Abstract
The yeasts of the genus Malassezia are part of the normal skin microbiota of a wide range of warm-blooded animals including humans. Within this genus, Malassezia pachydermatis is commonly found in the normal skin microbiota of a variety of animal hosts. Malassezia yeasts are considered lipid-dependent due to their inability to synthesize long chain fatty acids de novo. While M. pachydermatis is typically able to grow on Sabouraud’s agar (SGA) without lipid supplementation, certain strains display an atypical lipid dependency and are unable to grow on SGA. The aim of this work was to study the genomic differences between atypical M. pachydermatis strains unable to grow on SGA and the reference strain. The genomes of three atypical lipid-dependent M. pachydermatis strains were sequenced using Illumina technology and compared with the reference genome of M. pachydermatis neotype strain CBS 1879. A total of 397 small variants with a high or moderate impact on the protein were observed in genes involved in lipid metabolism. Of those small variants observed we highlight the ones observed in 12 out of the 13 genes encoding secretory lipases and in the CKI1 gene that is unique to M. pachydermatis within the genus. The analysis of those small variations suggested a variation in their ability to adapt to environmental changes and their requirements to grow in different culture media.
Keywords: M. pachydermatis, Genome, Resequencing, Lipid dependency
Introduction
The yeasts of the genus Malassezia are part of the normal skin microbiota of a wide range of warm-blooded animals including humans [1, 2]. The genus includes 20 species, some of which have been isolated from various animal hosts while others tend to be more host specific as M. vespertilionis isolated from bats [3], M. cuniculi isolated from rabbits [4] or M. gallinae isolated from chickens [5]. Even though they are considered commensals, under certain circumstances some of these species may transition to opportunistic pathogens causing common skin disorders [2].
Lipid metabolism plays a crucial role in Malassezia’s metabolism. Malassezia yeasts can metabolize fatty acids to carry out a variety of biological processes such as energy homeostasis and storage, molecular signalling, membrane biogenesis, mediation of cell apoptosis and fusion, metabolism and even contribute to their pathogenicity [6, 7]. However, Malassezia species are unable to synthetize long-chain (C14-C16) fatty acids de novo due to the lack of the gene encoding for the fatty acid synthase [8, 9]. Therefore, they rely on the environment as a source of fatty acids. For this reason, Malassezia yeasts secrete several hydrolases such as lipases, esterases and phospholipases to supply their lipid requirements. It has been observed an expansion of phospholipase and secretory lipase gene families in Malassezia’s genome, especially in M. pachydermatis, where a high number of lipase gene gain events have occurred [8, 10]. The released fatty acids are then metabolized and modified by other lipid metabolism enzymes. They can then be used for the synthesis of triglycerides and sterols, for the synthesis of essential membrane lipids or can be degraded by β-oxidation enzymes [11]. This loss and gain of genes are possibly a consequence of the adaptation of these yeasts to the host’s skin and mucosae which provide at least lipids containing the essential fatty acids required for their growth [8, 12]. Therefore, for the isolation of Malassezia species enriched culture media with a specific lipid composition such as modified Dixon agar (mDA) [13] or Leeming and Notman agar (LNA) [14] are necessary. There is some variability in the lipid-dependency among species of the genus, thus conferring them different metabolic versatility [10, 15]. In fact, M. pachydermatis, was considered the only Malassezia species non-lipid-dependent due to its unique ability to grow on Sabouraud’s agar (SGA) [16]. Nevertheless, genomic analysis revealed that M. pachydermatis’ genome also lacks fatty acid synthase but, it is uniquely able to utilize lipid fractions within the peptone component of SGA [8, 9]. On the other hand, several M. pachydermatis isolates from dogs have shown some inconsistent lipid dependence and were able to grow on SGA after some subsequent transfers on this medium [17]. However, a few M. pachydermatis strains unable to grow on SGA have been reported [18–20]. In our laboratory, we characterized three lipid-dependent M. pachydermatis strains isolated from domestic animals [20]. The finding of these peculiar strains exemplifies the diversity within M. pachydermatis which involves atypical strains with particular growth requirements.
The aim of this work was to study the genomic differences between atypical M. pachydermatis strains unable to grow on SGA and the reference strain. To assess these growth limitations, the genomes of three atypical lipid-dependent M. pachydermatis strains were sequenced using Illumina technology and compared with the reference genome of M. pachydermatis neotype strain CBS 1879. We focused on the analyses of structural and small variants common to these atypical strains that were involved in lipid metabolism.
