selection in Bd are enriched in repeat-rich regions, suggesting these regions are a cradle for the evolution of chytrid pathogenicity. These are the hallmarks of two-speed genome evolution, and this study provides evidence of two-speed genomes in an animal pathogen, shedding light on the evolution of fungal pathogens of vertebrates driving global declines and extinctions.

A Satellite dsRNA Attenuates the Induction of Helper Virus-Mediated Symptoms in Aspergillus flavus.

Aspergillus flavus is an important fungal pathogen of animals and plants. Previously, we reported a novel partitivirus, Aspergillus flavus partitivirus 1 (AfPV1), infecting A. flavus. In this study, we obtained a small double-stranded (ds) RNA segment (734 bp), which is a satellite RNA of the helper virus, AfPV1. The presence of AfPV1 altered the colony morphology, decreased the number of conidiophores, created significantly larger vacuoles, and caused more sensitivity to osmotic, oxidative, and UV stresses in A. flavus, but the small RNA segment could attenuate the above symptoms caused by the helper virus AfPV1 in A. flavus. Moreover, AfPV1 infection reduced the pathogenicity of A. flavus in corn (Zea mays), honeycomb moth (Galleria mellonella), mice (Mus musculus), and the adhesion of conidia to host epithelial cells, and increased conidial death by macrophages. However, the small RNA segment could also attenuate the above symptoms caused by the helper virus AfPV1 in A. flavus, perhaps by reducing the genomic accumulation of the helper virus AfPV1 in A. flavus. We used this model to investigate transcriptional genes regulated by AfPV1 and the small RNA segment in A. flavus, and their role in generating different phenotypes. We found that the pathways of the genes regulated by AfPV1 in its host were similar to those of retroviral viruses. Therefore, some pathways may be of benefit to non-retroviral viral integration or endogenization into the genomes of its host. Moreover, some potential antiviral substances were also found in A. flavus using this system.

Characterization of fungal communities on shared bicycles in Southwest China

BackgroundThe widespread use of shared bicycles has increased the demand and sanitary requirements for shared bicycles. Previous studies have identified potentially pathogenic bacteria on the surfaces of shared bicycles, but fungal communities have not been investigated.MethodsWe sampled shared-bicycle handles and saddles from five selected locations in a metropolis (Chengdu, China, n = 98) and used surrounding air deposition samples as controls (n = 12). Full-length ITS sequencing and multiple bioinformatic analyses were utilized to reveal fungal community structures and differences.ResultsAspergillus was dominant on both the handles and saddles of shared bicycles, and Alternaria and Cladosporium were the most abundant families in the air samples. Significant differences in fungal community structures were found among the three groups. The handle samples contained higher abundances of Aureobasidium melanogenum and Filobasidium magnum than the saddle and air samples. The saddle samples had a higher abundance of Cladosporium tenuissimum than the other two sample types (P < 0·05). A higher abundance of fungal animal pathogens on shared-bicycle surfaces than in air by FUNGuild (P < 0·05). Moreover, the co-occurrence network of fungi on handles was more stable than that on saddles.ConclusionThere were more potential pathogens, including Aspergillus pseudoglaucus, Aureobasidium melanogenum, Kazachstania pintolopesii, Filobasidium magnum, Candida tropicalis, and Malassezia globose were found on shared bicycles than in air, suggesting that hands should not contact mucous membrane after cycling, especially in susceptible individuals, and hygiene management of shared bicycles should be given more attention by relevant organizations worldwide.

Hybridization Facilitates Adaptive Evolution in Two Major Fungal Pathogens.

Hybridization is increasingly recognized as an important force impacting adaptation and evolution in many lineages of fungi. During hybridization, divergent genomes and alleles are brought together into the same cell, potentiating adaptation by increasing genomic plasticity. Here, we review hybridization in fungi by focusing on two fungal pathogens of animals. Hybridization is common between the basidiomycete yeast species Cryptococcus neoformans × Cryptococcus deneoformans, and hybrid genotypes are frequently found in both environmental and clinical settings. The two species show 10–15% nucleotide divergence at the genome level, and their hybrids are highly heterozygous. Though largely sterile and unable to mate, these hybrids can propagate asexually and generate diverse genotypes by nondisjunction, aberrant meiosis, mitotic recombination, and gene conversion. Under stress conditions, the rate of such genetic changes can increase, leading to rapid adaptation. Conversely, in hybrids formed between lineages of the chytridiomycete frog pathogen Batrachochytrium dendrobatidis (Bd), the parental genotypes are considerably less diverged (0.2% divergent). Bd hybrids are formed from crosses between lineages that rarely undergo sex. A common theme in both species is that hybrids show genome plasticity via aneuploidy or loss of heterozygosity and leverage these mechanisms as a rapid way to generate genotypic/phenotypic diversity. Some hybrids show greater fitness and survival in both virulence and virulence-associated phenotypes than parental lineages under certain conditions. These studies showcase how experimentation in model species such as Cryptococcus can be a powerful tool in elucidating the genotypic and phenotypic consequences of hybridization.

