HGT in the human and skin commensal Malassezia: A bacterially derived flavohemoglobin is required for NO resistance and host interaction Giuseppe Ianiria,1, Marco A. Coelhoa, Fiorella Ruchtib, Florian Sparberb, Timothy J. McMahonc,CiFua, Madison Bolejackd,e, Olivia Donovane,f, Hayden Smutneye,f, Peter Mylere,g,h,i, Fred Dietricha, David Fox IIId,e, Salomé LeibundGut-Landmannb, and Joseph Heitmana,2 aDepartment of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710; bSection of Immunology, Vetsuisse Faculty, University of Zürich, CH-8057 Zürich, Switzerland; cDepartment of Medicine, Veterans Affairs Medical Center, Durham, NC 27705; dUnion Chimique Belge Pharma, Bainbridge Island, WA 98110; eSeattle Structural Genomics Center for Infectious Disease, Seattle, WA 98101; fUnion Chimique Belge Pharma, Bedford, MA 01730; gCenter for Global Infectious Disease Research, Seattle Children’s Research Institute, Seattle, WA 98101; hDepartment of Biomedical Information and Medical Education, University of Washington, Seattle, WA 98195; and iDepartment of Global Health, University of Washington, Seattle, WA 98195 Edited by Jay C. Dunlap, Geisel School of Medicine, Dartmouth College, Hanover, NH, and approved May 18, 2020 (received for review February 26, 2020) The skin of humans and animals is colonized by commensal and disease in patients with CARD9 mutations, and in accelerating pathogenic fungi and bacteria that share this ecological niche and the progression of pancreatic adenocarcinoma in murine models have established microbial interactions. Malassezia are the most and in humans, and cystic fibrosis pulmonary exacerbation (6–8). abundant fungal skin inhabitant of warm-blooded animals and Nitric oxide (NO) is a reactive compound of central importance have been implicated in skin diseases and systemic disorders, in- in biological systems and it functions both as a signaling and toxic cluding Crohn’s disease and pancreatic cancer. Flavohemoglobin is molecule. While little is known about NO synthesis in fungi, in a key enzyme involved in microbial nitrosative stress resistance mammals NO is synthesized by NO synthases (NOS isoforms). and nitric oxide degradation. Comparative genomics and phyloge- NOS1 and NOS3 are constitutively expressed in neurons and en- netic analyses within the Malassezia genus revealed that dothelium, respectively, and produce NO to promote S-nitrosylation flavohemoglobin-encoding genes were acquired through indepen- and transcriptional regulation. S-nitrosylation is a posttranslational MICROBIOLOGY dent horizontal gene transfer events from different donor bacteria mechanism involving oxidative modification of cysteine by NO, that are part of the mammalian microbiome. Through targeted gene deletion and functional complementation in Malassezia sym- Significance podialis, we demonstrated that bacterially derived flavohemoglo- bins are cytoplasmic proteins required for nitric oxide detoxification Malassezia and nitrosative stress resistance under aerobic conditions. RNA- species are the main fungal components of the sequencing analysis revealed that endogenous accumulation of mammalian skin microbiome and are associated with a number Malassezia nitric oxide resulted in up-regulation of genes involved in stress of skin disorders. Recently, has also been found in ’ response and down-regulation of the MalaS7 allergen-encoding association with Crohn s disease and with pancreatic cancer. genes. Solution of the high-resolution X-ray crystal structure of The elucidation of the molecular bases of skin adaptation by Malassezia Malassezia flavohemoglobin revealed features conserved with both is critical to understand its role as commensal and bacterial and fungal flavohemoglobins. In vivo pathogenesis is in- pathogen. In this study we employed evolutionary, molecular, dependent of Malassezia flavohemoglobin. Lastly, we identified an biochemical, and structural analyses to demonstrate that the Malassezia additional 30 genus- and species-specific horizontal gene transfer bacterially derived flavohemoglobins acquired by candidates that might have contributed to the evolution of this through horizontal gene transfer resulted in a gain of function genus as the most common inhabitants of animal skin. critical for nitric oxide detoxification and resistance to nitro- sative stress. Our study underscores horizontal gene transfer as Malassezia Malassezia | flavohemoglobin | horizontal gene transfer an important force modulating evolution and niche adaptation. he skin microbiome includes numerous microorganisms that Author contributions: G.I., S.L.-L., and J.H. designed research; G.I., M.A.C., F.R., F.S., C.F., Testablish a variety of direct and indirect interactions char- M.B., O.D., and H.S. performed research; G.I., T.J.M., P.M., D.F., and S.L.