Materials and Methods
Strains
Three M. pachydermatis haploid strains (MA366, MA374 and MA380) from our collection were studied. These strains were described as atypical lipid-dependent M. pachydermatis strains as they were unable to grow on SGA without lipid supplementation. Their lipid-dependency has been previously evaluated, and their identity has been confirmed by DNA sequencing [20]. The three strains were isolated from the external ear canal of healthy animals. Strain MA374 was isolated from a cow and MA366 and MA380 were isolated from dogs.
Malassezia Culture and DNA Extraction
The three atypical M. pachydermatis strains were cultured on modified SGA supplemented with 4 ml/L Tween 40 and 1 ml/L Tween 20, for 4–5 days at 35 °C. Genomic DNA extraction for Illumina sequencing was done with a phenol:chloroform-based protocol previously described [21]. The eluted DNA was stored at − 20 °C until used.
Genomic DNA Sequencing and Variant Analysis
The three M. pachydermatis strains (MA366, MA374 and MA380) were sequenced using an Illumina NovaSeq 6000 machine to produce short-paired end reads of 150 bp after the generation of libraries using the Illumina TruSeq PCR Free protocol. Prior to further analysis, a quality check was performed on the raw sequencing data, removing low quality portions while preserving the longest high-quality part of NGS reads. A minimum length of 35 bp and a quality score of 25 were set using BBDuk software [22]. The resulting high-quality reads were mapped against the M. pachydermatis reference genome downloaded from the NCBI database (GCF_001278385.1) together with the genome annotation. The mapping was performed with the software minimap2 [23] with the -x sr option. The resulting BAM files were sorted and filtered to remove optical duplicates using the software sambamba [24].
The identification of small variant sites (variant calling; VCs) was performed with the software Freebayes [25] using only uniquely mapping reads (MAPQ > 30), genotyping sites with a coverage of at least 10X and setting a ploidy of 1. In the following step, VCFtools [26] was used to keep only variants with a quality higher than 30 and a read depth of at least 30X. The identification of structural variant sites (SVs) as big deletions, insertions, inversions, duplications and translocations was performed using the MANTA algorithm [27]. The SVs are identified using an approach based on the distance of the paired-end and on the detection of split reads. The obtained variants were then filtered to include only those with a minimum quality of 30 and a minimum depth of 30X. The filtered variants were annotated using the software VEP (Variant Effect Predictor) [28]. During the annotation step, the effect of each variant on the genes in the genome was predicted by the software VEP to infer the presence of low, moderate, or high impact variants.
Results
Resequencing Study
The general resequencing genome data of the three atypical M. pachydermatis strains is summarized in Table 1. On average 95% of the reads passed the initial quality control. Thus, we obtained a mean of 16,904,854 reads per strain, of which 95.25% (16,095,120) mapped on the reference genome with a depth coverage between 264 and 322X. The GC content of the genomes ranged from 52.17 to 52.65%. The resulting mapping of the reads from the atypical M. pachydermatis strains to the CBS1879 reference genome allowed the study of the genomic differences that could explain the lack of growth in SGA. The genome resequencing information of M. pachydermatis MA366, MA374, and MA380 have been deposited in the NCBI Sequence Read Archive (SRA) database under accession PRJNA1293882.
Table 1.
Statistics of resequencing results
| MA366 | MA374 | MA380 | |
|---|---|---|---|
| Raw data | 8,556,457 | 10,002,459 | 8,080,955 |
| Trimmed data | 7,985,743 | 9,617,819 | 7,753,719 |
| % Passed | 93.33 | 96.15 | 95.95 |
| Number of reads | 15,971,486 | 19,235,638 | 15,507,438 |
| Mapped reads | 15,220,961 | 18,206,465 | 14,857,935 |
| % Mapping | 95.3 | 94.65 | 95.81 |
| Average coverage (X) | 269 | 322 | 264 |
| % GC | 52.17 | 52.65 | 52.47 |
SV Analysis
A total of 279 different SVs were observed across the three strains from which 31 were common to the three strains (Table 2). Of those common SVs, 64.52% were breakpoints, 19.35% insertions and 16.13% were deletions (Fig. 1). Focusing on their effect, 41.77% were upstream gene variants, 25.32% were downstream gene variants, 13.92% were intergenic variants, and 6.33% were coding sequence variants (Fig. 2). For further analysis we only focused on those SVs that were common to the three strains and produced a moderate or high impact. Therefore, six SVs with a moderate or high impact were found common to the three strains and were distributed into six different genes (Table 3). Although none of those were related to lipid metabolism, a high impact 1,092 bp deletion was identified. The rest of the common SVs observed had a low impact or an undefined impact on the resulting protein.
