Malassezia commensal yeasts are associated with a number of skin disorders, such as atopic eczema/dermatitis and dandruff, and they also can cause systemic infections. Here we describe the 7.67-Mbp genome of Malassezia sympodialis, a species associated with atopic eczema, and contrast its genome repertoire with that of Malassezia globosa, associated with dandruff, as well as those of other closely related fungi. Ninety percent of the predicted M. sympodialis protein coding genes were experimentally verified by mass spectrometry at the protein level. We identified a relatively limited number of genes related to lipid biosynthesis, and both species lack the fatty acid synthase gene, in line with the known requirement of these yeasts to assimilate lipids from the host. Malassezia species do not appear to have many cell wall-localized glycosylphosphatidylinositol (GPI) proteins and lack other cell wall proteins previously identified in other fungi. This is surprising given that in other fungi these proteins have been shown to mediate interactions (e.g., adhesion and biofilm formation) with the host. The genome revealed a complex evolutionary history for an allergen of unknown function, Mala s 7, shown to be encoded by a member of an amplified gene family of secreted proteins. Based on genetic and biochemical studies with the basidiomycete human fungal pathogen Cryptococcus neoformans, we characterized the allergen Mala s 6 as the cytoplasmic cyclophilin A. We further present evidence that M. sympodialis may have the capacity to undergo sexual reproduction and present a model for a pseudobipolar mating system that allows limited recombination between two linked MAT loci.

Acetolactate synthase catalyses the first common step in isoleucine and valine biosynthesis and is the target of several classes of inhibitors. The Cryptococcus neoformans ILV2 gene, encoding acetolactate synthase, was identified by complementation of a Saccharomyces cerevisiae ilv2 mutant. C. neoformans is highly resistant to the commercially available acetolactate synthase inhibitor, sulfometuron methyl (SM). Expression of C. neoformans ILV2 in S. cerevisiae conferred SM resistance, indicating that the SM resistance of C. neoformans is due, at least in part, to C. neoformans Ilv2p. The C. neoformans ILV2 gene was disrupted. The ilv2 mutants were auxotrophic for isoleucine and valine and the auxotrophy was satisfied by these amino acids only when proline, and not ammonium, was the nitrogen source, indicating nitrogen regulation of amino acid transport. ilv2 mutants rapidly lost viability at 37 °C and when starved for isoleucine and valine. Consistent with these phenotypes, an ilv2 mutant was avirulent and unable to survive in mice. Because C. neoformans Ilv2p is required for virulence and survival in vivo, inhibitors of branched-chain amino acid biosynthesis may make valuable antifungal agents.

This paper describes (i) the expression profile of the methionine synthase gene (MET6) in the human pathogenic fungus Cryptococcus neoformans and (ii) the phenotypes of a C. neoformans met6 mutant. In contrast to the MET3 gene, which showed no significant change in expression in any environmental condition tested, the MET6 gene showed a substantial induction in response to methionine and a dramatic transcriptional induction in response to homocysteine. Like a met3 mutant, the met6 mutant was a methionine auxotroph. However, relative to a met3 mutant, the met6 mutant grew very slowly and was less heat-shock resistant. In contrast to a met3 mutant, the met6 mutant lost viability when starved of methionine, and it was deficient in capsule formation. Like a met3 mutant, the met6 mutant was avirulent. In contrast to a met3 mutant, the met6 mutant was hypersensitive to fluconazole and to the calcineurin inhibitors FK506 and cyclosporin A. A synergistic fungicidal effect was also found between each of these drugs and met6. The phenotypic differences between the met3 and met6 mutants may be due to the accumulation in met6 mutants of homocysteine, a toxic metabolic intermediate that inhibits sterol biosynthesis.

