HGT in the Human and Skin Commensal Malassezia: a Bacterially Derived Flavohemoglobin Is Required for NO Resistance and Host Interaction

Home , Allantoicase, Formamidase, Isopenicillin N synthase, Malassezia dermatis, Malassezia nana, Malassezia sympodialis

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 flavohemoglobins 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 nitrosative 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 phy- Imbalance in cellular NO levels leads to altered redox ho- logenetic support for the two Malassezia flavohemoglobin clades meostasis, resulting in the production of reactive nitrogen species whose distribution was not monophyletic, we performed ap- that are responsible for nitrosative stress (10). NO dioxygenases proximately unbiased (AU) comparative topology tests. The are enzymes that living cells use to actively consume poisonous constrained ML phylogeny, in which all Malassezia flavohemoglobins NO by converting it to inert nitrate, a source of nitrogen (16). Red were forced to be monophyletic was significantly rejected (AU blood cell hemoglobin is the main mammalian dioxygenase that test, P value = 0.001, Fig. 1D), thus not supporting the null metabolizes NO in the vascular lumen, whereas a type I flavo- hypothesis that all flavohemoglobin genes in Malassezia have a hemoglobin constitutes the main enzyme deployed by microbes to single origin. counteract NO toxicity (10, 11). Some fungi within the Aspergillus To validate this further, a region of ∼30 kb surrounding the genus have two type I flavohemoglobins, one that is cytosolic and flavohemoglobin-encoding gene in all sequenced Malassezia spe- protects the cells against exogenous NO, and another that is mi- cies was subjected to synteny comparison (Fig. 2). Overall, this tochondrial and is putatively involved in detoxification of region was highly syntenic across species, with the exception of NO derived from nitrite metabolism (17, 18). A type II flavo- some lineage-specific rearrangements located upstream of YHB1 hemoglobin has also been identified in Mycobacterium tuberculosis (Fig. 2B). Remarkably, while the same regions were also highly and other actinobacteria, but it lacks NO consuming activity conserved in M. yamatoensis and M. slooffiae, they both lacked the and it utilizes D-lactate as an electron donor to mediate elec- flavohemoglobin-encoding gene, which is instead located in dif- tron transfer (19). ferent, nonsyntenic, regions of their genomes (Fig. 2B and SI Evolution of flavohemoglobins in microbes has been pre- Appendix,Fig.S1). Therefore, both phylogenic and synteny com- viously investigated, revealing a dynamic distribution across bac- parisons strongly support the hypothesis that Malassezia flavo- teria and eukaryotes characterized by frequent gene loss, gene hemoglobin genes were acquired through independent HGT duplication, and horizontal gene transfer (HGT) events (20–22). events from different bacterial donor species. We named the An interesting finding of these phylogenetic studies was the HGT- flavohemoglobin of clade 1 Yhb1 following the S. cerevisiae no- mediated acquisition of a bacterial flavohemoglobin-encoding menclature (23), and that of M. yamatoensis and M. slooffiae gene, YHB1,byMalassezia globosa and Malassezia sympodialis Yhb101 (clade 2). The two different flavohemoglobin protein se- (20, 21). In the present study we employed evolutionary, molec- quences share 38% identity (SI Appendix,Fig.S2). ular, biochemical, and structural analyses to characterize the HGT- Because there is evidence that genomic regions flanking hor- mediated acquisition of bacterial flavohemoglobin-encoding genes izontally acquired genes are enriched in DNA transposons within the Malassezia genus. Moreover, because the HGT- and retrotransposons (24, 25), a 5-kb region surrounding the mediated acquisition of the flavohemoglobin-encoding genes con- flavohemoglobin-encoding genes was analyzed in two Malassezia ferred the ability to metabolize NO, Malassezia genomes were species representative of clade 1 (M. sympodialis) and clade 2 searched for other HGT that could represent important (M. slooffiae). Dot plot comparisons revealed overall high co- gain-of-function events. Thirty additional genus- and species- linearity with the common flanking genes encoding a hypothet- specific HGT events were identified, with the donors being pre- ical protein and Nsr1 (SI Appendix, Fig. S3A). Interestingly, a dominantly Actinobacteria and Proteobacteria. Similar to Malassezia, highly repetitive sequence that shares similarity with the long these donor species are some of the most common members of terminal repeat (LTR) Gypsy was identified in the NSR1 gene human and mammalian microbiomes, suggesting that niche overlap flanking YHB1 (SI Appendix, Fig. S3 A and B), and we speculate may have enhanced the opportunity for interkingdom HGT. that this LTR-like region might have facilitated the non- homologous end joining (NHEJ) integration of the bacterial Results YHB1 gene into the Malassezia common ancestor. Malassezia Flavohemoglobin-Encoding Genes Were Ancestrally Acquired The flavohemoglobin-encoding gene YHB101 in M. yamatoensis from Bacteria through Independent HGT Events. Previous studies and M. slooffiae seems to have been acquired in a single HGT reported that the flavohemoglobin-encoding gene YHB1 was event from the same bacterial donor lineage (Fig. 1 A–C), al- acquired by M. globosa and M. sympodialis through HGT from though its genomic location is not syntenic in these two species Corynebacterium, a bacterial genus within the Actinobacteria that (Fig. 2B). In M. slooffiae, YHB101 is located in a region that is includes species that are part of the human microbiome otherwise highly conserved across Malassezia (SI Appendix, Fig. (20, 21). S1 A and B) and is devoid of transposable elements or repetitive Because flavohemoglobins are widespread in both bacteria regions that could have facilitated NHEJ of YHB101 (SI Ap- and eukaryotes, we examined whether the remaining 13 sequenced pendix, Fig. S1B). In contrast, in M. yamatoensis, YHB101 is lo- Malassezia species also contain a flavohemoglobin-encoding gene cated at the end of a chromosome and the adjacent genes are not

2of11 | www.pnas.org/cgi/doi/10.1073/pnas.2003473117 Ianiri et al. Downloaded by guest on September 25, 2021 A B Actinobacteria (46 collapsed leaves) Actinobacteria Actinobacteria-Dietzia timorensis WP 067476380 Clade 2 Actinobacteria-Dietzia alimentaria WP 010539839 other Bacteria Actinobacteria-Dietzia papillomatosis WP 061914237 other Fungi Actinobacteria-Dietzia alimentaria WP 083249821 Malassezia Actinobacteria (111 collapsed leaves) Actinobacteria-Corynebacterium sphenisci WP 075693348 Actinobacteria-Corynebacterium sp. WP 070833536 Actinobacteria-Corynebacterium vitaeruminis WP 048760194 Actinobacteria-Corynebacterium sputi WP 027019717 Actinobacteria-Corynebacterium sp. WP 070519831 Actinobacteria-Corynebacterium xerosis WP 046650400 Actinobacteria-Corynebacterium halotolerans WP 048739615 Actinobacteria-Brevibacterium sp. WP 070575005 Clade 1 Actinobacteria-Brevibacterium ravenspurgense WP 061941653 Malassezia cuniculi CBS11721 g2655 Malassezia vespertilionis CBS15041 PKI83671 Malassezia japonica CBS9431 g532 Malassezia furfur CBS14141 g2042 Malassezia obtusa CBS7876 g2779 0.1 Malassezia pachydermatis CBS1879 XP 017991035 Malassezia restricta KCTC27527 MRES 14465 Malassezia globosa CBS7966 XP 001730006 Malassezia sympodialis ATCC42132 XP 018739873 Malassezia caprae CBS10434 g1277 Malassezia dermatis CBS9169 g2857 Malassezia equina CBS9969 g1952 Clade 1 Malssezia nana CBS9557 g2008 0.1 Malassezia nana CBS9560 SS C Actinobacteria (72 collapsed leaves) Actinobacteria-Plantibacter flavus WP 085511486 Actinobacteria-Curtobacterium sp. Leaf261 WP 055955513 Actinobacteria-Curtobacterium sp. UNCCL20 WP 092091368 Actinobacteria-Kocuria palustris PEL EME36996 Actinobacteria-Kocuria sp. ZOR0020 WP 047692158 Actinobacteria-Raineyella antarctica WP 092609914 Actinobacteria-Bacterium sp SIT5 WP 040160721 Actinobacteria-Mycobacterium abscessus subsp. abscessus SIK27392 Actinobacteria-Mycobacterium abscessus subsp. abscessus SIJ95761 Actinobacteria-Kocuria kristinae WP 058730278