Table 2.
Structural variant analysis summarized per strain
| Structural variants (SV) | MA366 | MA374 | MA380 |
|---|---|---|---|
| Total SVs | 198 | 147 | 312 |
| Number of insertions | 67 | 47 | 108 |
| Number of deletions | 90 | 72 | 157 |
| Number of breakpoints | 40 | 26 | 44 |
| Number of duplications | 1 | 2 | 3 |
| Different SVs | 279 | ||
| Common SVs | 31 |
Fig. 1.
Structural variant types observed common to the three strains
Fig. 2.
Most severe consequences common to the three strains produced by structural variants
Table 3.
Common structural variants observed
| ID | Gene description | Function | Impact type | VEP prediction |
|---|---|---|---|---|
| 2872812 | Protein kinase | Intracellular signal transduction, microtubule cytoskeleton organization and protein phosphorylation | Moderate DEL (− 55 bp) | Protein altering variant |
| 28730611 | Nucleoside | Transmembrane transporter activity | Moderate INS (228 bp) | Protein altering variant |
| 28730102 | Tripeptidyl peptidase | Serine-type endopeptidase activity and tripeptidyl-peptidase activity | High DEL (− 1092 bp) | Stop lost, coding sequence variant, 3 prime UTR variant and feature truncation |
| 28728297 | DEAD-domain-containing protein | rRNA processing | Moderate DEL (− 24 bp) | In-frame deletion |
| 28726826 | Histone-lysine n-methyltransferase SETD1 | Nucleic acid binding, histone H3K4 methyltransferase activity and protein binding | Moderate INS (29 bp) | In-frame insertion |
| 28730018 | Essential component of the nuclear pore | Nuclear import and export | Moderate DEL (− 66pb) | In-frame deletion |
VC Analysis
A total of 344,667 different small variants were observed across the three strains, of which 65,672 variants were detected common to the three strains (Table 4). Of those common small variants, 92.74% were single nucleotide variants, 4.71% were complex substitutions, 1.4% were indels and 1.14% were multiple nucleotide polymorphisms (Fig. 3). Focusing on their effect, 43.03% were downstream gene variants, 42.08% upstream gene variants, 10.85% were synonymous variants and 3.24% were missense variants (Fig. 4). Regarding their impact on the protein, 5,097 small variants with a moderate or high impact were found common to the three strains and were distributed into 1,383 genes. For further analysis, this study only focused on common small variants that had a moderate or high impact on the protein and were related to lipid metabolism. Therefore, a total of 397 small variants were observed in genes involved in lipid metabolism (Table 5). Only two small variants showed a high impact on the resulting protein, producing the loss of a start and a stop codon, respectively. Both high impact SNPs were found in genes encoding secretory lipases TGL3 (ID 28726643 and ID 28728420). The remaining alterations produced a moderate impact on the protein and were considered missense variants. Only two of these moderate impact small variants were splice region missense variants. These 397 small variants were distributed into 99 genes, of which 23% were lipases and esterases, 11% were genes related to sterol metabolism, 33% were genes involved in phospholipid metabolism and 20% were genes of fatty acid metabolism. Also, variants were observed in other genes related to the lipid metabolism (12%) as transferases or lipid binding enzymes. Within the group of lipases (Table 5), 23 lipase-like genes including secretory lipases and esterase lipases were found to have 73 moderate and 2 high impact small variants altering the protein function. A total of 12 genes encoding secretory lipases showed moderate and high impact small variants. Also, several esterase and esterase lipase encoding genes were altered. Within the group of sterol synthesis genes, 11 essential genes such as the ERG2, ERG5, ERG9, ERG10, ERG13, ERG24 or the HMG1/2 genes, related to sterol and ergosterol synthesis showed 32 moderate impact small variants. Regarding the group of genes involved in phospholipid metabolism, 33 genes of importance for the synthesis of phospholipids such as phospholipases, the CHO2 gene and the CKI1 gene showed 163 alterations. Nevertheless, 20 essential genes involved in fatty acid metabolism showed 86 moderate impact small variants. Within this group of fatty acid metabolism genes, the alterations observed in the Δ-9 and Δ-12 desaturase genes, the Acyl-CoA oxidase gene, the Acetyl-CoA synthetase gene and both copies of the polyketide gene stand out due to their relevance for the lipid metabolism. A total of 44 small variants were also observed in other 12 genes involved in lipid metabolism, such as genes related to lipid binding, homeostasis and other metabolic processes.
