The Evolution of Sexual Reproduction and the Mating-Type Locus: Links to Pathogenesis of Cryptococcus Human Pathogenic Fungi.

Cryptococcus species utilize a variety of sexual reproduction mechanisms, which generate genetic diversity, purge deleterious mutations, and contribute to their ability to occupy myriad environmental niches and exhibit a range of pathogenic potential. The bisexual and unisexual cycles of pathogenic Cryptococcus species are stimulated by properties associated with their environmental niches and proceed through well-characterized signaling pathways and corresponding morphological changes. Genes governing mating are encoded by the mating-type (MAT) loci and influence pathogenesis, population dynamics, and lineage divergence in Cryptococcus. MAT has undergone significant evolutionary changes within the Cryptococcus genus, including transition from the ancestral tetrapolar state in nonpathogenic species to a bipolar mating system in pathogenic species, as well as several internal reconfigurations. Owing to the variety of established sexual reproduction mechanisms and the robust characterization of the evolution of mating and MAT in this genus, Cryptococcus species provide key insights into the evolution of sexual reproduction.

Genetic transformation of the frog-killing chytrid fungus Batrachochytrium dendrobatidis.

Batrachochytrium dendrobatidis (Bd), a causative agent of chytridiomycosis, is decimating amphibian populations around the world. Bd belongs to the chytrid lineage, a group of early-diverging fungi that are widely used to study fungal evolution. Like all chytrids, Bd develops from a motile form into a sessile, growth form, a transition that involves drastic changes in its cytoskeletal architecture. Efforts to study Bd cell biology, development, and pathogenicity have been limited by the lack of genetic tools with which to test hypotheses about underlying molecular mechanisms. Here, we report the development of a transient genetic transformation system for Bd. We used electroporation to deliver exogenous DNA into Bd cells and detected transgene expression for up to three generations under both heterologous and native promoters. We also adapted the transformation protocol for selection using an antibiotic resistance marker. Finally, we used this system to express fluorescent protein fusions and, as a proof of concept, expressed a genetically encoded probe for the actin cytoskeleton. Using live-cell imaging, we visualized the distribution and dynamics of polymerized actin at each stage of the Bd life cycle, as well as during key developmental transitions. This transformation system enables direct testing of key hypotheses regarding mechanisms of Bd pathogenesis. This technology also paves the way for answering fundamental questions of chytrid cell, developmental, and evolutionary biology.

Beyond morphogenesis and secondary metabolism: function of Velvet proteins and LaeA in fungal pathogenesis.

Velvet proteins, as well as the epigenetic regulator LaeA, are conserved in numerous fungal species, where, in response to environmental cues, they control several crucial cellular processes, including sexual and asexual morphogenesis, secondary metabolism, response to oxidative stress, and virulence. During the last two decades, knowledge of their mechanism of action as well as understanding their functional roles, has greatly increased, particularly in Aspergillus species. Research efforts from multiple groups followed, leading to the characterization of other Velvet and LaeA homologs in species of other fungal genera, including important opportunistic plant and animal pathogens. This review focuses mainly on the current knowledge of the role of Velvet and LaeA function in fungal pathogenesis. Velvet proteins and LaeA are unique to fungi, and for this reason, additional knowledge of these critical regulatory proteins will be important in the development of targeted control strategies to decrease the detrimental impact of fungal pathogens capable of causing disease in plants and animals.

Siderophore of plant growth promoting rhizobacterium origin reduces reactive oxygen species mediated injury in Solanum spp. caused by fungal pathogens.