D Malassezia yamatoensis MY9725 g1976 YHB101 MICROBIOLOGY Malassezia sloffiae CBS7856 g3296 YHB101 Clade 2 Actinobacteria-Rothia nasimurium WP 083091450 AU test of monophyly Actinobacteria-Mycobacterium abscessus subsp. bolletii SLE76150 Actinobacteria-Rothia sp. HMSC065D02 WP 070740240 Tree lnL *Diff lnL P value Actinobacteria-Rothia mucilaginosa DY-18 BAI65576 Actinobacteria-Rothia mucilaginosa WP 049361801 Unconstrained -607815.172 Actinobacteria-Kocuria sp. ICS0012 WP 064845130 Actinobacteria-Kocuria rhizophila WP 019309939 Actinobacteria-Kocuria rhizophila WP 058954447 Constrained -608369.863 554.6 0.001 Actinobacteria-Kocuria WP 055086260 Actinobacteria-Kocuria varians WP 068469182 *Diff lnL, difference in log likelihood between the best topology and Actinobacteria-Kocuria sp. SM24M-10 WP 047803737 the constrained topology that forced Malassezia to be monophyletic. Actinobacteria-Kocuria turfanensis WP 062735233 Actinobacteria-Kocuria flava WP 058857164 Actinobacteria-Kocuria polaris WP 035927729 0.1 Actinobacteria-Kocuria polaris WP 058873918

Fig. 1. Evidence for independent HGT events of the flavohemoglobin-encoding genes in Malassezia from the Actinobacteria. (A) Maximum likelihood phylogeny consisting of 2,155 flavohemoglobin protein sequences. Two groups (clades 1 and 2) of horizontally transferred flavohemoglobin genes (YHB1 and YHB101)inMalassezia are colored in orange. Other tree branches are colored according to the key on the Top Left, representing other major groups of organisms. The phylogeny was visualized using iTOL v3.6.1 (61) and rooted at the midpoint. (B and C) Zoomed views of the ML phylogeny showing in more detail the position of Malassezia flavohemoglobins from clades 1 and 2, and their putative bacterial donor lineages. (D) Results of topological constraint tests that significantly rejected the monophyletic origin for both Malassezia flavohemoglobins clades, providing additional support for independent HGT events.

syntenic in other Malassezia species, with the exception of a more Fig. 2A), the YHB1 gene was lost in M. slooffiae, which instead distant group of five genes (from JLP1 to MSS1)(SI Appendix, retained the YHB101 gene, presumably in its ancestral location; Fig. S1C). In other Malassezia species (e.g., Malassezia japonica, conversely, Malassezia cuniculi lost the YHB101 gene and retained M. slooffiae,andM. sympodialis) these five genes are sub- the YHB1 gene in the ancestral location. Lastly, all species within telomeric, suggesting that chromosomal reshuffling might have the Malassezia furfur lineage (clade C, Fig. 2A)havetheYHB1 contributed to generate the unique arrangement of genes sur- gene in its ancestral location, with the exception of M. yamatoensis rounding M. yamatoensis YHB101 (Fig. 2 and SI Appendix, that has lost this gene and retained instead the YHB101 gene, Fig. S1C). which was then relocated from its original position to a subtelomeric Based on the analyses performed and on the availability of region (Fig. 2B). This model implies that Malassezia ves- Malassezia genomes, we propose the following evolution- pertilionis, M. japonica, Malassezia obtusa,andM. furfur have ary model of flavohemoglobin-mediated HGT in Malassezia. independently lost the YHB101 gene during their evolution First, the YHB1 and YHB101 genes were independently ac- (Fig. 2A). Because none of the Malassezia species has the two quired by the Malassezia common ancestor via HGT from a flavohemoglobin genes (YHB1 and YHB101) in their genomes, we Brevibacterium-related and a Kocuria-related bacterial donor, posit that loss of one or the other flavohemoglobin may be a respectively. An early loss of the YHB101 subsequently occurred consequence of different selection pressures across descendant in the common ancestor of the lineages that include M. sympo- lineages of the HGT recipient. dialis and M. globosa (clades A and B, Fig. 2A), which retained the YHB1 gene in its ancestral location; lack of synteny in the Bacterially Derived Flavohemoglobin-Encoding Genes Are Required region upstream of the YHB1 gene in M. globosa and Malassezia for Nitrosative Stress Resistance and NO Detoxification in Malassezia. restricta represents a more recent chromosomal rearrangement Flavohemoglobins are critical for NO detoxification and coun- (Fig. 2B). In the early-branching Malassezia lineage (clade D, teract nitrosative stress (10). To assess whether this HGT event in

Ianiri et al. PNAS Latest Articles | 3of11 Downloaded by guest on September 25, 2021 AB

0057 0058 00590060 0061 0062 tRNA0063 YHB1 2993 2994 2995 2996 2997 2998 2999 YHB1 M. caprae CBS10434 YHB101 HGT M. dermatis CBS9169 Gene loss 3748 3747 3746 n.a. 37453744 3743 tRNA3742 3741 3740 3739 3738 3737 3736 3735 3734 Gene M. sympodialis ATCC42132 relocation M. equina CBS9969 A  M. nana CBS9557

M. pachydermatis CBS1879

2985 2986 2987 2988 2989 2990 2991 2992 M. globosa CBS7966

B M. restricta KCTC27527

30223023302430253026 M. furfur CBS14141

M. yamatoensis MY9725 CuAO CAR1 HNM1 UGA4 MATE CAR1 MCH4 YHB101 5 kb

hyp M. obtusa CBS7876

M. japonica JCM11963 C M. vespertilionis CBS15041

2577 2578 2579 2580 25812582 2583 2584 2585 2586 2587 2588 2589 2590 2591 2592 M. cuniculi CBS11721

IDO1 hyp D M. slooffiae CBS7956

0.08 3487 3488 3489 2579 2580 2581 YHB101 2582 2583 2584 258525862587 2588 2589

3748 3747 3746 0059 3745 3744 3743 tRNA3742 3740 3739 3738 3737 3736 hyp 3734

Fig. 2. Evolutionary trajectory of flavohemoglobin-encoding genes in Malassezia after their acquisition via HGT from different donor bacteria lineages. (A) Phylogenetic relationship of Malassezia species with available genome sequence inferred from the concatenation of 246 single-copy proteins. Color codes assigned to the different phylogenetic clades (named A to D) are kept consistent in all figures. The tree was rooted at the midpoint and white circles in the tree nodes indicate full UFboot and SH-aLRT branch support. The proposed evolutionary events that led to the final arrangement of the flavohemoglobin- encoding genes reported in B are shown in the phylogenetic tree, as given in the key; double arrows indicate relocation of the YHB101 gene in subtelomeric position. (B) Chromosomal regions encompassing the YHB1 gene in Malassezia. Genes are shown as arrows denoting the direction of transcription, and orthologs are represented in the same color. Nonsyntenic genes are shown in white, and small arrows in black represent tRNAs. The YHB1 gene is shown as red arrows outlined in bold in the center. The end of a scaffold is represented by a forward slash. For M. yamatoensis and M. slooffiae, yellow bars indicate the absence of the YHB1 gene in otherwise syntenic regions, and those in green indicate instances where another flavohemoglobin-encoding gene, named YHB101 and represented as orange arrows outlined in bold, was acquired by an independent HGT event. A defective YHB1 gene in Malassezia nana CBS9557 is denoted by the Greek symbol ψ. Gene codes in red or blue are as they appear in M. globosa (prefix “MGL_”)orM. sympodialis (prefix “MSYG_”) genome annotations, respectively; those in black were named based on top BLASTp hits in S. cerevisiae; and “hyp” represent hypothetical proteins. Black circle represents the end of a chromosome. Scaffold/chromosomal locations and accession numbers are given for each region in SI Appendix, Table S1.

