HGT in the human and skin commensal Malassezia: A bacterially derived flavohemoglobin is required for NO resistance and host interaction

Significance

Malassezia species are the main fungal components of the mammalian skin microbiome and are associated with a number of skin disorders. Recently, Malassezia has also been found in association with Crohn’s disease and with pancreatic cancer. The elucidation of the molecular bases of skin adaptation by Malassezia is critical to understand its role as commensal and pathogen. In this study we employed evolutionary, molecular, biochemical, and structural analyses to demonstrate that the bacterially derived flavohemoglobins acquired by Malassezia through horizontal gene transfer resulted in a gain of function critical for nitric oxide detoxification and resistance to nitrosative stress. Our study underscores horizontal gene transfer as an important force modulating Malassezia evolution and niche adaptation.

Abstract

The skin of humans and animals is colonized by commensal and pathogenic fungi and bacteria that share this ecological niche and have established microbial interactions. Malassezia are the most abundant fungal skin inhabitant of warm-blooded animals and have been implicated in skin diseases and systemic disorders, including Crohn’s disease and pancreatic cancer. Flavohemoglobin is a key enzyme involved in microbial nitrosative stress resistance and nitric oxide degradation. Comparative genomics and phylogenetic analyses within the Malassezia genus revealed that flavohemoglobin-encoding genes were acquired through independent horizontal gene transfer events from different donor bacteria that are part of the mammalian microbiome. Through targeted gene deletion and functional complementation in Malassezia sympodialis, we demonstrated that bacterially derived flavohemoglobins are cytoplasmic proteins required for nitric oxide detoxification and nitrosative stress resistance under aerobic conditions. RNA-sequencing analysis revealed that endogenous accumulation of nitric oxide resulted in up-regulation of genes involved in stress response and down-regulation of the MalaS7 allergen-encoding genes. Solution of the high-resolution X-ray crystal structure of Malassezia flavohemoglobin revealed features conserved with both bacterial and fungal flavohemoglobins. In vivo pathogenesis is independent of Malassezia flavohemoglobin. Lastly, we identified an additional 30 genus- and species-specific horizontal gene transfer candidates that might have contributed to the evolution of this genus as the most common inhabitants of animal skin.

The skin microbiome includes numerous microorganisms that establish a variety of direct and indirect interactions characterized by the exchange of genetic material that impact microbial biology, contributing to their speciation and evolution. Malassezia is the most abundant fungal genus resident on human skin, representing more than 90% of the skin mycobiome (1). This genus presently consists of 18 diverse species (2), each with an unusually compact genome that underwent extensive gene turnover events as a result of evolutionary adaptation and colonization to a nutrient-limited ecological niche such as the skin (3). Although commensals, Malassezia species are also associated with a number of clinical skin disorders, including pityriasis versicolor, dandruff, and atopic dermatitis (AD) (4). A recently developed epicutaneous murine model revealed that the host responses to Malassezia are dominated by proinflammatory cytokine IL-17 and related factors that prevent fungal overgrowth and exacerbate inflammation under atopy-like conditions (5). Furthermore, Malassezia species have also been implicated recently as causal agents of Crohn’s disease/inflammatory bowel disease in patients with CARD9 mutations, and in accelerating the progression of pancreatic adenocarcinoma in murine models and in humans, and cystic fibrosis pulmonary exacerbation (6–8).

Nitric oxide (NO) is a reactive compound of central importance in biological systems and it functions both as a signaling and toxic molecule. While little is known about NO synthesis in fungi, in mammals NO is synthesized by NO synthases (NOS isoforms). NOS1 and NOS3 are constitutively expressed in neurons and endothelium, respectively, and produce NO to promote S-nitrosylation and transcriptional regulation. S-nitrosylation is a posttranslational mechanism involving oxidative modification of cysteine by NO, and this is the central NO-mediated signaling mechanism that affects myriad of cellular physiological and pathophysiological processes (9). On the other hand, NOS2 is not constitutively expressed but is induced in inflammatory cells in response to infection and is involved in wound healing, immune regulation, and host defense (10).

In fungi, NO is synthesized through a reductive denitrification pathway from nitrite, and through an oxidative pathway from l-arginine, although the detailed biochemical mechanisms have not yet been fully elucidated (11–13). Compared to mammals, plants, and bacteria, the role of NO in fungal biology is understudied. In Saccharomyces cerevisiae NO is important for activation of transcription factors that are involved in resistance to a variety of environmental stress conditions, such as oxidative stress, heat shock, and hydrostatic pressure (11). Other studies report an involvement of NO in pathogenesis of Botrytis cinerea and Magnaporthe oryzae, in morphogenesis and reproduction in Aspergillus nidulans, and in the yeast-to-hyphae dimorphic transition in Candida albicans (12, 14, 15).