AIMS: The study aims to explore antifungal properties of bacillibactin siderophore produced by the plant growth-promoting rhizobacterium (PGPR) Bacillus subtilis against fungal phytopathogens Alternaria porri and Fusarium equiseti isolated from Solanum lycopersicum and Solanum melongena plants. METHODS AND RESULTS: Alternaria porri and F. equiseti were isolated from infected plants of eggplant and tomato, respectively. A plate assay was employed to assess the effect of bacillibactin against the phytopathogens. The antifungal potential of the PGPR was evaluated by estimation of dry fungal biomass, visualization of cellular deformity using compound and scanning electron microscopy, antioxidative enzyme assay and analysis of membrane damage via using lipid peroxidation. Inductively coupled plasma atomic emission spectroscopy (ICP-AES) analysis was employed to investigate changes in intracellular iron content. The impact of bacillibactin on pathogenesis was evaluated by infecting detached leaves of S. lycopersicum and S. melongena plants with both the pathogens and treating the infected leaves with bacillibactin. Leaves were further investigated for ROS accumulation, extent of necrosis and cell death. Our findings revealed significant damage to the hyphal structure of A. porri and F. equiseti following treatment with bacillibactin. Biomass reduction, elevated antioxidative enzyme levels, and membrane damage further substantiated the inhibitory effects of the siderophore on fungal growth. ICP-AES analysis indicates an increase in intracellular iron content suggesting enhanced iron uptake facilitated by bacillibactin. Moreover, application of 1500 µg ml-1 bacillibactin on infected leaves demonstrated a substantial inhibition of ROS accumulation, necrosis, and cell death upon bacillibactin treatment. CONCLUSIONS: This study confirms the potent antagonistic activity of bacillibactin against both the phytopathogens A. porri and F. equiseti growth, supporting its potential as a promising biological control agent for fungal plant diseases. Bacillibactin-induced morphological, physiological, and biochemical alterations in the isolated fungi and pathogen-infected leaves highlight the prospects of bacillibactin as an effective and sustainable solution to mitigate economic losses associated with fungal infections in vegetable crops.

Evolution of pathogenicity-associated genes in Rhizoctonia solani AG1-IA by genome duplication and transposon-mediated gene function alterations.

BACKGROUND: Rhizoctonia solani is a polyphagous fungal pathogen that causes diseases in crops. The fungal strains are classified into anastomosis groups (AGs); however, genomic complexity, diversification into the AGs and the evolution of pathogenicity-associated genes remain poorly understood. RESULTS: We report a recent whole-genome duplication and sequential segmental duplications in AG1-IA strains of R. solani. Transposable element (TE) clusters have caused loss of synteny in the duplicated blocks and introduced differential structural alterations in the functional domains of several pathogenicity-associated paralogous gene pairs. We demonstrate that the TE-mediated structural variations in a glycosyl hydrolase domain and a GMC oxidoreductase domain in two paralogous pairs affect the pathogenicity of R. solani. Furthermore, to investigate the association of TEs with the natural selection and evolution of pathogenicity, we sequenced the genomes of forty-two rice field isolates of R. solani AG1-IA. The genomic regions with high population mutation rates and with the lowest nucleotide diversity are enriched with TEs. Genetic diversity analysis predicted the genes that are most likely under diversifying and purifying selections. We present evidence that a smaller variant of a glucosamine phosphate N-acetyltransferase (GNAT) protein, predicted to be under purifying selection, and an LPMP_AA9 domain-containing protein, predicted to be under diversifying selection, are important for the successful pathogenesis of R. solani in rice as well as tomato. CONCLUSIONS: Our study has unravelled whole-genome duplication, TE-mediated neofunctionalization of genes and evolution of pathogenicity traits in R. solani AG1-IA. The pathogenicity-associated genes identified during the study can serve as novel targets for disease control.

A small cysteine-rich fungal effector, BsCE66 is essential for the virulence of Bipolaris sorokiniana on wheat plants.