Malassezia resulted in a gain of function, we deleted the YHB1 complemented strains was confirmed by qPCR (SI Appendix, Fig. open reading frame (ORF) (MSYG_3741)ofM. sympodialis S4 C and D), fluorescence-activated cell sorting (FACS), and ATCC42132 through targeted mutagenesis using our recently fluorescence microcopy imaging of GFP expression, which developed transformation protocol based on transconjugation revealed that Malassezia flavohemoglobins are cytoplasmic mediated by Agrobacterium tumefaciens (26, 27) (SI Appendix, (Fig. 3B). Fig. S4A). Next, we tested whether Malassezia flavohemoglobins were The M. sympodialis yhb1Δ mutant exhibits hypersensitivity to able to actively detoxify NO, which could potentially account for the NO donors DETA NONOate and sodium nitrite (NaNO2), its involvement in nitrosative stress resistance. To this aim, we but not to hydrogen peroxide (H2O2) (Fig. 3A). The two iden- adapted a biochemical assay used for evaluating NO consumption tified Malassezia flavohemoglobins, Yhb1 and Yhb101, were by hemoglobin in red blood cells and plasma (28) to the com- used to generate GFP fusion proteins whose expression was mensal yeast Malassezia (SI Appendix,Fig.S5). As shown in driven by the respective endogenous promoter to complement Fig. 2C, while the M. sympodialis WT strain exhibited robust and the M. sympodialis yhb1Δ mutant phenotype and to assess dose-dependent NO degradation, the yhb1Δ mutant showed no protein localization (SI Appendix,Fig.S4B). Reintroduction of NO consumption. Complemented strains were able to actively either flavohemoglobin in the M. sympodialis yhb1Δ mutant consume NO, although M. sympodialis yhb1Δ + YHB101-GFP restored resistance to nitrosative stress at the wild-type (WT) displayed a lower NO consumption, corroborating the results level (Fig. 3A). In agreement, fusion protein expression in of the phenotypic assay, GFP expression, and FACS analysis

4of11 | www.pnas.org/cgi/doi/10.1073/pnas.2003473117 Ianiri et al. Downloaded by guest on September 25, 2021 Downloaded by guest on September 25, 2021 yhb1Δ upearw.I hseprmn,10 experiment, this In arrows. purple YHB101 yhb1Δ 3. Fig. aiie al. et Ianiri NaNO iiaino h Osga yfo yoer;sotnosfursec of fluorescence (* spontaneous differences cytometry; significant flow statistically by indicates Asterisks signal NO the of tification significant. ersnaiefursetsann fitaellrN ihDFF Ain DA DAF-FM with NO intracellular of staining fluorescent Representative C B A yhb1 +YHB101-GFP M. sympodialisyhb1 2 n ihhdoe eoie ( peroxide. hydrogen with and , uat n opeetn strains complementing and mutant, + Nitric Oxide Response - Millivolts yhb1 +YHB1-GFP n epcieGPsga nlzdtruhFC.( FACS. through analyzed signal GFP respective and , .sympodialis M. yhb1 + yhb1 + M. sympodialisWT YHB1 100 100 100 100 .smoilsyhb1 M. sympodialis yhb1 M. sympodialis yhb1 M. sympodialis M. sympodialis 40 60 80 40 60 80 40 60 80 40 60 80 yhb1 YHB101-GFP YHB1-GFP 0 and DIC 01 21 415 14 13 12 11 10 9 8 7 6 5 4 3 2 1 yhb1 lvhmgoisaeivle nntoaiesrs eitneadN erdto.(A degradation. NO and resistance stress nitrosative in involved are flavohemoglobins Δ + WT YHB101 F Merge GFP + YHB101-GFP + YHB1-GFP  Dxn1m M10mM 1mM 1mM mDixon h letaeidctsteN ee vrapro f1 i.N and NO min. 15 of period a over level NO the indicates trace blue the ; μL(Y),20 B Time (min) F xrsini the in expression GFP ) yhb1Δ μL( + YHB1 × )ad30 and 2) = P 0.5 mM and -10 < 10 10

GFP 10 .smoilsyhb1 M. sympodialis yhb1 M. sympodialis yhb1 M. sympodialis 0 .5 ** 0.05, 3 3 4 5 10 C yhb1 Deta NONOate Ocnupinasyby assay consumption NO ) 3 μL( .smoilsyhb1 sympodialis M. Δ × = + ) n 40 and 3), 10 P YHB101 4 < FSC-H .1 codn oteupie Student unpaired the to according 0.01) .sympodialis M. .sympodialis M. 10  backgroundsignal  +YHB101-GFP  +YHB1-GFP nmio grsplmne ihteN oo gn EANNaeand NONOate DETA agent donor NO the with supplemented agar mDixon on μL( 5 Nitrosative stress × )ad50 and 4) E 10 Δ

Nitric Oxide Nitric Oxide 6 uat n opeetn strains complementing and mutant, 10 10 10 10 10 10 10 10 10 10 a sda akrudt eetseiial A-MD signal. DA DAF-FM specifically detect to background as used was .sympodialis M. Tadtoindependent two and WT 1 2 3 4 5 1 2 3 4 5 10 10 3 3 μL( yhb1 -unstained F WT -unstained D × 10 10 )of 5) FSC-H S- FSC-H FSC-H DAF-FM DA 4 4 yhb1-2 yhb1-1 WT

Malassezia stained cells (%) Malassezia 10 20 30 WT, NaNO 10 10 0 I A-MD Merge DAF-FMDA DIC tessniiiyasyof assay sensitivity Stress ) 5 5 yhb1 ’ s 2 10 10 WT t etwt Welch with test Δ 6 6 ellrssesoswr netd ( injected. were suspensions cellular (Y yhb1 ** uat n opeetn strains complementing and mutant, yhb1-1 = 10 10 10 10 10 10 10 10 10 10 es)ijcin r niae by indicated are injections yeast) Δ 1 2 3 4 5 1 2 3 4 5 * 10 10 uat,ad( and mutants, NSLts Articles Latest PNAS yhb1Δ yhb1 -DAF-FMDA 3 3 ns WT -DAF-FMDA yhb1-2 10 10 ’ Oxidative stress correction. s + 4 4 FSC-H H YHB1 .sympodialis M. 2 O 10 10 2 E 5 5 1 mM and and “ ns | 10 10 yhb1Δ F quan- ) ” 5of11 6 6 snot is WT, D + )

MICROBIOLOGY A yhb1 vs WT B NO vs untreated 1.0 MSYG_1280 HEM1 2.0 MSYG_3126 MSYG_3135 0.0 1.5 DCG1 DAL2

1.0 −1.0 0.5 MSYG_0901 −2.0 0.0 Log fold change Log fold change −0.5 −3.0 −1.0 YHB1 MSYG_4236

1 1e+02 1e+04 1 1e+02 1e+04 1e+05 means of normalized counts means of normalized counts CDUpregulated Downregulated 30 Upregulated 20 NO yhb1 NO vsyhb1 untreated vs WT vs vs GCY1 10 untreated WT CTA1 0 MalaS7

Number of genes 1102 12 MalaS7 10 MSYG_0903* 20 MSYG_2464 MSYG_3146

Cytosol 4010 12 MSYG_0001 Allergens DNA repairProteolysis Ion binding Drug binding Nuclear part MSYG_0901* Protein folding CofactorCatalytic binding activity MSYG_2630** Hydrolase activity Mitochondrial part NO yhb1 Response to stress Transferase activity Plasma membrane Biosynthetic process Transcription factors Calcium metabolism vs vs MSYG_4238 Lipid metabolic process Oxidoreductase activity untreated WT Transmembrane transport Cellular catabolic process MSYG_4236 Cellular response to stimuli Oxidation-reduction process Intracellular organelle lumen Downregulated Heterocyclic compound binding Carbohydrate metabolicRegulation process of metabolic process Organic cyclic compoundIntegral binding component of membrane 0.5 -1.0 Biological process Molecular function Cellular components log2FC

Fig. 4. Transcriptomic profile of M. sympodialis strains under NO-accumulating conditions. (A) MA plot displaying the transcriptomic changes of the M. sympodialis yhb1Δ compared to the M. sympodialis WT strain. Red dots indicate differentially expressed genes for FDR < 0.05. The most up-regulated and down-regulated genes are indicated, along with the YHB1 gene, which represents an internal control as its down-regulation is expected because the gene is

deleted. (B) MA plot displaying the transcriptomic changes of M. sympodialis WT grown in the presence of NaNO2 compared to the untreated control. Red dots indicate differentially expressed genes for FDR < 0.05; the most up-regulated and down-regulated genes are indicated. (C) Gene Ontology classification relative to the RNA-seq condition reported in B. Up-regulated genes are indicated in red, and down-regulated genes are indicated in green. (D)Venndia- grams showing comparison of the up-regulated and down-regulated genes relative to RNA-seq conditions reported in A and B; the panel on the Right shows a heatmap of the log2 FC of the shared up-regulated (red) and down-regulated (green) genes. Predicted allergens are indicated with one asterisk, and two asterisks indicate a predicted secreted lipase.