Table 4.
Small variant analysis summarized per strain
| Small variants | MA366 | MA374 | MA380 |
|---|---|---|---|
| Total small variants | 220,731 | 221,029 | 224,711 |
| Number of SNPs | 169,552 | 169,690 | 173,265 |
| Number of indels | 7,333 | 7,409 | 7,351 |
| Insertion indels | 2,961 | 2,992 | 3,164 |
| Deletion indels | 4,372 | 4,417 | 4,187 |
| Large (> 10 bp) insertions | 148 | 155 | 163 |
| Large (> 10 bp) deletions | 221 | 240 | 258 |
| Number of hom ref loci | 120,050 | 120,064 | 116,494 |
| Number of hom var loci | 220,731 | 221,029 | 224,711 |
| Number of variant loci | 220,731 | 221,029 | 224,711 |
| Different | 334,667 | ||
| Common | 65,672 |
Fig. 3.
Small variant types observed common to the three strains
Fig. 4.
Most severe consequences produced by small variant sites common to the three strains
Table 5.
Genes involved in lipid metabolisms with a moderate or high impact small variants common to the three
| ID | Gene description | Function | VEP prediction |
|---|---|---|---|
| Lipases & esterases | |||
| 28726643 | Secretory lipase | Enables triglyceride lipase activity | Start lost|Missense variants |
| 28726875 | Secretory lipase | Enables triglyceride lipase activity | Missense variants |
| 28727692 | Secretory lipase | Enables triglyceride lipase activity | Missense variants |
| 28727697 | Secretory lipase | Enables triglyceride lipase activity | Missense variants |
| 28728326 | Secretory lipase | Enables triglyceride lipase activity | Missense variants |
| 28728420 | Secretory lipase | Enables triglyceride lipase activity | Stop lost |
| 28729307 | Secretory lipase | Enables triglyceride lipase activity | Missense variants |
| 28729308 | Secretory lipase | Enables triglyceride lipase activity | Missense variants |
| 28729753 | Secretory lipase | Enables triglyceride lipase activity | Missense variants |
| 28729754 | Secretory lipase | Enables triglyceride lipase activity | Missense variants |
| 28729086 | Secretory lipase | Enables triglyceride lipase activity | Missense variant |
| 28730532 | Secretory lipase | Enables triglyceride lipase activity | Missense variants |
| 28729104 | GDSL family lipase | Hydrolase activity, acting on ester bonds | Missense variants |
| 28729106 | GDSL family lipase | Hydrolase activity, acting on ester bonds | Missense variants |
| 28726738 | Triglyceride lipase-cholesterol esterase | Lipid metabolic process | Missense variants |
| 28726765 | Phospholipase carboxylesterase family protein | Hydrolase activity | Missense variants |
| 28726792 | PLC-like phosphodiesterase | Phosphatidylinositol phospholipase C activity | Missense variants |
| 28727315 | PLC-like phosphodiesterase | Phosphoric diester hydrolase activity | Missense variants |
| 28729016 | PLC-like phosphodiesterase | Phosphoric diester hydrolase activity | Missense variants |
| 28729037 | Palmitoyl-protein thioesterase 1 | Palmitoyl hydrolase activity and thioester hydrolase activity | Missense variants |
| 28729456 | Arylacetamide deacetylase | Hydrolase activity | Missense variants |
| 28729458 | Esterase lipase | Hydrolase activity | Missense variants |
| 28726990 | Acyl-thioesterase 2 | Acyl-CoA hydrolase activity | Missense variants |
| Sterol metabolism | |||
| 28727161 | ERG5 (C-22 sterol desaturase) | Sterol metabolic process | Missense variants |
| 28726452 | HMG1/2 (Hydroxymethylglutaryl-reductase) | Ergosterol biosynthetic process | Missense variants |
| 28727558 | ERG13 (Hydroxymethylglutaryl-synthase) | Ergosterol biosynthetic process and farnesyl diphosphate biosynthetic process | Missense variant |
| 28728578 | ERG24 (C-14 reductase) | Ergosterol biosynthetic process | Missense variants |
| 28728633 | Cytochrome-b5 reductase | Ergosterol biosynthetic process | Missense variant |
| 28729490 | ERG2 (C-8 sterol isomerase) | Ergosterol biosynthetic process | Missense variant |
| 28730546 | ERG9 (Farnesyl-diphosphate farnesyltransferase) | Ergosterol biosynthetic process and farnesyl diphosphate metabolic process | Missense variants |
| 28729142 | ERG10 (Acetyl-CoA C-acetyltransferase) | Fatty acid beta-oxidation, sterol synthesis | Missense variant |