-L. contributed acterized by the exchange of genetic material that impact mi- new reagents/analytic tools; G.I., M.A.C., F.R., F.S., C.F., M.B., F.D., D.F., and S.L.-L. ana- crobial biology, contributing to their speciation and evolution. lyzed data; and G.I. wrote the paper with contributions from M.A.C., M.B., D.F., S.L.-L., and J.H. Malassezia is the most abundant fungal genus resident on human The authors declare no competing interest. skin, representing more than 90% of the skin mycobiome (1). This genus presently consists of 18 diverse species (2), each with This article is a PNAS Direct Submission. an unusually compact genome that underwent extensive gene Published under the PNAS license. turnover events as a result of evolutionary adaptation and col- Data deposition: The sequence data generated in this study have been submitted to the National Center for Biotechnology Information under BioProject accession number onization to a nutrient-limited ecological niche such as the skin PRJNA626605. Individual accession numbers are SRR11574550 for RNA-seq reads of (3). Although commensals, Malassezia species are also associated Malassezia WT untreated control samples; SRR11574549 for RNA-seq reads of Malassezia with a number of clinical skin disorders, including pityriasis WT NO-treated samples; and SRR11574548 for RNA-seq Malassezia yhb1Δ mutant. The final structure factors and coordinates of the flavohemolgobin Yhb101 of Malassezia yamatoensis versicolor, dandruff, and atopic dermatitis (AD) (4). A recently have been deposited in the Protein Data Bank (PDB) under code 6O0A. developed epicutaneous murine model revealed that the host 1Present address: Department of Agricultural, Environmental and Food Sciences, Univer- responses to Malassezia are dominated by proinflammatory cy- sity of Molise, 86100 Campobasso, Italy. tokine IL-17 and related factors that prevent fungal overgrowth 2To whom correspondence may be addressed. Email: [email protected]. and exacerbate inflammation under atopy-like conditions (5). This article contains supporting information online at https://www.pnas.org/lookup/suppl/ Furthermore, Malassezia species have also been implicated re- doi:10.1073/pnas.2003473117/-/DCSupplemental. cently as causal agents of Crohn’s disease/inflammatory bowel www.pnas.org/cgi/doi/10.1073/pnas.2003473117 PNAS Latest Articles | 1of11 Downloaded by guest on September 25, 2021 and this is the central NO-mediated signaling mechanism that and whether it had a fungal or bacterial origin. BLAST analyses affects myriad of cellular physiological and pathophysiological with the M. globosa Yhb1 sequence as query identified a single processes (9). On the other hand, NOS2 is not constitutively copy of YHB1 in all Malassezia species and strains with sequenced expressed but is induced in inflammatory cells in response to in- genomes. Intriguingly, this comparative search revealed that the fection and is involved in wound healing, immune regulation, and best hits in Malassezia yamatoensis and Malassezia slooffiae had host defense (10). lower E values (5e-64 and 1e-59, respectively) compared to the In fungi, NO is synthesized through a reductive denitrification remaining Malassezia species (E values ranging from 0.0 to 7e- pathway from nitrite, and through an oxidative pathway from 161), possibly suggesting different origins or modifications of L-arginine, although the detailed biochemical mechanisms have these flavohemoglobin-encoding genes. To elucidate the evolu- not yet been fully elucidated (11–13). Compared to mammals, tionary trajectory of flavohemoglobin within the whole Malassezia plants, and bacteria, the role of NO in fungal biology is under- genus, a maximum likelihood (ML) phylogenetic tree was recon- studied. In Saccharomyces cerevisiae NO is important for acti- structed using >2,000 Yhb1 bacterial and fungal sequences vation of transcription factors that are involved in resistance to a retrieved from GenBank. This phylogenetic analysis revealed two variety of environmental stress conditions, such as oxidative clades of flavohemoglobin within the Malassezia genus: clade 1 stress, heat shock, and hydrostatic pressure (11). Other studies that includes 13 species and clusters together with Brevi- report an involvement of NO in pathogenesis of Botrytis cinerea bacterium species belonging to Actinobacteria; and clade 2 that and Magnaporthe oryzae, in morphogenesis and reproduction in includes M. yamatoensis and M. slooffiae and clusters together Aspergillus nidulans, and in the yeast-to-hyphae dimorphic tran- with different Actinobacteria, with the closest relative being sition in Candida albicans (12, 14, 15). Kocuria kristinae (Fig. 1 A–C). To evaluate the statistical