(Fig. 3 A–C). These differences might be due to less efficient sympodialis (Fig. 3D). NO staining was quantified by FACS cross-species complementation of the M. yamatoensis Yhb101- analysis, revealing significantly higher NO accumulation in the GFP fusion protein in the yhb1Δ mutant of M. sympodialis, which flavohemoglobin mutant yhb1Δ compared to the M. sympodialis was a strategy chosen because of the lack of protocols for gene WT (Fig. 3 E and F). Because DAF-FM DA and GFP have deletion in M. slooffiae and M. yamatoensis. Taken together, similar excitation/emission spectra, complemented strains could these genetic and biochemical analyses show that the bacterially not be tested for NO accumulation via flow cytometry, and derived flavohemoglobins protect Malassezia from nitrosative therefore an independent M. sympodialis yhb1Δ mutant was tested stress by decreasing toxic levels of NO. and yielded similar results. These results indicate that the lack of a To assess intracellular production of NO by M. sympodialis, functional flavohemoglobin leads to intracellular accumulation cells were stained with the NO-specific dye 4-amino-5-methylamino- of NO. 2ʹ,7ʹ-diaminofluorescein diacetate (DAF-FM DA), which passively Finally, a broader analysis was performed to assess other diffuses across membranes and emits increased fluorescence after functions of the Malassezia flavohemoglobins in response to a reacting with NO. Fluorescent microscopy revealed intracellular variety of environmental stresses and clinical antifungals, but in accumulation of NO in both the WT and yhb1Δ mutant of M. all cases the M. sympodialis yhb1Δ mutant phenotype was not

6of11 | www.pnas.org/cgi/doi/10.1073/pnas.2003473117 Ianiri et al. Downloaded by guest on September 25, 2021 significantly different from the WT (SI Appendix, Fig. S6). Sev- and CBS9559 from cows with otitis externa, and CBS9560 and eral studies also report the protective role of both bacterial and CBS9561 from healthy cows (30). fungal flavohemoglobins against NO under anaerobic conditions The M. nana strains CBS9557, CBS9559, and CBS9560 were (10, 23). However, we could not confirm this function for the used to investigate whether the pseudogenization event occurred Malassezia flavohemoglobin in our anaerobic experiments be- in a M. nana ancestor and whether it impacts nitrosative stress cause no phenotypic differences were observed between the WT, resistance and NO consumption. Because no genomes are avail- the yhb1Δ mutant, and the complemented strains (SI Appendix, able for the M. nana strains CBS9559 and CBS9560, their YHB1 Fig. S7). gene was amplified by PCR and Sanger sequenced using primers designed on the YHB1 of M. nana CBS9557 (SI Appendix,Table A Recent Inactivation of YHB1 in M. nana Results in Compromised NO S2). YHB1 sequence comparison confirmed a premature stop Enzymatic Consumption. Analysis of Yhb1 protein prediction across codon present in only CBS9557, with both Brazilian M. nana species revealed that Malassezia nana YHB1 underwent pseudo- isolates having a full-coding YHB1 gene (SI Appendix,Fig.S8A). genization [i.e., loss of gene function by disruption of its coding Phenotypic analysis revealed no significant difference in resistance sequence with generation of a pseudogene, which is usually in- to nitrosative stress by the three M. nana strains, with only a dicated as ψ (29)] following a G-to-T transversion in the glycine modest increased sensitivity displayed by CBS9557 exposed to codon GGA, generating a premature TGA stop codon at the 29th 10 mM of sodium nitrite (SI Appendix, Fig. S8B). Strikingly, M. amino acid (SI Appendix,Fig.S8A). Literature search revealed nana CBS9557 displayed undetectable NO consumption activity as observed for the M. sympodialis yhb1Δ mutant, while M. nana that the sequenced strain of M. nana CBS9557 was isolated in CBS9559 and CBS9560 showed regular dose-dependent NO Japan from a cat with otitis externa (30), while the other four consumption (SI Appendix, Fig. S8C). These data suggest that known M. nana strains were collected in Brazil: M. nana CBS9558 the inactivation of flavohemoglobin in M. nana CBS9557 impaired the ability to consume NO, but this does not impact the resistance to nitrosative stress, which might be compensated by other stress responsive pathways. Intriguingly, another pseudogenization event of a bacterial gene encoding an aliphatic amidase was also identified in M. nana CBS9557 (see Fig. 6). These nonsense mutations were identified only in the M. nana CBS9557 isolated in Japan, suggesting that the different origin of the M. nana strains might MICROBIOLOGY contribute to this intraspecies diversity. This hypothesis is further supported by different phenotypic traits displayed by the M. nana isolates (SI Appendix,Fig.S9). Exposure to several stress conditions revealed different responses to the most common antifungal drugs by M. nana strains, with strain CBS9557 displaying increased sensitivitytoamphotericinBandresistancetofluconazole,and the geographically related strains CBS9559 and CBS9560 displaying an opposite phenotype (SI Appendix, Fig. S9).

NO Accumulation in M. sympodialis Leads to Up-Regulation of Genes Involved in Nitrogen Metabolism, Ergosterol Biosynthesis, and Protein Folding, and Down-Regulation of Predicted Pathogenicity Factors. Because the M. sympodialis flavohemoglobin mutant yhb1Δ accumulates higher amounts of NO than the WT (Fig. 3 D and E), we compared their transcriptomic profile to elucidate any potential signaling role of endogenous NO. RNA-seq analysis revealed 36 differentially expressed genes for false discovery rate (FDR) < 0.05, of which 14 were up-regulated and 22 were down-regulated; using an additional threshold of log2 fold change (FC) ± 0.5, we found 3 up-regulated and 9 down-regulated genes (Fig. 4A and Datasets S1 and S2). Of these, the only up-regulated gene with log2 FC > 1 encodes an uncharacterized protein (MSYG_1280), while two others with 0.5 < log2 FC < 1 encode Nop56 (or Sik1), a nucleolar protein involved in pre-rRNA processing, and an uncharacterized protein (MSYG_0148) predicted to be involved in magnesium transport. Other known up-regulated genes with log2 FC < 0.5 are involved in response to stresses and transport. The majority of the down-regulated genes include those encoding hypothetical proteins (5 out of 9), the regulator of phospholipase D Srf1, two MalaS7 allergens, and an uncharacterized allergen. It is worth noting that a large number of differentially expressed genes (DEGs) are predicted to encode unknown proteins, suggesting Fig. 5. Three-dimensional X-ray crystal structure of the M. yamatoensis novel and unknown signaling pathways regulated by endogenous flavohemoglobin Yhb101. (A) The globin domain (cyan) binds a heme NO in Malassezia. molecule. The reductase domain consists of a FAD-binding domain (gray) M. and a NAD-binding domain (tan) that bind a FAD molecule. (B)Anoverlay Next, to elucidate the global transcriptomic response of of flavohemoglobin globin domains from fungus, bacteria, and yeast: sympodialis exposed to nitrosative stress, RNA-seq analysis for globin domains of M. yamatoensis (PDB ID 6O0A; blue), S. cerevisiae M. sympodialis WT cells treated with sodium nitrite was performed. (PDB ID 4G1V; yellow), and E. coli (PDB ID 1GVH; green) show structural Compared to the untreated control, 112 genes were up-regulated similarity. and 50 were down-regulated (FDR < 0.05; log2 FC ± 0.5)

Ianiri et al. PNAS Latest Articles | 7of11 Downloaded by guest on September 25, 2021 key: D A Presence Gene amplification B C Absence Yhb101