| 28727196 | Fatty acid hydroxylase | Ergosterol biosynthetic process | Missense variants |
| 28729939 | Hypothetical protein | Sterol binding | Missense variants |
| 28730398 | Isoprenoid biosynthesis-related protein | Farnesyl diphosphate biosynthetic process | 7 Missense variants|1 Missense variant and splice region variant |
| Phospholipid metabolism | |||
| 28726906 | Phospholipase C | Hydrolase activity, acting on ester bonds | Missense variants |
| 28726907 | Phospholipase C | Hydrolase activity, acting on ester bonds | Missense variants |
| 28730308 | Phospholipase C | Hydrolase activity, acting on ester bonds | Missense variants |
| 28730591 | Phospholipase C | Hydrolase activity, acting on ester bonds | Missense variants |
| 28727150 | SPO14-phospholipase D | Phosphatidylinositol binding, phospholipase D activity | Missense variants |
| 28726795 | Lysophospholipase PLB1 | Phospholipase A2 activity | Missense variants |
| 28728006 | Lysophospholipase | Carboxylic ester hydrolase activity and palmitoyl-(protein) hydrolase activity | Missense variants |
| 28728925 | Cytosolic phospholipase | Glycerophospholipid catabolic process | Missense variants |
| 28729637 | Hypothetical protein | Sphingomyelin phosphodiesterase activity | Missense variants |
| 28729683 | Aminophospholipid transporting P-type ATPase | Phospholipid translocation | Missense variants |
| 28730521 | P-type ATPase (amino-phospholipid-translocase) | Phospholipid translocation | 1 in-frame deletion | 7 Missense variants |
| 28729006 | Phospholipid-translocating ATPase | Phospholipid translocation | Missense variants |
| 28728579 | Hypothetical protein | Glycerophospholipid biosynthetic process | Missense variant & splice region variant | Missense variant |
| 28728584 | Phosphatidylserine decarboxylase PSD3 | Phospholipid biosynthetic process | Missense variants |
| 28726724 | EPT1-sn-diacylglycerol ethanolamine cholinephosphotransferase | Phospholipid biosynthetic process | Missense variants |
| 28727718 | Phosphatidyl synthase | Cardiolipin biosynthetic process | Missense variants |
| 28728940 | Acyltransferase-domain-containing protein | Cardiolipin acyl-chain remodelling | Missense variants |
| 28726864 | Choline Kinase (CKI1) | Phosphatidylcholine biosynthetic process and phosphatidylethanolamine biosynthetic process | Missense variants |
| 28727050 | Patatin domain containing protein | Phosphatidylcholine metabolic process | Missense variants |
| 28727422 | Phosphatidylethanolamine n-methyltransferase (CHO2) | Phosphatidylcholine biosynthetic process | Missense variants |
| 28727844 | Myo-inositol monophosphatase | Inositol metabolic process | Missense variant |
| 28729642 | Phosphatidylinositol-4-kinase | Phosphatidylinositol phosphate biosynthetic process and phosphatidylinositol-mediated signalling | Missense variants |
| 28728992 | Vacuole-associated enzyme activator complex component | Phosphatidylinositol biosynthetic process | Missense variants |
| 28728374 | Hypothetical protein | Inositol phosphoceramide metabolic process | Missense variants |
| 28728184 | Nicotinate-nucleotide diphosphorylase | Phosphatidylinositol phosphate biosynthetic process | Missense variants |
| 28727917 | Atypical PIKK PI3K protein kinase | Phosphatidylinositol-3-phosphate biosynthetic process and phosphatidylinositol mediated signalling | Missense variants |
| 28727417 | Inositol hexakisphosphate kinase inositol pyrophosphate synthase | Inositol metabolic process and inositol phosphate biosynthetic process | Missense variant |
| 28726664 | Cdc4 and related F-box and WD40 protein | Phosphatidylinositol binding | Missense variants |
| 28727015 | Diadenosine hexaphosphate hydrolase | Diphosphoinositol polyphosphate metabolic process | Missense variants |
| 28727024 | 1-phosphatidylinositol-3-phosphate 5-kinase | Phosphatidylinositol phosphate biosynthetic process | Missense variants |
| 28727350 | Saicar synthase-like protein | Phosphatidylinositol metabolic process | Missense variants |
| 28726868 | PX domain containing protein | Phosphatidylinositol binding | Missense variants |
| 28726871 | AUR1 (Inositol phosphorylceramide synthase) | Inositol phosphoceramide synthase activity | Missense variant |