The Spot Blotch (SB) caused by hemibiotrophic fungal pathogen Bipolaris sorokiniana is one of the most devastating wheat diseases leading to 15-100% crop loss. However, the biology of Triticum-Bipolaris interactions and host immunity modulation by secreted effector proteins remain underexplored. Here, we identified a total of 692 secretory proteins including 186 predicted effectors encoded by B. sorokiniana genome. Gene Ontology categorization showed that these proteins belong to cellular, metabolic and signaling processes, and exhibit catalytic and binding activities. Further, we functionally characterized a cysteine-rich, B. sorokiniana Candidate Effector 66 (BsCE66) that was induced at 24-96 hpi during host colonization. The Δbsce66 mutant did not show vegetative growth defects or stress sensitivity compared to wild-type, but developed drastically reduced necrotic lesions upon infection in wheat plants. The loss-of-virulence phenotype was rescued upon complementing the Δbsce66 mutant with BsCE66 gene. Moreover, BsCE66 does not form homodimer and conserved cysteine residues form intra-molecular disulphide bonds. BsCE66 localizes to the host nucleus and cytosol, and triggers a strong oxidative burst and cell death in Nicotiana benthamiana. Overall, our findings demonstrate that BsCE66 is a key virulence factor that is necessary for host immunity modulation and SB disease progression. These findings would significantly improve our understanding of Triticum-Bipolaris interactions and assist in the development of SB resistant wheat varieties.

Role of the anillin-like protein in growth of Cryptococcus neoformans at human host temperature.

Cryptococcus neoformans, a basidiomycete yeast, causes lethal meningitis in immunocompromised individuals. The ability of C. neoformans to proliferate at 37°C is essential for virulence. We identified anillin-like protein, CnBud4, as essential for proliferation of C. neoformans at 37°C and for virulence in a heterologous host Galleria mellonella at 25°C. C. neoformans cells lacking CnBud4 were inviable at 25°C in the absence of active calcineurin and were hypersensitive to membrane stress and an anti-fungal agent fluconazole, phenotypes previously described for C. neoformans mutants lacking septins. CnBud4 localized to the mother-bud neck during cytokinesis in a septin-dependent manner. In the absence of CnBud4, septin complex failed to transition from a collar-like single ring to the double ring during cytokinesis. In an ascomycete yeast, Saccharomyces cerevisiae, the anillin-like homologue ScBud4 participates in the organization of the septin ring at the mother-bud neck and plays an important role in specifying location for new bud emergence, known as axial budding pattern. In contrast to their role in S. cerevisiae, neither septins nor CnBud4 were needed to direct the position of the new bud in C. neoformans, suggesting that this function is not conserved in basidiomycetous yeasts. Our data suggest that the requirement of CnBud4 for growth at 37°C and pathogenicity in C. neoformans is based on its conserved role in septin complex organization.

Mechanisms of fungal dissemination.

Fungal infections are an increasing threat to global public health. There are more than six million fungal species worldwide, but less than 1% are known to infect humans. Most of these fungal infections are superficial, affecting the hair, skin and nails, but some species are capable of causing life-threatening diseases. The most common of these include Cryptococcus neoformans, Aspergillus fumigatus and Candida albicans. These fungi are typically innocuous and even constitute a part of the human microbiome, but if these pathogens disseminate throughout the body, they can cause fatal infections which account for more than one million deaths worldwide each year. Thus, systemic dissemination of fungi is a critical step in the development of these deadly infections. In this review, we discuss our current understanding of how fungi disseminate from the initial infection sites to the bloodstream, how immune cells eliminate fungi from circulation and how fungi leave the blood and enter distant organs, highlighting some recent advances and offering some perspectives on future directions.

Flying under the radar: Histoplasma capsulatum avoidance of innate immune recognition.

The dimorphic fungal pathogen Histoplasma capsulatum takes advantage of the innate immune system, utilizing host macrophages as a proliferative niche while largely avoiding stimulation of signaling host receptors. As a result, innate immune cells are unable to control H. capsulatum on their own. Not all host phagocytes respond to H. capsulatum in the same way, with neutrophils and dendritic cells playing important roles in impeding fungal growth and initiating a protective TH1 response, respectively. Dendritic cells prime T-cell differentiation after internalization of yeasts via VLA-5 receptors and subsequent degradation of the yeasts. Dendritic cell-expressed TLR7 and TLR9 promote a type I interferon response for TH1 polarization. In contrast to dendritic cells, macrophages provide a hospitable intracellular environment. H. capsulatum yeasts enter macrophages via binding to phagocytic receptors. Simultaneously, α-glucan masks immunostimulatory cell wall ß-glucans and a secreted endoglucanase removes exposed ß-glucans to minimize recognition of yeasts by Dectin-1. This review highlights how phagocytes interact with H. capsulatum yeasts and the mechanisms H. capsulatum uses to limit the innate immune response.