Which tree orders in southern Africa have the highest antimicrobial activity and selectivity against bacterial and fungal pathogens of animals?

BackgroundThe study randomly screened leaf extracts of several hundred southern African tree species against important microbial pathogens to determine which taxa have the highest activity and may yield useful products to treat infections in the animal health market.MethodsWe determined the antibacterial and antifungal activity of 714 acetone leaf extracts of 537 different tree species against Enterococcus faecalis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Candida albicans and Cryptococcus neoformans. A sensitive serial dilution microplate method was used.ResultsSeveral extracts had MICs as low as 0.02 mg/ml. We analysed 14 out of the 38 tree orders where we determined the activity of more than 8 different tree species representing 89% of all species examined. There were statistically significant differences in some cases. Celastrales, Rosales and Myrtales had the highest activity against Gram-positive bacteria, the Myrtales and Fabales against the Gram-negative bacteria and the Malvales and Proteales against the fungi. Species present in the Asterales followed by the Gentiales and Lamiales had the lowest activities against all the microorganisms tested. Fabales species had the highest activities against all the microorganisms tested. There was substantial selectivity in some orders. Proteales species had very high activity against the fungi but very low activity against the bacteria. The species in the Celastrales and Rosales had very low antifungal activity, low activity against Gram-negative bacteria and very high activity against Gram-positive bacteria.ConclusionAgainst all classes of microorganisms, the four orders containing species with the highest average antimicrobial activities also contained several species with low activities against different pathogens and vice versa. These results therefore should be used with circumspection in selecting tree orders that would yield the highest probability of finding species with promising activities. Nevertheless there was a twofold increase in probability of finding extracts with interesting antifungal activity from orders with high mean activity than from orders with low mean activity. The probability increased to threefold and fivefold for Gram-positive and Gram-negative bacteria respectively.

Genetic isolation among sympatric vegetative compatibility groups of the aflatoxin-producing fungus Aspergillus flavus

Aspergillus flavus, a fungal pathogen of animals and both wild and economically important plants, is most recognized for producing aflatoxin, a cancer-causing secondary metabolite that contaminates food and animal feed globally. Aspergillus flavus has two self/nonself recognition systems, a sexual compatibility system and a vegetative incompatibility system, and both play a role in directing gene flow in populations. Aspergillus flavus reproduces clonally in wild and agricultural settings, but whether a cryptic sexual stage exists in nature is currently unknown. We investigated the distribution of genetic variation in 243 samples collected over 4 years from three common vegetative compatibility groups (VCGs) in Arizona and Texas from cotton using 24 microsatellite loci and the mating type locus (MAT) to assess population structure and potential gene flow among A. flavus VCGs in sympatric populations. All isolates within a VCG had the same mating type with OD02 having MAT1-2 and both CG136 and MR17 having MAT1-1. Our results support the hypothesis that these three A. flavus VCGs are genetically isolated. We found high levels of genetic differentiation and no evidence of gene flow between VCGs, including VCGs of opposite mating-type. Our results suggest that these VCGs diverged before domestication of agricultural hosts (>10,000 yr bp).

Identification of ENA1 as a Virulence Gene of the Human Pathogenic Fungus Cryptococcus neoformans through Signature-Tagged Insertional Mutagenesis

A library of more than 4,500 signature-tagged insertion mutants of the human pathogenic fungus Cryptococcus neoformans was generated, and a subset was screened in a murine inhalation model to identify genes required for virulence. New genes that regulate aspects of C. neoformans virulence were also identified by screening the entire library for in vitro phenotypes related to the ability to cause disease, including melanin production, growth at high temperature, and growth under conditions of nutrient limitation. A screen of 10% of the strain collection in mice identified an avirulent mutant strain with an insertion in the ENA1 gene, which is predicted to encode a fungus-specific sodium or potassium P-type ATPase. The results of the deletion of the gene and complementation experiments confirmed its key role in mammalian virulence. ena1 mutant strains exhibited no change in sensitivity to high salt concentrations but were sensitive to alkaline pH conditions, providing evidence that the fungus may have to survive at elevated pH during infection of the mammalian host. The mutation of the well-characterized virulence factor calcineurin (CNA1) also rendered C. neoformans strains sensitive to elevated pH. ENA1 transcripts in wild-type and cna1 mutant strains were upregulated in response to high pH, and cna1 ena1 double mutant strains exhibited increased sensitivity to elevated pH, indicating that at least two pathways in the fungus mediate survival under alkaline conditions. Signature-tagged mutagenesis is an effective strategy for the discovery of new virulence genes in fungal pathogens of animals.