Imbalance in cellular NO levels leads to altered redox homeostasis, resulting in the production of reactive nitrogen species that are responsible for nitrosative stress (10). NO dioxygenases are enzymes that living cells use to actively consume poisonous NO by converting it to inert nitrate, a source of nitrogen (16). Red blood cell hemoglobin is the main mammalian dioxygenase that metabolizes NO in the vascular lumen, whereas a type I flavohemoglobin constitutes the main enzyme deployed by microbes to counteract NO toxicity (10, 11). Some fungi within the Aspergillus genus have two type I flavohemoglobins, one that is cytosolic and protects the cells against exogenous NO, and another that is mitochondrial and is putatively involved in detoxification of NO derived from nitrite metabolism (17, 18). A type II flavohemoglobin has also been identified in Mycobacterium tuberculosis and other actinobacteria, but it lacks NO consuming activity and it utilizes D-lactate as an electron donor to mediate electron transfer (19).

Evolution of flavohemoglobins in microbes has been previously investigated, revealing a dynamic distribution across bacteria and eukaryotes characterized by frequent gene loss, gene duplication, and horizontal gene transfer (HGT) events (20–22). An interesting finding of these phylogenetic studies was the HGT-mediated acquisition of a bacterial flavohemoglobin-encoding gene, YHB1, by Malassezia globosa and Malassezia sympodialis (20, 21). In the present study we employed evolutionary, molecular, biochemical, and structural analyses to characterize the HGT-mediated acquisition of bacterial flavohemoglobin-encoding genes within the Malassezia genus. Moreover, because the HGT-mediated acquisition of the flavohemoglobin-encoding genes conferred the ability to metabolize NO, Malassezia genomes were searched for other HGT that could represent important gain-of-function events. Thirty additional genus- and species-specific HGT events were identified, with the donors being predominantly Actinobacteria and Proteobacteria. Similar to Malassezia, these donor species are some of the most common members of human and mammalian microbiomes, suggesting that niche overlap may have enhanced the opportunity for interkingdom HGT.

Results

Malassezia Flavohemoglobin-Encoding Genes Were Ancestrally Acquired from Bacteria through Independent HGT Events.

Previous studies reported that the flavohemoglobin-encoding gene YHB1 was acquired by M. globosa and M. sympodialis through HGT from Corynebacterium, a bacterial genus within the Actinobacteria that includes species that are part of the human microbiome (20, 21).

Because flavohemoglobins are widespread in both bacteria and eukaryotes, we examined whether the remaining 13 sequenced Malassezia species also contain a flavohemoglobin-encoding gene and whether it had a fungal or bacterial origin. BLAST analyses with the M. globosa Yhb1 sequence as query identified a single copy of YHB1 in all Malassezia species and strains with sequenced genomes. Intriguingly, this comparative search revealed that the best hits in Malassezia yamatoensis and Malassezia slooffiae had lower E values (5e-64 and 1e-59, respectively) compared to the remaining Malassezia species (E values ranging from 0.0 to 7e-161), possibly suggesting different origins or modifications of these flavohemoglobin-encoding genes. To elucidate the evolutionary trajectory of flavohemoglobin within the whole Malassezia genus, a maximum likelihood (ML) phylogenetic tree was reconstructed using >2,000 Yhb1 bacterial and fungal sequences retrieved from GenBank. This phylogenetic analysis revealed two clades of flavohemoglobin within the Malassezia genus: clade 1 that includes 13 species and clusters together with Brevibacterium species belonging to Actinobacteria; and clade 2 that includes M. yamatoensis and M. slooffiae and clusters together with different Actinobacteria, with the closest relative being Kocuria kristinae (Fig. 1 A–C). To evaluate the statistical phylogenetic support for the two Malassezia flavohemoglobin clades whose distribution was not monophyletic, we performed approximately unbiased (AU) comparative topology tests. The constrained ML phylogeny, in which all Malassezia flavohemoglobins were forced to be monophyletic was significantly rejected (AU test, P value = 0.001, Fig. 1D), thus not supporting the null hypothesis that all flavohemoglobin genes in Malassezia have a single origin.

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) in Malassezia 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.

To validate this further, a region of ∼30 kb surrounding the flavohemoglobin-encoding gene in all sequenced Malassezia species was subjected to synteny comparison (Fig. 2). Overall, this region was highly syntenic across species, with the exception of some lineage-specific rearrangements located upstream of YHB1 (Fig. 2B). Remarkably, while the same regions were also highly conserved in M. yamatoensis and M. slooffiae, they both lacked the flavohemoglobin-encoding gene, which is instead located in different, nonsyntenic, regions of their genomes (Fig. 2B and SI Appendix, Fig. S1). Therefore, both phylogenic and synteny comparisons strongly support the hypothesis that Malassezia flavohemoglobin genes were acquired through independent HGT events from different bacterial donor species. We named the flavohemoglobin of clade 1 Yhb1 following the S. cerevisiae nomenclature (23), and that of M. yamatoensis and M. slooffiae Yhb101 (clade 2). The two different flavohemoglobin protein sequences share 38% identity (SI Appendix, Fig. S2).

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_”) or M. 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.