The conserved target of rapamycin complex 1 (TORC1) integrates nutrient signals to orchestrate cell growth and proliferation. Leucine availability is conveyed to control TORC1 activity via the leu-tRNA synthetase/EGOC-GTPase module in yeast and mammals, but the mechanisms sensing leucine remain only partially understood. We show here that both leucine and its α-ketoacid metabolite, α-ketoisocaproate, effectively activate the yeast TORC1 kinase via both EGOC GTPase-dependent and -independent mechanisms. Leucine and α-ketoisocaproate are interconverted by ubiquitous branched-chain aminotransferases (BCAT), which in yeast are represented by the mitochondrial and cytosolic enzymes Bat1 and Bat2, respectively. BCAT yeast mutants exhibit severely compromised TORC1 activity, which is partially restored by expression of Bat1 active site mutants, implicating both catalytic and structural roles of BCATs in TORC1 control. We find that Bat1 interacts with branched-chain amino acid metabolic enzymes and, in a leucine-dependent fashion, with the tricarboxylic acid (TCA)-cycle enzyme aconitase. BCAT mutation perturbed TCA-cycle intermediate levels, consistent with a TCA-cycle block, and resulted in low ATP levels, activation of AMPK, and TORC1 inhibition. We propose the biosynthetic capacity of BCAT and its role in forming multicomplex metabolons connecting branched-chain amino acids and TCA-cycle metabolism governs TCA-cycle flux to activate TORC1 signaling. Because mammalian mitochondrial BCAT is known to form a supramolecular branched-chain α-keto acid dehydrogenase enzyme complex that links leucine metabolism to the TCA-cycle, these findings establish a precedent for understanding TORC1 signaling in mammals.

The genus Malassezia includes 14 species that are found on the skin of humans and animals and are associated with a number of diseases. Recent genome sequencing projects have defined the gene content of all 14 species; however, to date, genetic manipulation has not been possible for any species within this genus. Here, we develop and then optimize molecular tools for the transformation of Malassezia furfur and Malassezia sympodialis using Agrobacterium tumefaciens delivery of transfer DNA (T-DNA) molecules. These T-DNAs can insert randomly into the genome. In the case of M. furfur, targeted gene replacements were also achieved via homologous recombination, enabling deletion of the ADE2 gene for purine biosynthesis and of the LAC2 gene predicted to be involved in melanin biosynthesis. Hence, the introduction of exogenous DNA and direct gene manipulation are feasible in Malassezia species.

The isoleucine and valine biosynthetic enzyme acetolactate synthase (Ilv2p) is an attractive antifungal drug target, since the isoleucine and valine biosynthetic pathway is not present in mammals, Saccharomyces cerevisiae ilv2Δ mutants do not survive in vivo, Cryptococcus neoformans ilv2 mutants are avirulent, and both S. cerevisiae and Cr. neoformans ilv2 mutants die upon isoleucine and valine starvation. To further explore the potential of Ilv2p as an antifungal drug target, we disrupted Candida albicans ILV2, and demonstrated that Ca. albicans ilv2Δ mutants were significantly attenuated in virulence, and were also profoundly starvation-cidal, with a greater than 100-fold reduction in viability after only 4 h of isoleucine and valine starvation. As fungicidal starvation would be advantageous for drug design, we explored the basis of the starvation-cidal phenotype in both S. cerevisiae and Ca. albicans ilv2Δ mutants. Since the mutation of ILV1, required for the first step of isoleucine biosynthesis, did not suppress the ilv2Δ starvation-cidal defects in either species, the cidal phenotype was not due to α-ketobutyrate accumulation. We found that starvation for isoleucine alone was more deleterious in Ca. albicans than in S. cerevisiae, and starvation for valine was more deleterious than for isoleucine in both species. Interestingly, while the target of rapamycin (TOR) pathway inhibitor rapamycin further reduced S. cerevisiae ilv2Δ starvation viability, it increased Ca. albicans ilv1Δ and ilv2Δ viability. Furthermore, the recovery from starvation was dependent on the carbon source present during recovery for S. cerevisiae ilv2Δ mutants, reminiscent of isoleucine and valine starvation inducing a viable but non-culturable-like state in this species, while Ca. albicans ilv1Δ and ilv2Δ viability was influenced by the carbon source present during starvation, supporting a role for glucose wasting in the Ca. albicans cidal phenotype.