Combatting the evolution of antifungal resistance in Cryptococcus neoformans.

Fungal infections are a global concern and the evolution of intrinsic resistance to current antifungals presents an alarming problem. For Cryptococcus neoformans, a human fungal pathogen of primarily immunocompromised individuals, resistance toward treatment strategies demands alternative approaches. Given the prevalence of virulence factor production during cryptococcal infection, an emerging and important field of research encompasses the development of novel antivirulence therapies proposed to improve host immune responses and promote fungal clearance. To accomplish this task, information regarding the presence and role of virulence factors, the mechanisms of action within the host, and the ability to influence fungal susceptibility to antifungals is pertinent. Research into mechanisms of antifungal resistance for C. neoformans is limited but extrapolation from successful studies in other fungal species can improve our understanding of mechanisms employed by C. neoformans and suggest targeted strategies to enhance our ability to combat the pathogen. In this Review, we highlight antifungal therapy options against Cryptococcus, explore current knowledge of underlying mechanisms promoting resistance, and present new opportunities for novel and effective strategies to overcome fungal infections and reduce, or possibly even reverse, the effects of resistance evolution.

The OxrA Protein of Aspergillus fumigatus Is Required for the Oxidative Stress Response and Fungal Pathogenesis.

An efficient reactive oxygen species (ROS) detoxification system is vital for the survival of the pathogenic fungus Aspergillus fumigatus within the host high-ROS environment of the host. Therefore, identifying and targeting factors essential for oxidative stress response is one approach to developing novel treatments for fungal infections. The oxidation resistance 1 (Oxr1) protein is essential for protection against oxidative stress in mammals, but its functions in pathogenic fungi remain unknown. The present study aimed to characterize the role of an Oxr1 homolog in A. fumigatus. The results indicated that the OxrA protein plays an important role in oxidative stress resistance by regulating the catalase function in A. fumigatus, and overexpression of catalase can rescue the phenotype associated with OxrA deficiency. Importantly, the deficiency of oxrA decreased the virulence of A. fumigatus and altered the host immune response. Using the Aspergillus-induced lung infection model, we demonstrated that the ΔoxrA mutant strain induced less tissue damage along with decreased levels of lactate dehydrogenase (LDH) and albumin release. Additionally, the ΔoxrA mutant caused inflammation at a lower degree, along with a markedly reduced influx of neutrophils to the lungs and a decreased secretion of cytokine usually associated with recruitment of neutrophils in mice. These results characterize the role of OxrA in A. fumigatus as a core regulator of oxidative stress resistance and fungal pathogenesis. IMPORTANCE Knowledge of ROS detoxification in fungal pathogens is useful in the design of new antifungal drugs and could aid in the study of oxidative stress resistance mechanisms. In this study, we demonstrate that OxrA protein localizes to the mitochondria and functions to protect against oxidative damage. We demonstrate that OxrA contributes to oxidative stress resistance by regulating catalase function, and overexpression of catalase (CatA or CatB) can rescue the phenotype that is associated with OxrA deficiency. Remarkably, a loss of OxrA attenuated the fungal virulence in a mouse model of invasive pulmonary aspergillosis and altered the host immune response. Therefore, our finding indicates that inhibition of OxrA might be an effective approach for alleviating A. fumigatus infection. The present study is, to the best of our knowledge, a pioneer in reporting the vital role of Oxr1 protein in pathogenic fungi.

Proteome and secretome profiling of zinc availability in Cryptococcus neoformans identifies Wos2 as a subtle influencer of fungal virulence determinants.