HGT candidates in multiple species Species-specific HGT candidates M. cap. M. equ. M. nan. M. res. M. yam. M. obt. M. ves. M. cun. M. slo. M. der. M. sym. M. pac. M. glo. M. fur. M. jap. Flavohemoglobin (Yhb1) Catalase A / Catalase HPII (Cta1) 2222 2 22 NAD(P)/FAD-dependent oxidoreductase 2 2 Isopenicillin N synthase / Flavonol synthase 2 2 3 Deoxyribodipyrimidine photo-lyase (Phr1) 2 Arylamine N-acetyltransferase 2 Phenazine biosynthesis protein (PhzF/Yhi9) Class I SAM-dependent methyltransferase 2 2 Septicolysin-like 5 2 Prolyl aminopeptidase Twin-arginine translocation pathway protein Sorbitol dehydrogenase (Sor1) 4,5-DOPA dioxygenase extradiol NADPH-dependent quinone reductase (Zta1) Gamma-glutamyltranspeptidase (Ecm38) Nuclear transport factor 2-like protein (Ntf2) Bifunctional metallophosphatase/5'-nucleotidase Formamidase Amidohydrolase NAD(P)-dependent alcohol dehydrogenase 2 Cytosine deaminase (Fcy1) Aliphatic amidase \ Arsenate reductase (glutaredoxin) SDR family NAD(P)-dependent oxidoreductase Aldehyde reductase/epimerase (Gre2) 3 Bacterial low temp. requirement A protein (LtrA) Aspartate aminotransferase Lipase Serine protease / trypsin Alcohol dehydrogenase (Sfa1) Nicotinamidase/Isochorismatase (Pnc1)

Fig. 6. Malassezia genes acquired through HGT from bacteria. HGT candidates identified in the genomes of the 15 Malassezia species (represented on the Top according to their phylogenetic classification) are shown as different lines in the presence–absence matrix, with the closest ortholog in S. cerevisiae indicated in parenthesis, where available. For each HGT candidate, the presence of the gene in a genome is indicated by orange square, and the intensityof the color is correlated with the gene copy number (numbers in white). HGT candidates occurring in multiple Malassezia species are shown in the Top half of the matrix, whereas those that are species-specific HGT candidates are shown in the Bottom half of the matrix, and color coded as shown in the key. Asterisks indicate HGT candidate genes identified in the previous study (3). The bacterially derived gene encoding an aliphatic amidase identified in M. nana CBS9557 seems to be another instance of a pseudogene in this strain (indicated as ψ).

(Fig. 4B and Datasets S3 and S4). The most expressed genes cerevisiae and Schizosaccharomyces pombe (31, 34), and by the included HEM1 encoding a 5-aminolevulinate synthase involved transcription factor Cta4 and the Hog1 kinase in C. albicans (35), in heme biosynthesis, MSYG_3126 encoding a hypothetical with the consequent activation of genes known to be required for secreted lipase, the allantoicase-encoding gene DAL2, MSYG_3153 oxidative stress response, such as those involved in glutathione encoding an uncharacterized NAD(P)/FAD-dependent oxidore- turnover and other antioxidant/detoxification systems. M. sympo- ductase, and DCG1 encoding a protein with unknown function dialis CTA1 and CCP1 are the only oxidative stress responsive predicted to be related to nitrogen metabolism (Fig. 4B). The genes activated in response to nitrosative stress (SI Appendix, Fig. flavohemoglobin-encoding gene YHB1 was significantly up- < S10 and Dataset S3). regulated for FDR 0.05, but it had low expression level (log2 The most represented GO category of down-regulated genes = FC 0.33). Low expression of YHB1 was also observed in S. encodes integral components of membrane, which includes trans- cerevisiae cells exposed to nitrosative stress (31), although its role porters and putative Malassezia allergens; other down-regulated in NO consumption has been well characterized (23). The most genes are involved in calcium metabolism, protein folding, and represented Gene Ontology (GO) classes of up-regulated genes proteolysis. Two transcription factors were down-regulated and they are involved in stress resistance, cellular detoxification and transport, and metabolism (Fig. 4C). Functional protein associ- include the pH responsive Rim101, and an uncharacterized bZIP ation network analysis revealed enrichment of genes involved in transcription factor (Fig. 4C and Dataset S4). nitrogen metabolism and regulation, ergosterol biosynthesis, and Comparison of the two different RNA-seq datasets revealed heat shock response (SI Appendix,Fig.S10); we speculate that two common up-regulated genes, encoding the glycerol dehy- among the up-regulated transcription factor encoding genes drogenase Gcy1 and the catalase Cta1, and 10 down-regulated (HSF1, UPC2, BAS1,andHMS1), the heat shock factor Hsf1 genes that include four Malassezia allergens, a putative secreted might be the key candidate that activates nitrosative stress re- lipase, and five hypothetical proteins (Fig. 4D). While it is not sponsive genes, given its known role in response to stresses in surprising to find up-regulation of a detoxifying enzyme such as other fungi (32, 33). Conversely, response to nitrosative stress is catalase, it is intriguing to find down-regulation of genes encoding mediated by the transcription factors Yap1 and Msn2/Msn4 in S. predicted pathogenicity factors.

8of11 | www.pnas.org/cgi/doi/10.1073/pnas.2003473117 Ianiri et al. Downloaded by guest on September 25, 2021 In conclusion, our transcriptomic data indicate that the re- previous findings in Cryptococcus neoformans (38), but it corrob- sponse of Malassezia to NO and nitrosative stress is mostly dif- orates results obtained in Aspergillus fumigatus (18). Furthermore, ferent from other studied fungi, and it involves metabolic pathways the recently developed murine model for Malassezia skin infection and genes that were not known to be relevant to overcome (5) was utilized to test pathogenicity of the flavohemoglobin nitrosative stress. strains and the induction of host response. Corroborating ex vivo data, we found no differences both in terms of host tissue colo- Malassezia Flavohemoglobin Has Characteristic Features of Both nization and host inflammatory response for the yhb1Δ mutant Bacterial and Fungal Flavohemoglobins. We hypothesized that the compared to the complemented strains (SI Appendix, Figs. S13 structure of a protein acquired by HGT will likely remain similar C–E and S14). In agreement, there were no differences between − − to that of the donor organism in order to retain its original WT and Nos2 / mice when challenged with M. sympodialis WT function. Attempts to resolve the crystal structures of both (SI Appendix,Fig.S13F–H). These results suggest that fla- Malassezia flavohemoglobins were carried out, but only the M. vohemoglobin is not required for pathogenesis of Malassezia in an yamatoensis flavohemoglobin Yhb101 formed crystals to be an- experimental skin model. alyzed. The structure was determined de novo by single-wave- length anomalous diffraction phasing off the heme-iron bound to Analysis of Malassezia Genomes Revealed Extensive HGT Events from the globin domain of the protein (SI Appendix, Table S3). The Bacteria. Given the gain of function due to acquisition of the flavohemoglobin structure is highly conserved with previously bacterially derived flavohemoglobins, we sought to identify addi- characterized structures of this enzyme family, and it consists of tional HGT candidate genes in Malassezia species. In a previous an N-terminal globin domain coordinating an iron-bound study, eight HGT events were identified in M. sympodialis,and (Fe2+) heme and a C-terminal reductase domain with both then their presence was assessed in other species within the genus FAD- and NAD-binding subdomains, of which only FAD is (3). In the present study, we applied a previously described ana- bound (Fig. 5A). An overlay of a flavohemoglobin structure from lytical pipeline (40) based on three HGT metrics—the HGT in- Escherichia coli and S. cerevisiae on the M. yamatoensis crystal dex, the Alien Index (AI), and the Consensus Hit Support structure highlights conserved binding sites between the proteins (CHS)—to identify novel genus and species-specific HGT events. (Fig. 5B). Alignment of the globin domains between literature Our goal was not to explicitly establish the evolutionary history and experimental structures resulted in an rmsd value of 1.532 Å of individual genes, but rather to estimate bacteria-derived HGT for E. coli and 1.434 Å for S. cerevisiae, mostly resulting from candidates for the complete set of Malassezia genomes. Besides slight shifts in the D-loop and E-helix between the structures recovering the YHB1 and YHB101 genes as HGT candidates, this