The number of plastic-degrading microorganisms reported is rapidly increasing, making it possible to explore the conservation and distribution of presumed plastic-degrading traits across the diverse microbial tree of life. Putative degraders of conventional high-molecular-weight polymers, including polyamide, polystyrene, polyvinylchloride, and polypropylene, are spread widely across bacterial and fungal branches of the tree of life, although evidence for plastic degradation by a majority of these taxa appears limited. In contrast, we found strong degradation evidence for the synthetic polymer polylactic acid (PLA), and the microbial species related to its degradation are phylogenetically conserved among the bacterial family Pseudonocardiaceae. We collated data on genes and enzymes related to the degradation of all types of plastic to identify 16,170 putative plastic degradation orthologs by mining publicly available microbial genomes. The plastic with the largest number of putative orthologs, 10,969, was the natural polymer polyhydroxybutyrate (PHB), followed by the synthetic polymers polyethylene terephthalate (PET) and polycaprolactone (PCL), with 8,233 and 6,809 orthologs, respectively. These orthologous genes were discovered in the genomes of 6,000 microbial species, and most of them are as yet not identified as plastic degraders. Furthermore, all these species belong to 12 different microbial phyla, of which just 7 phyla have reported degraders to date. We have centralized information on reported plastic-degrading microorganisms within an interactive and updatable phylogenetic tree and database to confirm the global and phylogenetic diversity of putative plastic-degrading taxa and provide new insights into the evolution of microbial plastic-degrading capabilities and avenues for future discovery. IMPORTANCE We have collated the most complete database of microorganisms identified as being capable of degrading plastics to date. These data allow us to explore the phylogenetic distribution of these organisms and their enzymes, showing that traits for plastic degradation are predominantly not phylogenetically conserved. We found 16,170 putative plastic degradation orthologs in the genomes of 12 different phyla, which suggests a vast potential for the exploration of these traits in other taxa. Besides making the database available to the scientific community, we also created an interactive phylogenetic tree that can display all of the collated information, facilitating visualization and exploration of the data. Both the database and the tree are regularly updated to keep up with new scientific reports. We expect that our work will contribute to the field by increasing the understanding of the genetic diversity and evolution of microbial plastic-degrading traits.

Global plastic production has increased exponentially since manufacturing commenced in the 1950’s, including polymer types infused with diverse additives and fillers. While the negative impacts of plastics are widely reported, particularly on marine vertebrates, impacts on microbial life remain poorly understood. Plastics impact microbiomes directly, exerting toxic effects, providing supplemental carbon sources and acting as rafts for microbial colonisation and dispersal. Indirect consequences include increased environmental shading, altered compositions of host communities and disruption of host organism or community health, hormone balances and immune responses. The isolation and application of plastic-degrading microbes are of substantial interest yet little evidence supports the microbial biodegradation of most high molecular weight synthetic polymers. Over 400 microbial species have been presumptively identified as capable of plastic degradation, but evidence for the degradation of highly prevalent polymers including polypropylene, nylon, polystyrene and polyvinyl chloride must be treated with caution; most studies fail to differentiate losses caused by the leaching or degradation of polymer monomers, additives or fillers. Even where polymer degradation is demonstrated, such as for polyethylene terephthalate, the ability of microorganisms to degrade more highly crystalline forms of the polymer used in commercial plastics appears limited. Microbiomes frequently work in conjunction with abiotic factors such as heat and light to impact the structural integrity of polymers and accessibility to enzymatic attack. Consequently, there remains much scope for extremophile microbiomes to be explored as a source of plastic-degrading enzymes and microorganisms. We propose a best-practice workflow for isolating and reporting plastic-degrading taxa from diverse environmental microbiomes, which should include multiple lines of evidence supporting changes in polymer structure, mass loss, and detection of presumed degradation products, along with confirmation of microbial strains and enzymes (and their associated genes) responsible for high molecular weight plastic polymer degradation. Such approaches are necessary for enzymatic degraders of high molecular weight plastic polymers to be differentiated from organisms only capable of degrading the more labile carbon within predominantly amorphous plastics, plastic monomers, additives or fillers.