| Fatty acid metabolism | |||
| 28730094 | 17-beta-hydroxysteroid dehydrogenase | Fatty acid elongation | Missense variants |
| 28730159 | Hypothetical protein | Fatty acid biosynthetic process | Missense variants |
| 28727736 | Hypothetical protein | Fatty acid catabolic process and in triglyceride biosynthetic process | Missense variants |
| 28728011 | Peroxisomal acyl-CoA thioester hydrolase 1 | Fatty acid catabolic process | Missense variants |
| 28729494 | Hypothetical protein | Fatty acid elongation, sphingolipid biosynthetic process and very long-chain fatty acid biosynthetic process | Missense variants |
| 28728787 | ATP-binding subfamily d member 2 | Fatty acid beta-oxidation, long-chain fatty acid import into peroxisome and very long-chain fatty acid catabolic process | Missense variants |
| 28726827 | Acyl-CoA oxidase | Fatty acid beta-oxidation using acyl-CoA oxidase and lipid homeostasis | Missense variants |
| 28727363 | Acetyl-CoA synthetase-like protein | Long-chain fatty acid metabolic process | Missense variants |
| 28726921 | Acyl-CoA dehydrogenase | Fatty acid beta-oxidation, involved in medium-chain fatty acid catabolic process | Missense variants |
| 28727005 | Peroxisomal half ABC transporter | Fatty acid beta-oxidation, long-chain fatty acid import into peroxisome, and very long-chain fatty acid catabolic process | Missense variants |
| 28729045 | Acetyl-CoA acyltransferase 2 | Fatty acid beta-oxidation | Missense variant |
| 28728338 | Acetyl carboxylase | Fatty acid biosynthetic process | Missense variants |
| 28728375 | Fatty acid elongase | Fatty acid elongation in monounsaturated, polyunsaturated and saturated fatty acid, in sphingolipid biosynthetic process, in very long-chain fatty acid biosynthetic process | Missense variant |
| 28729115 | ATP-citrate synthase | Fatty acid biosynthetic process | Missense variants |
| 28729865 | Polyketide synthase | Fatty acid biosynthetic process | Missense variants |
| 28728046 | Polyketide synthase | Fatty acid biosynthetic process | Missense variants |
| 28729055 | Δ12 fatty acid desaturase | Synthesis of polyunsaturated fatty acids | Missense variants |
| 28729274 | Δ9 desaturase | Synthesis of unsaturated fatty acids | Missense variant |
| 28726876 | Carnitine acyl carnitine carrier | Carnitine transmembrane transport | Missense variant |
| 28728234 | Acyltransferase CTAse COT CPT | Carnitine metabolic process | Missense variants |
| Other genes related to lipid metabolism | |||
| 28729152 | Sphingosine hydroxylase | Lipid biosynthetic process | Missense variants |
| 28727194 | Protein of class 3 family | Lipid metabolic process | Missense variants |
| 28726435 | Acid sphingomyelinase | Hydrolase activity | Missense variants |
| 28726734 | ABC1 domain containing protein | Lipid homeostasis | Missense variants |
| 28726824 | Mitochondrial distribution and morphology protein | Lipid binding | Missense variants |
| 28728172 | Hypothetical protein | Lipid binding | Missense variant |
| 28727246 | Phosphatidate cytidylyltransferase | Phosphatidate cytidylyltransferase activity | Missense variants |
| 28727719 | Orm1 type endoplasmic reticulum protein | Ceramide metabolic process, intracellular sphingolipid homeostasis and negative regulation of ceramide biosynthetic process | Missense variants |
| 28729010 | Serine palmitoyltransferase | Ceramide biosynthetic process and sphingosine biosynthetic process | Missense variants |
| 28728237 | Glycerol-3-phosphate acyltransferase | Cellular lipid metabolic process | Missense variants |
| 28729395 | Glycosyltransferase family 57 protein | Oligosaccharide-lipid intermediate biosynthetic process | Missense variants |
| 28727906 | Glycosyltransferase family 4 protein | Oligosaccharide-lipid intermediate biosynthetic process | Missense variant |
Discussion