Because there is evidence that genomic regions flanking horizontally acquired genes are enriched in DNA transposons and retrotransposons (24, 25), a 5-kb region surrounding the flavohemoglobin-encoding genes was analyzed in two Malassezia species representative of clade 1 (M. sympodialis) and clade 2 (M. slooffiae). Dot plot comparisons revealed overall high colinearity with the common flanking genes encoding a hypothetical protein and Nsr1 (SI Appendix, Fig. S3A). Interestingly, a highly repetitive sequence that shares similarity with the long terminal repeat (LTR) Gypsy was identified in the NSR1 gene flanking YHB1 (SI Appendix, Fig. S3 A and B), and we speculate that this LTR-like region might have facilitated the nonhomologous end joining (NHEJ) integration of the bacterial YHB1 gene into the Malassezia common ancestor.

The flavohemoglobin-encoding gene YHB101 in M. yamatoensis and M. slooffiae seems to have been acquired in a single HGT event from the same bacterial donor lineage (Fig. 1 A–C), although its genomic location is not syntenic in these two species (Fig. 2B). In M. slooffiae, YHB101 is located in a region that is otherwise highly conserved across Malassezia (SI Appendix, Fig. S1 A and B) and is devoid of transposable elements or repetitive regions that could have facilitated NHEJ of YHB101 (SI Appendix, Fig. S1B). In contrast, in M. yamatoensis, YHB101 is located at the end of a chromosome and the adjacent genes are not syntenic in other Malassezia species, with the exception of a more distant group of five genes (from JLP1 to MSS1) (SI Appendix, Fig. S1C). In other Malassezia species (e.g., Malassezia japonica, M. slooffiae, and M. sympodialis) these five genes are subtelomeric, suggesting that chromosomal reshuffling might have contributed to generate the unique arrangement of genes surrounding M. yamatoensis YHB101 (Fig. 2 and SI Appendix, Fig. S1C).

Based on the analyses performed and on the availability of Malassezia genomes, we propose the following evolutionary model of flavohemoglobin-mediated HGT in Malassezia. First, the YHB1 and YHB101 genes were independently acquired by the Malassezia common ancestor via HGT from a Brevibacterium-related and a Kocuria-related bacterial donor, respectively. An early loss of the YHB101 subsequently occurred in the common ancestor of the lineages that include M. sympodialis and M. globosa (clades A and B, Fig. 2A), which retained the YHB1 gene in its ancestral location; lack of synteny in the region upstream of the YHB1 gene in M. globosa and Malassezia restricta represents a more recent chromosomal rearrangement (Fig. 2B). In the early-branching Malassezia lineage (clade D, Fig. 2A), the YHB1 gene was lost in M. slooffiae, which instead retained the YHB101 gene, presumably in its ancestral location; conversely, Malassezia cuniculi lost the YHB101 gene and retained the YHB1 gene in the ancestral location. Lastly, all species within the Malassezia furfur lineage (clade C, Fig. 2A) have the YHB1 gene in its ancestral location, with the exception of M. yamatoensis that has lost this gene and retained instead the YHB101 gene, which was then relocated from its original position to a subtelomeric region (Fig. 2B). This model implies that Malassezia vespertilionis, M. japonica, Malassezia obtusa, and M. furfur have independently lost the YHB101 gene during their evolution (Fig. 2A). Because none of the Malassezia species has the two flavohemoglobin genes (YHB1 and YHB101) in their genomes, we posit that loss of one or the other flavohemoglobin may be a consequence of different selection pressures across descendant lineages of the HGT recipient.

Bacterially Derived Flavohemoglobin-Encoding Genes Are Required for Nitrosative Stress Resistance and NO Detoxification in Malassezia.

Flavohemoglobins are critical for NO detoxification and counteract nitrosative stress (10). To assess whether this HGT event in Malassezia resulted in a gain of function, we deleted the YHB1 open reading frame (ORF) (MSYG_3741) of M. sympodialis ATCC42132 through targeted mutagenesis using our recently developed transformation protocol based on transconjugation mediated by Agrobacterium tumefaciens (26, 27) (SI Appendix, Fig. S4A).

The M. sympodialis yhb1Δ mutant exhibits hypersensitivity to the NO donors DETA NONOate and sodium nitrite (NaNO₂), but not to hydrogen peroxide (H₂O₂) (Fig. 3A). The two identified Malassezia flavohemoglobins, Yhb1 and Yhb101, were used to generate GFP fusion proteins whose expression was driven by the respective endogenous promoter to complement the M. sympodialis yhb1Δ mutant phenotype and to assess protein localization (SI Appendix, Fig. S4B). Reintroduction of either flavohemoglobin in the M. sympodialis yhb1Δ mutant restored resistance to nitrosative stress at the wild-type (WT) level (Fig. 3A). In agreement, fusion protein expression in complemented strains was confirmed by qPCR (SI Appendix, Fig. S4 C and D), fluorescence-activated cell sorting (FACS), and fluorescence microcopy imaging of GFP expression, which revealed that Malassezia flavohemoglobins are cytoplasmic (Fig. 3B).