compared. Common to all structures analyzed is the histidine analysis additionally identified a total of 30 HGT candidate genes MICROBIOLOGY residue coordinating with the heme-iron from the proximal side. (Fig. 6 and Dataset S5), 7 of which are in common with the pre- This member of the catalytic triad is supported by tyrosine vious study. HGT candidates found in the majority of the Malas- (Tyr98) and glutamate (Glu140) residues conserved in sequence and sezia species include genes involved in broad resistance to stresses, structure between bacterial and fungal/yeast flavohemoglobins (36, including three that were up-regulated in M. sympodialis exposed 37). In M. yamatoensis, as also observed in E. coli,theheme-ironis to nitrosative stress (Dataset S3), such as the NAD(P)/FAD- ligated by five atoms: four from the heme and His88 from the dependent oxidoreductase-encoding gene MSYG_3153,thecatalase- F-helix. Substrates commonly bind on the distal side of the encoding gene MSYG_3147, and the sorbitol dehydrogenase- heme and lead to a conformational change in the planarity of encoding gene MSYG_0932 (Fig. 6). Other HGT candidates the heme molecule. The E-helix on the distal side of the heme include a deoxyribodipyrimidine photolyase predicted to be involved molecule contributes Leu58, a conserved residue which ap- in repair of ultraviolet (UV) radiation-induced DNA damage, and a proaches the heme-bound iron from 3.7 Å away. At this position, class I SAM-dependent methyltransferase potentially modifying a the sixth coordination site for the iron is occluded, again similar variety of biomolecules, including DNA, proteins, and small‐molecule to the E. coli crystal structure, but unlike the yeast structure secondary metabolites. Another interesting HGT candidate is the where a three-atom small molecule cocrystallized. gene encoding a septicolysin-like protein, which is known as a pore- In M. yamatoensis Yhb101, the D-loop acts as a bridge between forming bacterial toxin that might play a role as a virulence factor the C- and E-helices and the interface between the bound FAD (41). This gene is absent in all Malassezia species of clade A, and is and heme. Comparison of the D-loops from these structures present as five copies in M. globosa.Furthermore,alargenumberof shows the M. yamatoensis D-loop adopts a nearly identical helical HGT events unique to Malassezia species of clade A were found, and structure as that of S. cerevisiae, in contrast to the E. coli D-loop, the acquired genes encode a variety of proteins with different func- which is more extended. The M. yamatoensis E-helix also adopts a tions, such as hydrolysis, protein transport and folding, detoxification ∼30° bend immediately following Leu58, which may straighten out of xenobiotics, and resistance to stresses. Finally, 12 of the HGT once a substrate is bound. This structural adjustment likely com- candidates identified were unique to certain Malassezia species. An municates substrate binding near the heme to the reductase do- intriguing case is M. japonica for which we found 4 unique HGT main through movements in the D-loop as the heme B pyrrole candidates, 1 of them in three copies. These genes encode orthologs propionate forms a hydrogen bond with the main-chain NH of of the fungal Gre2 protein, which is known to be involved in re- Ser45, the first residue in the D-loop (SI Appendix,Fig.S11). sponses to a variety of environmental stresses (42). Lastly, in SI Appendix,Fig.S12a detailed comparison of the functional residues is shown between the Malassezia Discussion flavohemoglobins with those of the closer HGT donor bacteria In the present study we report the functional characterization of Brevibacterium ravenspurgense, K. kristinae, Rothia nasimurium, two Malassezia flavohemoglobin-encoding genes that were inde- and with the model yeast S. cerevisiae. pendently acquired through HGT from different Actinobacteria donors. Our experimental analyses demonstrate that both bacte- Malassezia Flavohemoglobins Are Not Required for Survival on the rially derived flavohemoglobins are involved in nitrosative stress Host. Previous studies in human fungal pathogens indicate that resistance and NO degradation, consistent with its known func- flavohemoglobins are required for pathogenesis (38, 39). In our tions in bacteria and fungi (10). experiments, we found that M. sympodialis WT, yhb1Δ mutant, We propose an evolutionary HGT model in which extant and yhb1Δ + YHB1 and yhb1Δ + YHB101 complemented strains flavohemoglobin-encoding genes in Malassezia result from a have similar levels of survival within activated macrophages complex pattern of gene retention/loss after being both acquired (SI Appendix,Fig.S13A and B). This result is in contrast with by a Malassezia common ancestor. Nevertheless, other evolutionary

Ianiri et al. PNAS Latest Articles | 9of11 Downloaded by guest on September 25, 2021 scenarios could also be hypothesized, such as: 1) the acquisition microbiota of animals, but also others that are known to inhabit a of the YHB1 in a Malassezia common ancestor via HGT from a variety of terrestrial and marine habitats, raising questions about Brevibacterium-related donor; 2) followed by more recent ac- a possible wider environmental distribution of a Malassezia an- quisitions of YHB101 by M. yamatoensis and M. slooffiae via in- cestor. This could be correlated with the presence of Malassezia dependent HGT events from a common, or closely related, DNA in a number of unexpected areas, such as in association bacterial donor(s) (Kocuria). In this scenario, the “resident” with corals and sea sponges in the ocean (53). Moreover, most of YHB1 in M. yamatoensis and M. slooffiae could have been dis- the HGT candidate genes identified in Malassezia operate as a placed upon secondary acquisition of the YHB101 gene [a phe- self-contained metabolic unit, which has been proposed to fa- nomenon termed as xenolog gene displacement (43)], or the cilitate HGT (21). Intriguingly, the high number of HGT events acquisition of YHB101 by HGT could have been preceded by suggests also a predisposition of Malassezia to bacterial conju- the loss of the cognate YHB1 copy. The identification of novel gation, in line with our previous findings that A. tumefaciens- Malassezia species and the analysis of their genomes will be key mediated transformation is the only effective technique for mo- for the elucidation of these complex models of gene evolution in lecular manipulation of Malassezia (54). There are a number of Malassezia. identified HGT that are predicted to be important for Malassezia Although the mechanisms of HGT in fungi are not fully un- pathophysiology and that can be characterized using the meth- derstood, several possible mechanisms have been reported odologies reported in the present study. (24, 44). One such mechanism is gene acquisition through con- jugation, which requires contact between bacterial donor and Materials and Methods fungal recipient (44). For the HGT events that mediated Detailed procedures of the materials and methods used are provided in SI flavohemoglobin acquisition by Malassezia, the closest phyloge- Appendix, SI Material and Methods. netic donors are Actinobacteria that are part of the mammalian microbiome and hence share the same ecological niche with Bioinformatics Analyses. The Malassezia flavohemoglobin sequences Malassezia. A dilemma that is common to all HGT events is that were identified by BLAST analyses. Regions of ∼32 kb surrounding the if a gene is required for survival in a certain condition, its transfer flavohemoglobin-encoding genes were used for synteny analysis between under that condition might in theory be difficult if not impossible Malassezia species. Flavohemoglobin protein sequences from Malassezia (45). Because NO is synthesized by mammals, including by the species, other fungi, and bacteria, were subjected to phylogenetic analysis skin (46), we speculate that the presence of NO enhanced the using IQ-TREE v1.5.5 (55). Tests of monophyly were performed using the AU test (56). The phylogenetic relationship among the 15 Malassezia species HGT transfer of bacterial flavohemoglobins to a fungal Malas- selected was performed using a consensus set of 246 protein sequences; the sezia ancestor that acquired the ability to actively consume NO. phylogenetic tree was constructed with IQ-TREE. To assess the extent of Notably, a large number of eukaryotic organisms including horizontal transfer into Malassezia genomes, we applied a previously de- fungi lack a flavohemoglobin-encoding gene, suggesting the ex- scribed pipeline (40) to the set of 15 available Malassezia proteomes. Tran- istence of alternative pathways for nitrosative stress resistance scriptomic analysis was carried out using HiSat and StringTie (57). DESeq2 < ± and NO utilization. For example, in Histoplasma capsulatum, the was used to determine the DEGs as having FDR 0.05 and log2 FC 0.5, etiologic agent of histoplasmosis, Yhb1 is replaced by a P450- which are common parameters used to define relevant genes in RNA-seq type NO reductase (47), whereas in other cases, such as for the experiments (58). Functional annotation of the DEGs was performed using basidiomycetous fungi Moniliella, Ustilago, and Puccinia,NO Blast2GO (59). metabolism in the absence of flavohemoglobin has yet to be M. sympodialis elucidated. The evolution and diversification of flavohemoglobin- Molecular Manipulation of . For targeted mutagenesis of M. sympodialis YHB1, ∼1.5-kb regions flanking the YHB1 gene were fused encoding genes has been a dynamic and complex process char- – – with the NAT marker and cloned in a binary vector for A. tumefaciens- acterized by several prokaryote prokaryote and prokaryote mediated transformation. For yhb1Δ functional complementation, two dif- eukaryote HGT events (21), hence suggesting its significant ferent flavohemoglobin GFP-fusion proteins were generated. M. sympodialis contribution for habitat colonization by a species but likely a was transformed through A. tumefaciens-mediated transformation as pre- dispensable role in evolutionary divergence. viously reported (26, 27). Is the bacterial flavohemoglobin required for Malassezia interaction with the host? While a number of studies in bacteria NO Quantification and NO Consumption Assay. Intracellular levels of NO in and fungi reported a role for flavohemoglobin in microbial patho- M. sympodialis WT and yhb1Δ mutant were measured by flow cytometry genesis (10), we surprisingly found that Malassezia flavohemoglobins using DAF-FM DA (14). The NO consumption assay was performed as previously are dispensable for survival within macrophages and for skin in- reported (60) using DETA NONOate as a NO source and a NO chemiluminescence fection in our experimental conditions. Conversely, we propose analyzer (TEA 810, Ellutia). that Malassezia flavohemoglobins are important for the commensal lifestyle of Malassezia through regulation of NO homeo- Flavohemoglobin Purification and Crystal Structure. A construct expressing His-Tev-YHB101 was cloned into E. coli BL21(DE3) cells for expression studies. stasis, a hypothesis corroborated by down-regulation of genes- For protein purification, a Ni2+ charged HiTrap Chelating High Performance encoding putative virulence factors (i.e., allergens and lipases) in was used. M. yamatoensis flavohemoglobin crystals were obtained with our transcriptomic analyses. Another hypothesis is that the HGT- Morpheus B12: 12.5% (wt/vol) PEG1000, 12.5% (wt/vol) PEG3350, 12.5% mediated acquisition of flavohemoglobins might be important to (vol/vol) 2-methyl-2,4-pentanediol, 0.03 M each sodium fluoride, sodium mediate Malassezia response to NO that is produced by sympatric bromide, and sodium iodide, and 0.1 M bicine/Trizma base pH 8.5. microbial communities and acts as a quorum signaling molecule, as reported in bacteria (48) and in S. cerevisiae (49). Interaction of M. sympodialis Strains with the Host. For studying interaction of Horizontal gene transfer is thought to occur much less fre- M. sympodialis strains with the host, ex vivo experiments were carried out using J774 A.1 macrophages. For in vivo murine experiments, WT C57BL/6j quently in eukaryotes than in prokaryotes (50), but there are −/− notable cases that invoke HGT as a prominent mechanism of and Nos2 mice were used. All mouse experiments in this study were eukaryotic evolution, such as in the transition of green plants conducted in strict accordance with the guidelines of the Swiss Animals Protection Law and were performed under the protocols approved by the from aquatic to terrestrial environments (51), and in the colo- Veterinary office of the Canton Zurich, Switzerland (license number 168/2018). nization of the animal digestive tracts by rumen fungi and ciliates All efforts were made to minimize suffering and ensure the highest ethical and (45, 52). Analysis of Malassezia genomes revealed a large num- humane standards. ber of HGT events, suggesting that they may also have played a substantial contribution in Malassezia evolution and niche ad- Data Availability. The sequence data generated in this study were submitted to aptation. Donor bacteria include those that are part of the the National Center for Biotechnology Information under BioProject accession no.