Biological activity of two related triterpenes isolated from Combretum nelsonii (Combretaceae) leaves

Antifungal bioassay-guided fractionation of Combretum nelsonii leaf extracts afforded two closely related triterpenes, asiatic acid and arjunolic acid. Antifungal activities of the mixture of asiatic acid and arjunolic acid were determined against five fungal animal pathogens. The minimum inhibitory concentrations of the mixture to the different pathogens varied from 0.2 to 1.6 µg mL−1; Candida albicans (0.9 µg mL−1), Cryptococcus neoformans (0.4 µg mL−1), Aspergillus fumigatus (1.6 µg mL−1), Microsporum canis (0.2 µg mL−1) and Sporothrix schenckii (0.2 µg mL−1). Microsporum canis and S. schenckii were the most susceptible followed by C. neoformans. Aspergillus fumigatus was the most resistant. The Rf value of the mixture of asiatic acid and arjunolic acid was 0.27 in CEF (chloroform : ethylacetate : formic acid), 0.09 (BEA; benzene : ethanol : ammonium hydroxide) and 0.55 (EMW; ethylacetate : methanol : water) which was active against all pathogens. In vitro cytotoxicity of mixture gave an LC50 of 10.58 µg mL−1 towards Vero monkey kidney cells.

The antifungal activity of twenty-four southern African Combretum species (Combretaceae)

The antifungal activities of acetone, hexane, dichloromethane and methanol leaf extracts of 24 South African Combretum species were determined against five fungal animal pathogens (Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus, Microsporum canis and Sporothrix schenckii) representing yeasts, moulds and dimorphic fungi. MIC's determined after 48 h were usually two times higher than values determined after 24 h. Most of the antifungal extracts had MIC values of c. 0.08 mg/ml, some with MIC values as low as 0.02 mg/ml. These are substantially better values that reported in the literature to date. M. canis was the most susceptible microorganism followed by S. schenckii. A. fumigatus was the most resistant of the pathogens tested. Methanol extracted the highest quantity from leaves, but the acetone extracts had the highest antifungal activity in practically all cases. The methanolic extracts of C. moggii and C. petrophilum were however most active against all the pathogens. All extracts of C. nelsonii were also very effective against all the pathogens. Based on these results and work done earlier, C. nelsonii was selected for fractionation and bioassay-guided isolation of the antifungal compounds followed by C. albopuntactum, and C. imberbe.

Antifungal activities of six South African Terminalia species (Combretaceae)

A serial microplate dilution method developed for bacteria was modified slightly and gave good results with several fungi. The antifungal activity of acetone, hexane, dichloromethane and methanol leaf extracts of six Terminalia species ( Terminalia prunioides, Terminalia brachystemma, Terminalia sericea, Terminalia gazensis, Terminalia mollis and Terminalia sambesiaca) were tested against five fungal animal pathogens ( Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus, Microsporum canis and Sporothrix schenkii). Methanol extracted the highest quantity, but the acetone extracts had the highest antifungal activity. Some of the extracts had antioxidant activity. Most of the antifungal extracts had MIC values of c. 0.08 mg/ml, some were with MIC values as low as 0.02 mg/ml. Microsporum canis was the most susceptible microorganism and Terminalia sericea extracts were the most active against nearly all microorganisms tested.

The diversity of antifungal compounds of six South African Terminalia species (Combretaceae) determined by bioautography

A bioautography method was developed to determine the number of antifungal compounds in Terminalia species extracts. Acetone, hexane, dichloromethane and methanol leaf extracts of six Terminalia species (T. prunioides, T. brachystemma, T. sericea, T. gazensis, T. mollis and T. sambesiaca) were tested against five fungal animal pathogens (Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus, Microsporum canis and Sporothrix schenkii). The Rf values and relative activities of separated compounds were determined. Hexane and dichloromethane extracts had at least three times more antifungal compounds than the other extracts indicating the nonpolar character of the antifungal compounds. From the Rf values, the non-polar character of the antifungal compounds was confirmed indicating that the antifungal activity is not due to tannins. M. canis had the highest number, up to ten, of antifungal compounds. All Terminalia species contained a compound (Rf =0.46 in benzene/ethanol/ammonium hydroxide (90/10/1) active against all tested pathogens. T. sericea and T. brachystemma were the most promising candidates for isolating antifungal compounds. The results demonstrate the value of bioautography in examining plant extracts with antifungal activity, selecting species for further study and dereplicating the isolation of compounds.