Fig. 3.

M. sympodialis flavohemoglobins are involved in nitrosative stress resistance and NO degradation. (A) Stress sensitivity assay of M. sympodialis WT, yhb1Δ mutant, and complementing strains yhb1Δ + YHB1 and yhb1Δ + YHB101 on mDixon agar supplemented with the NO donor agent DETA NONOate and NaNO₂, and with hydrogen peroxide. (B) GFP expression in the M. sympodialis yhb1Δ mutant, and complementing strains yhb1Δ + YHB1 and yhb1Δ + YHB101, and respective GFP signal analyzed through FACS. (C) NO consumption assay by M. sympodialis WT, yhb1Δ mutant, and complementing strains yhb1Δ + YHB1 and yhb1Δ + YHB101; the blue trace indicates the NO level over a period of 15 min. NO and Malassezia (Y = yeast) injections are indicated by purple arrows. In this experiment, 10 µL (Y), 20 µL (×2) and 30 µL (×3), and 40 µL (×4) and 50 µL (×5) of Malassezia cellular suspensions were injected. (D) Representative fluorescent staining of intracellular NO with DAF-FM DA in M. sympodialis WT and two independent yhb1Δ mutants, and (E and F) quantification of the NO signal by flow cytometry; spontaneous fluorescence of M. sympodialis was used as background to detect specifically DAF-FM DA signal. Asterisks indicates statistically significant differences (* = P < 0.05, ** = P < 0.01) according to the unpaired Student’s t test with Welch’s correction. “ns” is not significant.

Next, we tested whether Malassezia flavohemoglobins were able to actively detoxify NO, which could potentially account for its involvement in nitrosative stress resistance. To this aim, we adapted a biochemical assay used for evaluating NO consumption by hemoglobin in red blood cells and plasma (28) to the commensal yeast Malassezia (SI Appendix, Fig. S5). As shown in Fig. 2C, while the M. sympodialis WT strain exhibited robust and dose-dependent NO degradation, the yhb1Δ mutant showed no NO consumption. Complemented strains were able to actively consume NO, although M. sympodialis yhb1Δ + YHB101-GFP displayed a lower NO consumption, corroborating the results of the phenotypic assay, GFP expression, and FACS analysis (Fig. 3 A–C). These differences might be due to less efficient cross-species complementation of the M. yamatoensis Yhb101-GFP fusion protein in the yhb1Δ mutant of M. sympodialis, which was a strategy chosen because of the lack of protocols for gene deletion in M. slooffiae and M. yamatoensis. Taken together, these genetic and biochemical analyses show that the bacterially derived flavohemoglobins protect Malassezia from nitrosative stress by decreasing toxic levels of NO.

To assess intracellular production of NO by M. sympodialis, cells were stained with the NO-specific dye 4-amino-5-methylamino- 2ʹ,7ʹ-diaminofluorescein diacetate (DAF-FM DA), which passively diffuses across membranes and emits increased fluorescence after reacting with NO. Fluorescent microscopy revealed intracellular accumulation of NO in both the WT and yhb1Δ mutant of M. sympodialis (Fig. 3D). NO staining was quantified by FACS analysis, revealing significantly higher NO accumulation in the flavohemoglobin mutant yhb1Δ compared to the M. sympodialis WT (Fig. 3 E and F). Because DAF-FM DA and GFP have similar excitation/emission spectra, complemented strains could not be tested for NO accumulation via flow cytometry, and therefore an independent M. sympodialis yhb1Δ mutant was tested and yielded similar results. These results indicate that the lack of a functional flavohemoglobin leads to intracellular accumulation of NO.

Finally, a broader analysis was performed to assess other functions of the Malassezia flavohemoglobins in response to a variety of environmental stresses and clinical antifungals, but in all cases the M. sympodialis yhb1Δ mutant phenotype was not significantly different from the WT (SI Appendix, Fig. S6). Several studies also report the protective role of both bacterial and fungal flavohemoglobins against NO under anaerobic conditions (10, 23). However, we could not confirm this function for the Malassezia flavohemoglobin in our anaerobic experiments because no phenotypic differences were observed between the WT, the yhb1Δ mutant, and the complemented strains (SI Appendix, Fig. S7).

A Recent Inactivation of YHB1 in M. nana Results in Compromised NO Enzymatic Consumption.

Analysis of Yhb1 protein prediction across species revealed that Malassezia nana YHB1 underwent pseudogenization [i.e., loss of gene function by disruption of its coding sequence with generation of a pseudogene, which is usually indicated as ψ (29)] following a G-to-T transversion in the glycine codon GGA, generating a premature TGA stop codon at the 29th amino acid (SI Appendix, Fig. S8A). Literature search revealed that the sequenced strain of M. nana CBS9557 was isolated in Japan from a cat with otitis externa (30), while the other four known M. nana strains were collected in Brazil: M. nana CBS9558 and CBS9559 from cows with otitis externa, and CBS9560 and CBS9561 from healthy cows (30).