BACKGROUND: Fungal infections impact over 25% of the global population. For the opportunistic fungal pathogen, Cryptococcus neoformans, infection leads to cryptococcosis. In the presence of the host, disease is enabled by elaboration of sophisticated virulence determinants, including polysaccharide capsule, melanin, thermotolerance, and extracellular enzymes. Conversely, the host protects itself from fungal invasion by regulating and sequestering transition metals (e.g., iron, zinc, copper) important for microbial growth and survival. RESULTS: Here, we explore the intricate relationship between zinc availability and fungal virulence via mass spectrometry-based quantitative proteomics. We observe a core proteome along with a distinct zinc-regulated protein-level signature demonstrating a shift away from transport and ion binding under zinc-replete conditions towards transcription and metal acquisition under zinc-limited conditions. In addition, we revealed a novel connection among zinc availability, thermotolerance, as well as capsule and melanin production through the detection of a Wos2 ortholog in the secretome under replete conditions. CONCLUSIONS: Overall, we provide new biological insight into cellular remodeling at the protein level of C. neoformans under regulated zinc conditions and uncover a novel connection between zinc homeostasis and fungal virulence determinants.

Protective effect of fungal extracellular vesicles against murine candidiasis.

Extracellular vesicles (EVs) are lipid bilayered compartments released by virtually all living cells, including fungi. Among the diverse molecules carried by fungal EVs, a number of immunogens, virulence factors and regulators have been characterised. Within EVs, these components could potentially impact disease outcomes by interacting with the host. From this perspective, we previously demonstrated that EVs from Candida albicans could be taken up by and activate macrophages and dendritic cells to produce cytokines and express costimulatory molecules. Moreover, pre-treatment of Galleria mellonella larvae with fungal EVs protected the insects against a subsequent lethal infection with C. albicans yeasts. These data indicate that C. albicans EVs are multi-antigenic compartments that activate the innate immune system and could be exploited as vaccine formulations. Here, we investigated whether immunisation with C. albicans EVs induces a protective effect against murine candidiasis in immunosuppressed mice. Total and fungal antigen-specific serum IgG antibodies increased by 21 days after immunisation, confirming the efficacy of the protocol. Vaccination decreased fungal burden in the liver, spleen and kidney of mice challenged with C. albicans. Splenic levels of cytokines indicated a lower inflammatory response in mice immunised with EVs when compared with EVs + Freund's adjuvant (ADJ). Higher levels of IL-12p70, TNFα and IFNγ were detected in mice vaccinated with EVs + ADJ, while IL-12p70, TGFß, IL-4 and IL-10 were increased when no adjuvants were added. Full protection of lethally challenged mice was observed when EVs were administered, regardless the presence of adjuvant. Physical properties of the EVs were also investigated and EVs produced by C. albicans were relatively stable after storage at 4, -20 or -80°C, keeping their ability to activate dendritic cells and to protect G. mellonella against a lethal candidiasis. Our data suggest that fungal EVs could be a safe source of antigens to be exploited in vaccine formulations.

Host Gamma-Aminobutyric Acid Metabolic Pathway Is Involved in Resistance Against Rhizoctonia solani.

Rhizoctonia solani is a highly destructive necrotrophic fungal pathogen having a diverse host range, including rice and tomato. Previously R. solani infection has been found to cause large-scale readjustment in host primary metabolism and accumulation of various stress-associated metabolites such as gamma-aminobutyric acid (GABA) in rice. In this study, we report upregulation of GABA pathway genes during pathogenesis of R. solani in rice and tomato. The exogenous application of GABA provided partial resistance against R. solani infection in both the hosts. Furthermore, by using the virus-induced gene silencing approach, we knocked down the expression of some of the tomato genes involved in GABA biosynthesis (glutamate decarboxylase) and GABA catabolism (GABA-transaminase and succinic semialdehyde dehydrogenase) to study their role in host defense against R. solani infection. The silencing of each of these genes increased disease susceptibility in tomato. Overall the results from gene expression analysis, exogenous chemical application, and gene silencing studies suggest that the GABA pathway plays a positive role in plant defense against necrotrophic pathogen R. solani.

