Dataset Information

Gene Function Analysis in the Ubiquitous Human Commensal and Pathogen Malassezia Genus.

ABSTRACT: 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.

Importance

Species in the genus Malassezia are a defining component of the microbiome of the surface of mammals. They are also associated with a wide range of skin disease symptoms. Many species are difficult to culture in vitro, and although genome sequences are available for the species in this genus, it has not been possible to assess gene function to date. In this study, we pursued a series of possible transformation methods and identified one that allows the introduction of DNA into two species of Malassezia, including the ability to make targeted integrations into the genome such that genes can be deleted. This research opens a new direction in terms of now being able to analyze gene functions in this little understood genus. These tools will contribute to define the mechanisms that lead to the commensalism and pathogenicity in this group of obligate fungi that are predominant on the skin of mammals.

SUBMITTER: Ianiri G

PROVIDER: S-EPMC5137500 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Staphylococcus hominis is frequently isolated from human skin, and we hypothesize that it may protect the cutaneous barrier from opportunistic pathogens. We determined that S. hominis makes six unique autoinducing peptide (AIP) signals that inhibit the major virulence factor accessory gene regulator (agr) quorum sensing system of Staphylococcus aureus. We solved and confirmed the structures of three novel AIP signals in conditioned medium by mass spectrometry and then validated synthetic AIP activity against all S. aureus agr classes. Synthetic AIPs also inhibited the conserved agr system in a related species, Staphylococcus epidermidis. We determined the distribution of S. hominis agr types on healthy human skin and found S. hominis agr-I and agr-II were highly represented across subjects. Further, synthetic AIP-II was protective in vivo against S. aureus-associated dermonecrotic or epicutaneous injury. Together, these findings demonstrate that a ubiquitous colonizer of human skin has a fundamentally protective role against opportunistic damage. IMPORTANCE Human skin is home to a variety of commensal bacteria, including many species of coagulase-negative staphylococci (CoNS). While it is well established that the microbiota as a whole maintains skin homeostasis and excludes pathogens (i.e., colonization resistance), relatively little is known about the unique contributions of individual CoNS species to these interactions. Staphylococcus hominis is the second most frequently isolated CoNS from healthy skin, and there is emerging evidence to suggest that it may play an important role in excluding pathogens, including Staphylococcus aureus, from colonizing or infecting the skin. Here, we identified that S. hominis makes 6 unique peptide inhibitors of the S. aureus global virulence factor regulation system (agr). Additionally, we found that one of these peptides can prevent topical or necrotic S. aureus skin injury in a mouse model. Our results demonstrate a specific and broadly protective role for this ubiquitous, yet underappreciated skin commensal.

Project description:UNLABELLED: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. IMPORTANCE:Malassezia commensal yeasts are associated with a number of skin disorders. The previously published genome of M. globosa provided some of the first insights into Malassezia biology and its involvement in dandruff. Here, we present the genome of M. sympodialis, frequently isolated from patients with atopic eczema and healthy individuals. We combined comparative genomics with sequencing and functional characterization of specific genes in a population of clinical isolates and in closely related model systems. Our analyses provide insights into the evolution of allergens related to atopic eczema and the evolutionary trajectory of the machinery for sexual reproduction and meiosis. We hypothesize that M. sympodialis may undergo sexual reproduction, which has important implications for the understanding of the life cycle and virulence potential of this medically important yeast. Our findings provide a foundation for the development of genetic and genomic tools to elucidate host-microbe interactions that occur on the skin and to identify potential therapeutic targets.

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Gene Function Analysis in the Ubiquitous Human Commensal and Pathogen Malassezia Genus.

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