The M. nana strains CBS9557, CBS9559, and CBS9560 were used to investigate whether the pseudogenization event occurred in a M. nana ancestor and whether it impacts nitrosative stress resistance and NO consumption. Because no genomes are available for the M. nana strains CBS9559 and CBS9560, their YHB1 gene was amplified by PCR and Sanger sequenced using primers designed on the YHB1 of M. nana CBS9557 (SI Appendix, Table S2). YHB1 sequence comparison confirmed a premature stop codon present in only CBS9557, with both Brazilian M. nana isolates having a full-coding YHB1 gene (SI Appendix, Fig. S8A). Phenotypic analysis revealed no significant difference in resistance to nitrosative stress by the three M. nana strains, with only a modest increased sensitivity displayed by CBS9557 exposed to 10 mM of sodium nitrite (SI Appendix, Fig. S8B). Strikingly, M. nana CBS9557 displayed undetectable NO consumption activity as observed for the M. sympodialis yhb1Δ mutant, while M. nana CBS9559 and CBS9560 showed regular dose-dependent NO consumption (SI Appendix, Fig. S8C). These data suggest that 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 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 sensitivity to amphotericin B and resistance to fluconazole, and the geographically related strains CBS9559 and CBS9560 displaying an opposite phenotype (SI Appendix, Fig. S9).

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 intensity of 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).

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 novel and unknown signaling pathways regulated by endogenous NO in Malassezia.

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) Venn diagrams 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.

Next, to elucidate the global transcriptomic response of M. sympodialis exposed to nitrosative stress, RNA-seq analysis for M. sympodialis WT cells treated with sodium nitrite was performed. Compared to the untreated control, 112 genes were up-regulated and 50 were down-regulated (FDR < 0.05; log₂ FC ± 0.5) (Fig. 4B and Datasets S3 and S4). The most expressed genes included HEM1 encoding a 5-aminolevulinate synthase involved in heme biosynthesis, MSYG_3126 encoding a hypothetical secreted lipase, the allantoicase-encoding gene DAL2, MSYG_3153 encoding an uncharacterized NAD(P)/FAD-dependent oxidoreductase, and DCG1 encoding a protein with unknown function predicted to be related to nitrogen metabolism (Fig. 4B). The flavohemoglobin-encoding gene YHB1 was significantly up-regulated for FDR < 0.05, but it had low expression level (log₂ FC = 0.33). Low expression of YHB1 was also observed in S. cerevisiae cells exposed to nitrosative stress (31), although its role in NO consumption has been well characterized (23). The most represented Gene Ontology (GO) classes of up-regulated genes are involved in stress resistance, cellular detoxification and transport, and metabolism (Fig. 4C). Functional protein association network analysis revealed enrichment of genes involved in nitrogen metabolism and regulation, ergosterol biosynthesis, and heat shock response (SI Appendix, Fig. S10); we speculate that among the up-regulated transcription factor encoding genes (HSF1, UPC2, BAS1, and HMS1), the heat shock factor Hsf1 might be the key candidate that activates nitrosative stress responsive genes, given its known role in response to stresses in other fungi (32, 33). Conversely, response to nitrosative stress is mediated by the transcription factors Yap1 and Msn2/Msn4 in S. cerevisiae and Schizosaccharomyces pombe (31, 34), and by the transcription factor Cta4 and the Hog1 kinase in C. albicans (35), with the consequent activation of genes known to be required for oxidative stress response, such as those involved in glutathione turnover and other antioxidant/detoxification systems. M. sympodialis CTA1 and CCP1 are the only oxidative stress responsive genes activated in response to nitrosative stress (SI Appendix, Fig. S10 and Dataset S3).

The most represented GO category of down-regulated genes encodes integral components of membrane, which includes transporters and putative Malassezia allergens; other down-regulated genes are involved in calcium metabolism, protein folding, and proteolysis. Two transcription factors were down-regulated and they include the pH responsive Rim101, and an uncharacterized bZIP transcription factor (Fig. 4C and Dataset S4).

Comparison of the two different RNA-seq datasets revealed two common up-regulated genes, encoding the glycerol dehydrogenase Gcy1 and the catalase Cta1, and 10 down-regulated genes that include four Malassezia allergens, a putative secreted lipase, and five hypothetical proteins (Fig. 4D). While it is not surprising to find up-regulation of a detoxifying enzyme such as catalase, it is intriguing to find down-regulation of genes encoding predicted pathogenicity factors.

In conclusion, our transcriptomic data indicate that the response of Malassezia to NO and nitrosative stress is mostly different from other studied fungi, and it involves metabolic pathways and genes that were not known to be relevant to overcome nitrosative stress.

Malassezia Flavohemoglobin Has Characteristic Features of Both Bacterial and Fungal Flavohemoglobins.