10 of 11 | www.pnas.org/cgi/doi/10.1073/pnas.2003473117 Ianiri et al. Downloaded by guest on September 25, 2021 PRJNA626605. Individual accession nos. are given in SI Appendix, SI Material and on improving heme incorporation during recombinant expression of the Methods. The final structure factors and coordinates of the flavohemolgobin crystallography constructs, and Tom Edwards and Don Lorimer for their Yhb101 of M. yamatoensis were deposited in the Protein Data Bank with overall support of the project. This work was in part supported by NIH/National code 6O0A. Institute of Allergy and Infectious Diseases (NIAID) R01 Grant AI50113-15, and NIH/NIAID R37 MERIT Award AI39115-22 (to J.H.), and by Swiss National ACKNOWLEDGMENTS. We thank Stephen Rogers for assistance with the NO Science Foundation Grant 310030_189255 (to S.L.-L.). VA Merit BX-003478 −/− ’ consumption assay, Nicolas Fasel for Nos2 mice, Ellen Wallace for her supported T.J.M. s work. Crystallization work was funded by the NIH/NIAID (Con- contribution to cloning the Malassezia crystallography constructs, Jan tracts HHSN272200700057, HHSN272201200025C, and HHSN272201700059C to Abendroth for his contributions to solving the structure of Malassezia by P.M.). J.H. is co-director and fellow of the Canadian Institute for Advanced Re- phasing off the bound iron, Jason Yano and Rana Sidhu for consultation search program “Fungal Kingdom: Threats & Opportunities.”

1. K. Findley et al., Topographic diversity of fungal and bacterial communities in human 33. A. O. Veri et al., Tuning Hsf1 levels drives distinct fungal morphogenetic programs skin. Nature 498, 367–370 (2013). with depletion impairing Hsp90 function and overexpression expanding the target 2. B. Theelen et al., Malassezia ecology, pathophysiology, and treatment. Med. Mycol. space. PLoS Genet. 14, e1007270 (2018). 56 (suppl. 1), S10–S25 (2018). 34. P. Biswas, S. Ghosh, Global transcriptomic profiling of Schizosaccharomyces pombe in 3. G. Wu et al., Genus-wide comparative genomics of Malassezia delineates its phylogeny, response to nitrosative stress. Gene 558, 241–253 (2015). physiology, and niche adaptation on human skin. PLoS Genet. 11, e1005614 (2015). 35. C. Herrero-de-Dios et al., Redox regulation, rather than stress-induced phosphoryla- 4. G. Gaitanis, P. Magiatis, M. Hantschke, I. D. Bassukas, A. Velegraki, The Malassezia tion, of a Hog1 mitogen-activated protein kinase modulates its nitrosative-stress- genus in skin and systemic diseases. Clin. Microbiol. Rev. 25, 106–141 (2012). specific outputs. mBio 9, e02229-17 (2018). 5. F. Sparber et al., The skin commensal yeast Malassezia triggers a type 17 response that 36. E. El Hammi et al., Active site analysis of yeast flavohemoglobin based on its structure coordinates anti-fungal immunity and exacerbates skin inflammation. Cell Host Mi- with a small ligand or econazole. FEBS J. 279, 4565–4575 (2012). crobe 25, 389–403.e6 (2019). 37. A. Ilari, A. Bonamore, A. Farina, K. A. Johnson, A. Boffi, The X-ray structure of ferric 6. J. J. Limon et al., Malassezia is associated with Crohn’s disease and exacerbates colitis Escherichia coli flavohemoglobin reveals an unexpected geometry of the distal heme in mouse models. Cell Host Microbe 25, 377–388.e6 (2019). pocket. J. Biol. Chem. 277, 23725–23732 (2002). 7. B. Aykut et al., The fungal mycobiome promotes pancreatic oncogenesis via activation 38. M. de Jesús-Berríos et al., Enzymes that counteract nitrosative stress promote fungal – of MBL. Nature 574, 264 267 (2019). virulence. Curr. Biol. 13, 1963–1968 (2003). 8. P. Soret et al., Respiratory mycobiome and suggestion of inter-kingdom network 39. B. S. Hromatka, S. M. Noble, A. D. Johnson, Transcriptional response of Candida al- during acute pulmonary exacerbation in cystic fibrosis. Sci. Rep. 10, 3589 (2020). bicans to nitric oxide and the role of the YHB1 gene in nitrosative stress and virulence. 9. C. T. Stomberski, D. T. Hess, J. S. Stamler, Protein S-nitrosylation: Determinants of Mol. Biol. Cell 16, 4814–4826 (2005). specificity and enzymatic regulation of S-nitrosothiol-based signaling. Antioxid. Redox 40. R. W. Nowell et al., Comparative genomics of bdelloid rotifers: Insights from desic- – Signal. 30, 1331 1351 (2019). cating and nondesiccating species. PLoS Biol. 16, e2004830 (2018). 10. M. T. Forrester, M. W. Foster, Protection from nitrosative stress: A central role for 41. N. Mosqueda et al., Characterization of plasmids carrying the blaOXA-24/40 carba- – microbial flavohemoglobin. Free Radic. Biol. Med. 52, 1620 1633 (2012). penemase gene and the genes encoding the AbkA/AbkB proteins of a toxin/antitoxin