Due to the lack of the fatty acid synthase, Malassezia yeasts are unable to synthesize fatty acids de novo and rely on the environment as a main source. Thus, changes in the environment and its external fatty acid composition could represent a challenge for Malassezia yeasts [6, 8]. Malassezia species have a high number of enzymes related to the lipid metabolism to supply their lipid requirements. These enzymes may vary between species, conferring different metabolic versability to adapt to different environments [6, 8, 10, 29]. Malassezia pachydermatis is the only species within the genus able to grow on SGA without lipid supplementation, being a more versatile lipid-dependent yeast [8]. However, recently atypical M. pachydermatis strains that cannot grow on SGA have been observed [18–20]. To assess the basis of this atypical lipid dependency of some M. pachydermatis strains, the genome of three strains was sequenced and compared with the neotype strain. As the strains were unable to grown on SGA, they were cultured on SGA supplemented with Tween 20 and Tween 40. These lipid dependent M. pachydermatis strains have shown good growth on SGA with the addition of Tween 20 and Tween 40 [20] and they grew well in our study.
The analysis of the genome of these atypical M. pachydermatis strains revealed shared moderate to high impact SVs and small variants. These genetic variants were predicted to impact protein function, although the exact effects are not fully understood due to limited knowledge of the species' lipid metabolism. Although no SVs targeted lipid metabolism genes, a high-impact deletion was identified in the gene encoding the tripeptidyl peptidase enzyme. This enzyme may be involved in the degradation of peptides derived from peptone, a component of SGA known to contain fatty acids. The disruption of this enzyme could potentially alter the processing of peptone-derived substrates, thereby influencing fatty acid availability and metabolic responses in this medium [30].
Regarding small variants, we focused on those that affect lipid metabolism related genes. In our study, these atypical M. pachydermatis strains showed small variants in 23 lipase and esterase genes. The M. pachydermatis reference genome has 13 genes encoding secretory lipases, and in our strains, 12 out of these 13, showed moderate or high impact variants. Lipase genes are involved in the release of fatty acids from a variety of lipid compounds found in the environment, thus enabling lipid synthesis in Malassezia species [29, 31]. The two lipases showing high impact small variants were TGL3 and catalyze the hydrolysis of ester linkages of triglycerides [6]. The ability to uptake those fatty acids for their use in lipid synthesis is essential to sustain the growth of Malassezia [29].
Alterations were observed in genes encoding phospholipases, phosphodiesterases and other enzymes related to the phospholipid metabolism. Phospholipids are structural components of fungal cell membranes and play essential roles in their biology [7]. Moreover, phosphatidylcholine is an abundant glycerophospholipid found on eukaryotic membranes. In the presence of external choline, M. pachydermatis could synthetize phosphatidylcholine through both the Kennedy and the CDP-choline pathways [6, 7]. Within the genus Malassezia, M. pachydermatis is the only species able to synthesize phosphatidylcholine via the Kennedy pathway due to the presence of the choline kinase (CKI1) gene [6, 32]. However, our three atypical M. pachydermatis strains showed 5 moderated impact VCs in this gene, potentially altering phosphatidylcholine production through the Kennedy pathway. Nevertheless, another gene encoding a phosphatidylethanolamine n-methyltransferase (CHO2 gene) related to the synthesis of phosphatidylcholine through the CDP-choline pathway showed ten moderate impact small variants. The depletion of phosphatidylcholine in yeasts has been linked to a shift in the fatty acid composition of the cells, leading to an increase of ratios of C16 over C18 and saturated over unsaturated acyl chains. This shifting relies on the fatty acid composition of the environment and the adaptation mechanisms of the yeasts [32]. These mechanisms of adaptation are available in yeasts with an intact fatty acid synthesis machinery as Saccharomyces cerevisiae [32], whereas our atypical M. pachydermatis strains may face some difficulties adapting to the growth in a media without lipid supplementation.