We hypothesized that the structure of a protein acquired by HGT will likely remain similar to that of the donor organism in order to retain its original function. Attempts to resolve the crystal structures of both Malassezia flavohemoglobins were carried out, but only the M. yamatoensis flavohemoglobin Yhb101 formed crystals to be analyzed. The structure was determined de novo by single-wavelength anomalous diffraction phasing off the heme-iron bound to the globin domain of the protein (SI Appendix, Table S3). The flavohemoglobin structure is highly conserved with previously characterized structures of this enzyme family, and it consists of an N-terminal globin domain coordinating an iron-bound (Fe²⁺) heme and a C-terminal reductase domain with both FAD- and NAD-binding subdomains, of which only FAD is bound (Fig. 5A). An overlay of a flavohemoglobin structure from Escherichia coli and S. cerevisiae on the M. yamatoensis crystal structure highlights conserved binding sites between the proteins (Fig. 5B). Alignment of the globin domains between literature and experimental structures resulted in an rmsd value of 1.532 Å for E. coli and 1.434 Å for S. cerevisiae, mostly resulting from slight shifts in the D-loop and E-helix between the structures compared. Common to all structures analyzed is the histidine residue coordinating with the heme-iron from the proximal side. This member of the catalytic triad is supported by tyrosine (Tyr98) and glutamate (Glu140) residues conserved in sequence and structure between bacterial and fungal/yeast flavohemoglobins (36, 37). In M. yamatoensis, as also observed in E. coli, the heme-iron is ligated by five atoms: four from the heme and His88 from the F-helix. Substrates commonly bind on the distal side of the heme and lead to a conformational change in the planarity of the heme molecule. The E-helix on the distal side of the heme molecule contributes Leu58, a conserved residue which approaches the heme-bound iron from 3.7 Å away. At this position, the sixth coordination site for the iron is occluded, again similar to the E. coli crystal structure, but unlike the yeast structure where a three-atom small molecule cocrystallized.

Fig. 5.

Three-dimensional X-ray crystal structure of the M. yamatoensis flavohemoglobin Yhb101. (A) The globin domain (cyan) binds a heme molecule. The reductase domain consists of a FAD-binding domain (gray) and a NAD-binding domain (tan) that bind a FAD molecule. (B) An overlay of flavohemoglobin globin domains from fungus, bacteria, and yeast: globin domains of M. yamatoensis (PDB ID 6O0A; blue), S. cerevisiae (PDB ID 4G1V; yellow), and E. coli (PDB ID 1GVH; green) show structural similarity.

In M. yamatoensis Yhb101, the D-loop acts as a bridge between the C- and E-helices and the interface between the bound FAD and heme. Comparison of the D-loops from these structures shows the M. yamatoensis D-loop adopts a nearly identical helical structure as that of S. cerevisiae, in contrast to the E. coli D-loop, which is more extended. The M. yamatoensis E-helix also adopts a ∼30° bend immediately following Leu58, which may straighten out once a substrate is bound. This structural adjustment likely communicates substrate binding near the heme to the reductase domain through movements in the D-loop as the heme B pyrrole propionate forms a hydrogen bond with the main-chain NH of Ser45, the first residue in the D-loop (SI Appendix, Fig. S11).

Lastly, in SI Appendix, Fig. S12 a detailed comparison of the functional residues is shown between the Malassezia flavohemoglobins with those of the closer HGT donor bacteria Brevibacterium ravenspurgense, K. kristinae, Rothia nasimurium, and with the model yeast S. cerevisiae.

Malassezia Flavohemoglobins Are Not Required for Survival on the Host.

Previous studies in human fungal pathogens indicate that flavohemoglobins are required for pathogenesis (38, 39). In our experiments, we found that M. sympodialis WT, yhb1Δ mutant, and yhb1Δ + YHB1 and yhb1Δ + YHB101 complemented strains have similar levels of survival within activated macrophages (SI Appendix, Fig. S13 A and B). This result is in contrast with previous findings in Cryptococcus neoformans (38), but it corroborates results obtained in Aspergillus fumigatus (18). Furthermore, the recently developed murine model for Malassezia skin infection (5) was utilized to test pathogenicity of the flavohemoglobin strains and the induction of host response. Corroborating ex vivo data, we found no differences both in terms of host tissue colonization and host inflammatory response for the yhb1Δ mutant compared to the complemented strains (SI Appendix, Figs. S13 C–E and S14). In agreement, there were no differences between WT and Nos2^−/− mice when challenged with M. sympodialis WT (SI Appendix, Fig. S13 F–H). These results suggest that flavohemoglobin is not required for pathogenesis of Malassezia in an experimental skin model.

Analysis of Malassezia Genomes Revealed Extensive HGT Events from Bacteria.