Proteomics of Cryptococcus neoformans: From the Lab to the Clinic.

Fungal pathogens cause an array of diseases by targeting both immunocompromised and immunocompetent hosts. Fungi overcome our current arsenal of antifungals through the emergence and evolution of resistance. In particular, the human fungal pathogen, Cryptococcus neoformans is found ubiquitously within the environment and causes severe disease in immunocompromised individuals around the globe with limited treatment options available. To uncover fundamental knowledge about this fungal pathogen, as well as investigate new detection and treatment strategies, mass spectrometry-based proteomics provides a plethora of tools and applications, as well as bioinformatics platforms. In this review, we highlight proteomics approaches within the laboratory to investigate changes in the cellular proteome, secretome, and extracellular vesicles. We also explore regulation by post-translational modifications and the impact of protein-protein interactions. Further, we present the development and comprehensive assessment of murine models of cryptococcal infection, which provide valuable tools to define the dynamic relationship between the host and pathogen during disease. Finally, we explore recent quantitative proteomics studies that begin to extrapolate the findings from the bench to the clinic for improved methods of fungal detection and monitoring. Such studies support a framework for personalized medical approaches to eradicate diseases caused by C. neoformans.

The Paralogous Transcription Factors Stp1 and Stp2 of Candida albicans Have Distinct Functions in Nutrient Acquisition and Host Interaction.

Nutrient acquisition is a central challenge for all organisms. For the fungal pathogen Candida albicans, utilization of amino acids has been shown to be critical for survival, immune evasion, and escape, while the importance of catabolism of host-derived proteins and peptides in vivo is less well understood. Stp1 and Stp2 are paralogous transcription factors (TFs) regulated by the Ssy1-Ptr3-Ssy5 (SPS) amino acid sensing system and have been proposed to have distinct, if uncertain, roles in protein and amino acid utilization. We show here that Stp1 is required for proper utilization of peptides but has no effect on amino acid catabolism. In contrast, Stp2 is critical for utilization of both carbon sources. Commensurate with this observation, we found that Stp1 controls a very limited set of genes, while Stp2 has a much more extensive regulon that is partly dependent on the Ssy1 amino acid sensor (amino acid uptake and catabolism) and partly Ssy1 independent (genes associated with filamentous growth, including the regulators UME6 and SFL2). The ssy1Δ/Δ and stp2Δ/Δ mutants showed reduced fitness in a gastrointestinal (GI) colonization model, yet induced greater damage to epithelial cells and macrophages in a manner that was highly dependent on the growth status of the fungal cells. Surprisingly, the stp1Δ/Δ mutant was better able to colonize the gut but the mutation had no effect on host cell damage. Thus, proper protein and amino acid utilization are both required for normal host interaction and are controlled by an interrelated network that includes Stp1 and Stp2.

Cryptococcus neoformans Evades Pulmonary Immunity by Modulating Xylose Precursor Transport.

Cryptococcus neoformans is a fungal pathogen that kills almost 200,000 people each year and is distinguished by abundant and unique surface glycan structures that are rich in xylose. A mutant strain of C. neoformans that cannot transport xylose precursors into the secretory compartment is severely attenuated in virulence in mice yet surprisingly is not cleared. We found that this strain failed to induce the nonprotective T helper cell type 2 (Th2) responses characteristic of wild-type infection, instead promoting sustained interleukin 12p40 (IL-12p40) induction and increased IL-17A (IL-17) production. It also stimulated dendritic cells to release high levels of proinflammatory cytokines, a behavior we linked to xylose expression. We further discovered that inducible bronchus-associated lymphoid tissue (iBALT) forms in response to infection with either wild-type cryptococci or the mutant strain with reduced surface xylose; although iBALT formation is slowed in the latter case, the tissue is better organized. Finally, our temporal studies suggest that lymphoid structures in the lung restrict the spread of mutant fungi for at least 18 weeks after infection, which is in contrast to ineffective control of the pathogen after infection with wild-type cells. These studies demonstrate the role of xylose in modulation of host response to a fungal pathogen and show that cryptococcal infection triggers iBALT formation.