11. R. I. Astuti, R. Nasuno, H. Takagi, Nitric oxide signaling in yeast. Appl. Microbiol. system. J. Antimicrob. Chemother. 69, 2629–2633 (2014). MICROBIOLOGY – Biotechnol. 100,9483 9497 (2016). 42. M. Rep et al., The Saccharomyces cerevisiae Sko1p transcription factor mediates 12. D. Cánovas, J. F. Marcos, A. T. Marcos, J. Strauss, Nitric oxide in fungi: Is there NO light HOG pathway-dependent osmotic regulation of a set of genes encoding enzymes at the end of the tunnel? Curr. Genet. 62, 513–518 (2016). implicated in protection from oxidative damage. Mol. Microbiol. 40,1067–1083 (2001). 13. Y. Zhao, J. Lim, J. Xu, J. H. Yu, W. Zheng, Nitric oxide as a developmental and met- 43. E. V. Koonin, Orthologs, paralogs, and evolutionary genomics. Annu. Rev. Genet. 39, abolic signal in filamentous fungi. Mol. Microbiol. 113, 872–882 (2020). 309–338 (2005). 14. B. Koch et al., A metabolic checkpoint for the yeast-to-hyphae developmental switch 44. B. Lacroix, V. Citovsky, Transfer of DNA from bacteria to eukaryotes. mBio 7, e00863- regulated by endogenous nitric oxide signaling. Cell Rep. 25, 2244–2258.e7 (2018). 16 (2016). 15. A. T. Marcos, M. S. Ramos, T. Schinko, J. Strauss, D. Cánovas, Nitric oxide homeostasis 45. C. L. Murphy et al., Horizontal gene transfer as an indispensable driver for Neo- is required for light-dependent regulation of conidiation in Aspergillus. Fungal Genet. callimastigomycota evolution into a distinct gut-dwelling fungal lineage. Appli En- Biol. 137, 103337 (2020). viron. Microbiol. 85, e00988-19 (2019). 16. P. R. Gardner, Nitric oxide dioxygenase function and mechanism of flavohemoglobin, 46. M. M. Cals-Grierson, A. D. Ormerod, Nitric oxide function in the skin. Nitric Oxide: hemoglobin, myoglobin and their associated reductases. J. Inorg. Biochem. 99, Biol. Chem. 10, 179–193 (2004). 247–266 (2005). 47. M. P. Nittler, D. Hocking-Murray, C. K. Foo, A. Sil, Identification of Histoplasma cap- 17. S. Zhou et al., Functional analysis and subcellular location of two flavohemoglobins sulatum transcripts induced in response to reactive nitrogen species. Mol. Biol. Cell 16, from Aspergillus oryzae. Fungal Genet. Biol. 48, 200–207 (2011). 4792–4813 (2005). 18. K. Lapp et al., Characterization of the Aspergillus fumigatus detoxification systems 48. D. E. Williams, E. M. Boon, Towards understanding the molecular basis of nitric oxide- for reactive nitrogen intermediates and their impact on virulence. Front. Microbiol. 5, regulated group behaviors in pathogenic bacteria. J. Innate Immun. 11 ,205–215 (2019). 469 (2014). 49. L. Yang et al., Nitric oxide increases biofilm formation in Saccharomyces cerevisiae by 19. N. Thakur, A. Kumar, K. L. Dikshit, Type II flavohemoglobin of Mycobacterium activating the transcriptional factor Mac1p and thereby regulating the transmembrane smegmatis oxidizes d-lactate and mediate electron transfer. Int. J. Biol. Macromol. protein Ctr1. Biotechnol. Biofuels 12,30(2019). 112, 868–875 (2018). 50. P. J. Keeling, J. D. Palmer, Horizontal gene transfer in eukaryotic evolution. Nat. Rev. 20. D. Hoogewijs, S. Dewilde, A. Vierstraete, L. Moens, S. N. Vinogradov, A phylogenetic – analysis of the globins in fungi. PLoS One 7, e31856 (2012). Genet. 9, 605 618 (2008). 21. J. H. Wisecaver, W. G. Alexander, S. B. King, C. T. Hittinger, A. Rokas, Dynamic evo- 51. J. Yue, X. Hu, H. Sun, Y. Yang, J. Huang, Widespread impact of horizontal gene lution of nitric oxide detoxifying flavohemoglobins, a family of single-protein met- transfer on plant colonization of land. Nat. Commun. 3, 1152 (2012). abolic modules in bacteria and eukaryotes. Mol. Biol. Evol. 33, 1979–1987 (2016). 52. G. Ricard et al., Horizontal gene transfer from Bacteria to rumen Ciliates indicates 22. S. N. Vinogradov et al., A phylogenomic profile of globins. BMC Evol. Biol. 6,31(2006). adaptation to their anaerobic, carbohydrates-rich environment. BMC Genom. 7, 22 (2006). 23. L. Liu, M. Zeng, A. Hausladen, J. Heitman, J. S. Stamler, Protection from nitrosative 53. A. Amend, From dandruff to deep-sea vents: Malassezia-like fungi are ecologically stress by yeast flavohemoglobin. Proc. Natl. Acad. Sci. U.S.A. 97, 4672–4676 (2000). hyper-diverse. PLoS Pathog. 10, e1004277 (2014). 24. F. Husnik, J. P. McCutcheon, Functional horizontal gene transfer from bacteria to 54. G. Ianiri, A. F. Averette, J. M. Kingsbury, J. Heitman, A. Idnurm, Gene function analysis in the eukaryotes. Nat. Rev. Microbiol. 16,67–79 (2018). ubiquitous human commensal and pathogen Malassezia genus. mBio 7, e01853-16 (2016). 25. J. Ropars et al., Adaptive horizontal gene transfers between multiple cheese- 55. L. T. Nguyen, H. A. Schmidt, A. von Haeseler, B. Q. Minh, IQ-TREE: A fast and effective associated fungi. Curr. Biol. 25, 2562–2569 (2015). stochastic algorithm for estimating maximum-likelihood phylogenies. Mol. Biol. Evol. – 26. G. Ianiri, S. Applen Clancey, S. C. Lee, J. Heitman, FKBP12-dependent inhibition of 32, 268 274 (2015). calcineurin mediates immunosuppressive antifungal drug action in Malassezia. mBio 56. H. Shimodaira, An approximately unbiased test of phylogenetic tree selection. Syst. 8, e01752–e01717 (2017). Biol. 51, 492–508 (2002). 27. G. Ianiri, G. Dagotto, S. Sun, J. Heitman, Advancing functional genetics through 57. M. Pertea, D. Kim, G. M. Pertea, J. T. Leek, S. L. Salzberg, Transcript-level expression analysis of Agrobacterium-mediated insertional mutagenesis and CRISPR/Cas9 in the commensal RNA-seq experiments with HISAT, StringTie and Ballgown. Nat. Protoc. 11,1650–1667 (2016). and pathogenic yeast Malassezia. Genetics 212, 1163–1179 (2019). 58. N. J. Schurch et al., How many biological replicates are needed in an RNA-seq ex- 28. X. Wang et al., Biological activity of nitric oxide in the plasmatic compartment. Proc. periment and which differential expression tool should you use? RNA 22, 839–851 Natl. Acad. Sci. U.S.A. 101, 11477–11482 (2004). (2016). 29. Z. Zhang, M. Gerstein, Large-scale analysis of pseudogenes in the human genome. 59. A. Conesa et al., Blast2GO: A universal tool for annotation, visualization and analysis Curr. Opin. Genet. Dev. 14, 328–335 (2004). in functional genomics research. Bioinformatics 21, 3674–3676 (2005). 30. A. Hirai et al., Malassezia nana sp. nov., a novel lipid-dependent yeast species isolated 60. S. C. Rogers et al., Effect of plasma processing and storage on microparticle from animals. Int. J. Syst. Evol. Microbiol. 54, 623–627 (2004). abundance, nitric oxide scavenging, and vasoactivity. Transfusion 59,1568–1577 31. S. Horan, I. Bourges, B. Meunier, Transcriptional response to nitrosative stress in (2019). Saccharomyces cerevisiae. Yeast 23, 519–535 (2006). 61. I. Letunic, P. Bork, Interactive tree of life (iTOL) v3: An online tool for the display and 32. D.-H. Yang et al., Rewiring of signaling networks modulating thermotolerance in the annotation of phylogenetic and other trees. Nucleic Acids Res. 44,W242–W245 human pathogen Cryptococcus neoformans. Genetics 205, 201–219 (2017). (2016).

Ianiri et al. PNAS Latest Articles | 11 of 11 Downloaded by guest on September 25, 2021