Given the gain of function due to acquisition of the bacterially derived flavohemoglobins, we sought to identify additional HGT candidate genes in Malassezia species. In a previous study, eight HGT events were identified in M. sympodialis, and then their presence was assessed in other species within the genus (3). In the present study, we applied a previously described analytical pipeline (40) based on three HGT metrics—the HGT index, the Alien Index (AI), and the Consensus Hit Support (CHS)—to identify novel genus and species-specific HGT events. Our goal was not to explicitly establish the evolutionary history of individual genes, but rather to estimate bacteria-derived HGT candidates for the complete set of Malassezia genomes. Besides recovering the YHB1 and YHB101 genes as HGT candidates, this analysis additionally identified a total of 30 HGT candidate genes (Fig. 6 and Dataset S5), 7 of which are in common with the previous study. HGT candidates found in the majority of the Malassezia species include genes involved in broad resistance to stresses, including three that were up-regulated in M. sympodialis exposed to nitrosative stress (Dataset S3), such as the NAD(P)/FAD-dependent oxidoreductase-encoding gene MSYG_3153, the catalase-encoding gene MSYG_3147, and the sorbitol dehydrogenase-encoding gene MSYG_0932 (Fig. 6). Other HGT candidates include a deoxyribodipyrimidine photolyase predicted to be involved in repair of ultraviolet (UV) radiation-induced DNA damage, and a class I SAM-dependent methyltransferase potentially modifying a variety of biomolecules, including DNA, proteins, and small‐molecule secondary metabolites. Another interesting HGT candidate is the gene encoding a septicolysin-like protein, which is known as a pore-forming bacterial toxin that might play a role as a virulence factor (41). This gene is absent in all Malassezia species of clade A, and is present as five copies in M. globosa. Furthermore, a large number of HGT events unique to Malassezia species of clade A were found, and the acquired genes encode a variety of proteins with different functions, such as hydrolysis, protein transport and folding, detoxification of xenobiotics, and resistance to stresses. Finally, 12 of the HGT candidates identified were unique to certain Malassezia species. An intriguing case is M. japonica for which we found 4 unique HGT candidates, 1 of them in three copies. These genes encode orthologs of the fungal Gre2 protein, which is known to be involved in responses to a variety of environmental stresses (42).

Discussion

In the present study we report the functional characterization of two Malassezia flavohemoglobin-encoding genes that were independently acquired through HGT from different Actinobacteria donors. Our experimental analyses demonstrate that both bacterially derived flavohemoglobins are involved in nitrosative stress resistance and NO degradation, consistent with its known functions in bacteria and fungi (10).

We propose an evolutionary HGT model in which extant flavohemoglobin-encoding genes in Malassezia result from a complex pattern of gene retention/loss after being both acquired by a Malassezia common ancestor. Nevertheless, other evolutionary scenarios could also be hypothesized, such as: 1) the acquisition of the YHB1 in a Malassezia common ancestor via HGT from a Brevibacterium-related donor; 2) followed by more recent acquisitions of YHB101 by M. yamatoensis and M. slooffiae via independent HGT events from a common, or closely related, bacterial donor(s) (Kocuria). In this scenario, the “resident” YHB1 in M. yamatoensis and M. slooffiae could have been displaced upon secondary acquisition of the YHB101 gene [a phenomenon termed as xenolog gene displacement (43)], or the acquisition of YHB101 by HGT could have been preceded by the loss of the cognate YHB1 copy. The identification of novel Malassezia species and the analysis of their genomes will be key for the elucidation of these complex models of gene evolution in Malassezia.

Although the mechanisms of HGT in fungi are not fully understood, several possible mechanisms have been reported (24, 44). One such mechanism is gene acquisition through conjugation, which requires contact between bacterial donor and fungal recipient (44). For the HGT events that mediated flavohemoglobin acquisition by Malassezia, the closest phylogenetic donors are Actinobacteria that are part of the mammalian microbiome and hence share the same ecological niche with Malassezia. A dilemma that is common to all HGT events is that if a gene is required for survival in a certain condition, its transfer under that condition might in theory be difficult if not impossible (45). Because NO is synthesized by mammals, including by the skin (46), we speculate that the presence of NO enhanced the HGT transfer of bacterial flavohemoglobins to a fungal Malassezia ancestor that acquired the ability to actively consume NO.

Notably, a large number of eukaryotic organisms including fungi lack a flavohemoglobin-encoding gene, suggesting the existence of alternative pathways for nitrosative stress resistance and NO utilization. For example, in Histoplasma capsulatum, the etiologic agent of histoplasmosis, Yhb1 is replaced by a P450-type NO reductase (47), whereas in other cases, such as for the basidiomycetous fungi Moniliella, Ustilago, and Puccinia, NO metabolism in the absence of flavohemoglobin has yet to be elucidated. The evolution and diversification of flavohemoglobin-encoding genes has been a dynamic and complex process characterized by several prokaryote–prokaryote and prokaryote–eukaryote HGT events (21), hence suggesting its significant contribution for habitat colonization by a species but likely a dispensable role in evolutionary divergence.