Genome Sequence Resource for Elsinoë ampelina, the Causal Organism of Grapevine Anthracnose.

Elsinoë ampelina is an ascomycetous fungus that causes grape anthracnose, a potentially devastating disease worldwide. Here, we report a 28.29 Mb high-quality genome sequence of E. ampelina YL-1 that encodes 8,057 predicted protein-coding genes and represents the first sequenced genome assembly of E. ampelina. This study adds to the current genomic resources for the genus Elsinoë and paves the way for research on comparative genomic studies, E. ampelina-grape interactions, and improvement of management strategies.

Whole-Genome Approach to Understanding the Mechanism of Action of a Histatin 5-Derived Peptide.

The incidence of opportunistic fungal infections that threaten immunocompromised patients, along with the limited arsenal of antifungal drugs, calls for renewed efforts to develop novel antifungal therapies. Antimicrobial peptides have garnered interest as potential therapeutics. Among naturally occurring peptides, histatin 5 is a well-characterized 24-amino-acid peptide with strong antifungal activity. Our lab has identified a smaller histatin derivative, KM29, with stronger activity against multiple Candida spp., prompting us to investigate its fungicidal mechanism. A genetic screen was developed to test the Saccharomyces cerevisiae genomewide deletion collection for mutants with increased or decreased peptide sensitivity. The goal was to identify genes that would reveal insights into the mechanism of action of KM29, to be assessed in Candida albicans Several biological processes yielded increased sensitivity, with endosomal transport and vacuolar function appearing at high frequencies. Among the pathways involved in increased resistance, mitochondrial function showed the highest normalized genome frequency; hence, we focused on characterizing this pathway. KM29 localizes to mitochondria, and the killing activity depends on a functional electron transport chain. In addition, KM29 triggered reactive oxygen species (ROS) production, which was responsible for some cell death but insufficient to account for the complete killing activity. In agreement with this finding, we found that KM29 induced mitochondrial fragmentation and a mild loss of mitochondrial membrane potential. Furthermore, respiratory mutants exhibited severely diminished KM29 uptake. We confirmed this behavior in a C. albicans respiratory mutant. Taking our findings together, this work delineates the mitochondrial functions associated with KM29 fungicidal activity and provides additional pathways for further characterization in Candida spp.

ATG Genes Influence the Virulence of Cryptococcus neoformans through Contributions beyond Core Autophagy Functions.

The process of autophagy is conserved among all eukaryotes from yeast to humans and is mainly responsible for bulk degradation of cellular contents and nutrient recycling during starvation. Autophagy has been suggested to play a role in the pathogenesis of the opportunistic human fungal pathogen Cryptococcus neoformans, potentially through a contribution to the export of virulence factors. In this study, we showed that deletion of each of the ATG1, ATG7, ATG8, and ATG9 genes in C. neoformans leads to autophagy-related phenotypes, including impaired amino acid homeostasis under nitrogen starvation. In addition, the atgΔ mutants were hypersensitive to inhibition of the ubiquitin-proteasome system, a finding consistent with a role in amino acid homeostasis. Although each atgΔ mutant was not markedly impaired in virulence factor production in vitro, we found that all four ATG genes contribute to C. neoformans virulence in a murine inhalation model of cryptococcosis. Interestingly, these mutants displayed significant differences in their ability to promote disease development. A more detailed investigation of virulence for the atg1Δ and atg8Δ mutants revealed that both strains stimulated an exaggerated host immune response, which, in turn, contributed to disease severity. Overall, our results suggest that different ATG genes are involved in nonautophagic functions and contribute to C. neoformans virulence beyond their core functions in autophagy.