Additional small variants were found in genes essential for fatty acid metabolism in our atypical M. pachydermatis strains. Twenty altered genes affected fatty acid synthesis, elongation, degradation, and transport, suggesting reduced ability to utilize environmental fatty acids. Within this group of genes, the gene encoding for the Δ9-desaturase responsible for the synthesis of unsaturated fatty acids showed moderate impact small variants in the three atypical strains. This enzyme converts palmitic and stearic acids into palmitoleic and oleic acids, which are essential for membrane fluidity and growth. Malassezia globosa and Malassezia restricta lack this enzyme and rely entirely on external unsaturated fatty acids, so its alteration in atypical strains may reduce metabolic versatility [9, 11, 31]. Similarly, the Δ12-desaturase gene, involved in polyunsaturated fatty acid synthesis, showed two moderate impact variants [33]. It has been demonstrated that in Malassezia, growth is best stimulated by unsaturated fatty acids [10], so these changes may reduce metabolic versatility and could explain the need for additional lipid supplementation compared to the neotype strain. Furthermore, the polyketide synthase gene, which is structurally similar to fatty acid synthase and may contribute to lipid assimilation versatility, also showed moderate alterations [8, 29]. This could indicate a reduced ability to utilize certain environmental fatty acids and adapt to changing conditions.
In conclusion, sequencing the genome of three atypical lipid-dependent M. pachydermatis strains allowed a better understanding of this species and the growth differences observed between strains. The analysis of the small variations observed in genes related to lipid metabolism such as lipases, genes related to phospholipid metabolism and fatty acid metabolism suggested a variation in their ability to adapt to environmental changes and their requirements to grow in different culture media. We emphasize the small variations identified in 12 of the 13 genes encoding secretory lipases, as well as in the CKI1 gene, which is unique to M. pachydermatis within Malassezia genus. Thus, possibly explaining the lack of growth observed in SGA as they might not be able to utilize the lipid fractions within its peptone component and require additional lipid supplementation. Nevertheless, more studies would be necessary to further understand the lipid versatility of M. pachydermatis at the transcriptional level.
Finally, establishing genotype-phenotype-pathogenicity correlations and assessing host interaction and disease potential are critical next steps to take in future studies. However, it is important to note that the isolation of these atypical strains is extremely rare [20]. In our collection of more than 1,500 M. pachydermatis isolates, only three have shown this atypical lipid-dependent phenotype, and all were recovered from clinically healthy animals. This rarity suggests that these strains may represent uncommon auxotrophic mutants rather than a widespread pathogenic form, but further research is needed to clarify their biological significance.
Acknowledgements
The authors thank Carolina Gómez from the Veterinary Mycology Group of Universitat Autònoma de Barcelona (UAB) for valuable technical assistance.
Author Contributions
Leyna Díaz (Formal analysis, Methodology, Writing—original draft), Gemma Castellá (Conceptualization, Formal analysis, Methodology, Writing—review & editing), Riccardo Aiese Cigliano (Data curation, Formal analysis, Writing—review & editing), Walter Sanseverino (Data curation, Formal analysis, Writing—review & editing), and F. Javier Cabañes (Conceptualization, Formal analysis, Funding acquisition, Methodology, Supervision, Writing—review & editing).
Funding
Open Access Funding provided by Universitat Autonoma de Barcelona. This work was supported by Servei Veterinari de Bacteriologia i Micologia from the UAB.
Data Availability
The genome resequencing information of M. pachydermatis MA366, MA374, and MA380 have been deposited in the NCBI Sequence Read Archive (SRA) database under accession PRJNA1293882.
Declarations
Conflict of interest
Riccardo Aiese Cigliano and Walter Sanseverino were employed by the company Sequentia Biotech S.L. All other authors declare no conflict of interests.
Footnotes
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Associated Data
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Data Availability Statement
The genome resequencing information of M. pachydermatis MA366, MA374, and MA380 have been deposited in the NCBI Sequence Read Archive (SRA) database under accession PRJNA1293882.