Is the bacterial flavohemoglobin required for Malassezia interaction with the host? While a number of studies in bacteria and fungi reported a role for flavohemoglobin in microbial pathogenesis (10), we surprisingly found that Malassezia flavohemoglobins are dispensable for survival within macrophages and for skin infection in our experimental conditions. Conversely, we propose that Malassezia flavohemoglobins are important for the commensal lifestyle of Malassezia through regulation of NO homeostasis, a hypothesis corroborated by down-regulation of genes-encoding putative virulence factors (i.e., allergens and lipases) in our transcriptomic analyses. Another hypothesis is that the HGT-mediated acquisition of flavohemoglobins might be important to mediate Malassezia response to NO that is produced by sympatric microbial communities and acts as a quorum signaling molecule, as reported in bacteria (48) and in S. cerevisiae (49).

Horizontal gene transfer is thought to occur much less frequently in eukaryotes than in prokaryotes (50), but there are notable cases that invoke HGT as a prominent mechanism of eukaryotic evolution, such as in the transition of green plants from aquatic to terrestrial environments (51), and in the colonization of the animal digestive tracts by rumen fungi and ciliates (45, 52). Analysis of Malassezia genomes revealed a large number of HGT events, suggesting that they may also have played a substantial contribution in Malassezia evolution and niche adaptation. Donor bacteria include those that are part of the microbiota of animals, but also others that are known to inhabit a variety of terrestrial and marine habitats, raising questions about a possible wider environmental distribution of a Malassezia ancestor. This could be correlated with the presence of Malassezia DNA in a number of unexpected areas, such as in association with corals and sea sponges in the ocean (53). Moreover, most of the HGT candidate genes identified in Malassezia operate as a self-contained metabolic unit, which has been proposed to facilitate HGT (21). Intriguingly, the high number of HGT events suggests also a predisposition of Malassezia to bacterial conjugation, in line with our previous findings that A. tumefaciens-mediated transformation is the only effective technique for molecular manipulation of Malassezia (54). There are a number of identified HGT that are predicted to be important for Malassezia pathophysiology and that can be characterized using the methodologies reported in the present study.

Materials and Methods

Detailed procedures of the materials and methods used are provided in SI Appendix, SI Material and Methods.

Bioinformatics Analyses.

The Malassezia flavohemoglobin sequences were identified by BLAST analyses. Regions of ∼32 kb surrounding the flavohemoglobin-encoding genes were used for synteny analysis between Malassezia species. Flavohemoglobin protein sequences from Malassezia species, other fungi, and bacteria, were subjected to phylogenetic analysis 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 selected was performed using a consensus set of 246 protein sequences; the phylogenetic tree was constructed with IQ-TREE. To assess the extent of horizontal transfer into Malassezia genomes, we applied a previously described pipeline (40) to the set of 15 available Malassezia proteomes. Transcriptomic analysis was carried out using HiSat and StringTie (57). DESeq2 was used to determine the DEGs as having FDR < 0.05 and log₂ FC ± 0.5, which are common parameters used to define relevant genes in RNA-seq experiments (58). Functional annotation of the DEGs was performed using Blast2GO (59).

Molecular Manipulation of M. sympodialis.

For targeted mutagenesis of M. sympodialis YHB1, ∼1.5-kb regions flanking the YHB1 gene were fused with the NAT marker and cloned in a binary vector for A. tumefaciens-mediated transformation. For yhb1Δ functional complementation, two different flavohemoglobin GFP-fusion proteins were generated. M. sympodialis was transformed through A. tumefaciens-mediated transformation as previously reported (26, 27).

NO Quantification and NO Consumption Assay.

Intracellular levels of NO in M. sympodialis WT and yhb1Δ mutant were measured by flow cytometry using DAF-FM DA (14). The NO consumption assay was performed as previously reported (60) using DETA NONOate as a NO source and a NO chemiluminescence analyzer (TEA 810, Ellutia).

Flavohemoglobin Purification and Crystal Structure.

A construct expressing His-Tev-YHB101 was cloned into E. coli BL21(DE3) cells for expression studies. For protein purification, a Ni²⁺ charged HiTrap Chelating High Performance was used. M. yamatoensis flavohemoglobin crystals were obtained with Morpheus B12: 12.5% (wt/vol) PEG1000, 12.5% (wt/vol) PEG3350, 12.5% (vol/vol) 2-methyl-2,4-pentanediol, 0.03 M each sodium fluoride, sodium bromide, and sodium iodide, and 0.1 M bicine/Trizma base pH 8.5.

Interaction of M. sympodialis Strains with the Host.

For studying interaction of 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 and Nos2^−/− mice were used. All mouse experiments in this study were conducted in strict accordance with the guidelines of the Swiss Animals Protection Law and were performed under the protocols approved by the Veterinary office of the Canton Zurich, Switzerland (license number 168/2018). All efforts were made to minimize suffering and ensure the highest ethical and humane standards.

Data Availability.

The sequence data generated in this study were submitted to the National Center for Biotechnology Information under BioProject accession no. PRJNA626605. Individual accession nos. are given in SI Appendix, SI Material and Methods. The final structure factors and coordinates of the flavohemolgobin Yhb101 of M. yamatoensis were deposited in the Protein Data Bank with code 6O0A.