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In silico identification of prospective virulence factors associated with candidiasis in Meyerozyma guilliermondii strain SO from genome dataset

Abstract

Background

Meyerozyma guilliermondii is a prospective yeast that has extensively contributed to the biotechnology sector. In 2015, M. guilliermondii strain SO which was isolated from spoiled orange has successfully been developed as an inducer-free expression system and attained a significant impact in producing industrially important recombinant proteins. The species possesses high similarity to Candida albicans which may cause candidiasis. The industrial-benefiting M. guilliermondii strain SO has been underexplored for its virulence status. Thus, this study aimed to document the potential virulence factors through the comprehensive in silico analysis of M. guilliermondii strain SO genome. This analysis demonstrated the molecular characterization which could distinguish the pathogenicity status of M. guilliermondii.

Results

The genome data were generated from Illumina HiSeq 4000 sequencing platform and assembled into 51 scaffolds successfully accumulating a genome size of 10.63 Mbp. These enclosed 5,335 CDS genes and 5,349 protein sequences with 43.72% GC content. About 99.29% of them were annotated to public databases. Komagataella phaffii, Saccharomyces cerevisiae and the reference strain of M. guilliermondii (ATCC 6260) were used as the controls. They were compared with our in-house strain SO to identify the consensus domain or subdomain which could putatively be considered as virulence factors. Candida albicans was used as the pathogenic model. Hence, hidden Markov model against strain SO proteome had identified secreted aspartic proteases (SAP), phospholipase C (PLC) and phospholipase D (PLD) with an E-value of 2.4e−107, 9.5e−200 and 0.0e+00, respectively, in resemblance of C. albicans. The topology of the phylogenetic analysis indicated that these virulence factors in M. guilliermondii strain SO and C. albicans branched from the same node and clustered together as a clade, signifying their molecular relatedness and congeneric among these species, subsequently proposing the virulence status of M. guilliermondii.

Conclusion

The SAP, PLC and PLD genes’ features that were significant in expressing determinants of pathogenicity were successfully identified in M. guilliermondii strain SO genome dataset, thus concluding the virulency of this species. On account of this finding, the strategy of gene knockout through CRISPR-Cas9 or homologous recombination strategies is needed to engineer the feasible novel expression host system. Over and above, the genetically modified strain of M. guilliermondii allegedly may eradicate the risk of candidiasis infection.

Background

The universal inventory of microbial diversity by a congruence of the dominance scaling law was estimated upward of 1 trillion species [1]. Meanwhile to date, the assessment of global fungal diversity was predicted to range from 2.2 to 3.8 million species based on the improved statistical and molecular phylogenetic approach [2]. Of those fractions, about 650 species were reported as pathogens to cause human diseases [3] and relatively 150 species were discovered as opportunistic fungi to immunocompromised individuals [4].

Invasive candidiasis is a life-threatening fungal infection that is commonly caused by Candida spp. [5]. Candida albicans is the most predominant species that has immense potential of spreading and infecting human from superficial mucosal to systemic candidiasis [6, 7], indisputably, progressively demonstrating morbidity and mortality worldwide [8, 9]. The main pathogenic feature in Candida that obligates persistency in the human host is the mechanism of biofilm formation [10]. The yeast-to-hypha morphology transition manifests adherence ability to attach and invade human epithelial cells henceforth triggering pathogenesis [11]. The proteolytic activity from the proteinase enzyme is involved in pathogenicity [12] and possibly carries a virulence factor including secreted aspartyl proteinase (SAP) and phospholipases [13].

Candida guilliermondii (teleomorphic: Pichia guilliermondii) is currently renowned as Meyerozyma guilliermondii [14]. This fungi species is associated with Ascomycota phylum under Saccharomycotina CTG clade, where the genetic CUG codon is reassigned from leucine to serine [15, 16]. Natural habitat of this species is highly diverse in clinical and environmental samples, but most frequently found in oil-containing soil [17]. A strain of M. guilliermondii was isolated from a spoiled orange and identified as strain SO (GenBank JN084128) [18]. It had been developed as a prospective system for heterologous protein expression, providing an alternative to the intensively used species, Komagataella phaffii [19] (also referred to as Pichia pastoris [20]).

Veritably, this novel strain is capable of producing recombinant enzymes such as lipase [18], protease, diamine oxidase [21] and α-amylase [22]. Moreover, the competency of this microbial organism to facilitate expression mediated by alcohol oxidase (AOX) and formaldehyde dehydrogenase (FLD) promoters successfully demonstrates the independent commencement of mRNA transcription within a shorter time in the absence of any inducer such as methanol or methylamine [22]. The achievement may obliquely reduce the production cost, minimize methanol toxicity effects and would further innovate the technology of enzyme research. However, this potential yeast has been reported as a causative pathogen and associated with candidiasis [23]. An emergence incidence of candidiasis recently shifts toward non-albicans Candida spp. that demonstrates low intrinsic susceptibility to antimycotic drugs [24], subsequently denotes this opportunistic yeast [25,26,27] and probably associated with virulence determinants in C. albicans.

In fact, M. guilliermondii aligns the phylogenetic branch closely to C. albicans, signifying an affiliation among these two sensu stricto species [28]. True hyphae are absent in M. guilliermondii [29], and the haploid features of chromosome may exhibit less virulence in systemic infection model [30]. The composition of cell wall is also dissimilar, therefore exaggerating human innate immune cells to boost the production of cytokine and phagocytosis in dectin-1-dependent pathway, where C. albicans is incompetent to be recognized by the host due to its macromolecule structure [31]. The thickness of M. guilliermondii cell wall is about 160–170 nm [21] in comparison to the cell wall of C. albicans (approximately 400 nm) which is two times greater [32], emphasizing the establishment of defense mechanisms by the latter species [33]. Virulence factor proteins (enzymes) that can substantially be found in yeast are secreted aspartic proteases (SAP), phospholipases, lipases, agglutinin-like sequences (Als), 70-kDa heat shock protein, enolases and phytases [34]. In-depth microbiological characterization of isolates with M. guilliermondii candidiasis has produced biofilms, but with low metabolic activity and moderate biomass, supported by clinical presentation in human with the predisposing condition is less severe and lower mortality as compared to C. albicans [35].

Nevertheless, the production of virulence properties in M. guilliermondii may be detected by enzymatic activities of esterase (phospholipase C and D) and aspartic protease [36]. A comparative study of toxicity between Saccharomyces cerevisiae and M. guilliermondii on zebrafish embryos had shown 0% survival rate within 120 h post-exposure to the latter strain and the breakdown of lipid membrane revealed the activity of phospholipase enzyme in M. guilliermondii, meanwhile, the absence of scoliosis and 20% mortality rate of embryos treated with S. cerevisiae strengthened the status of S. cerevisiae as a non-toxic yeast [37]. Hence, it is critical to recognize the source of factors that may invoke the safety standards of the yeast expression system, especially yeasts that may possess a high similarity of pathogenic properties to C. albicans. To date, there has been limited published research documented on the virulence properties in M. guilliermondii as per commonly inspected in pathogenic fungal species like Candida spp. [38]. Thus, in this work, the presumed Candida-like virulence factors were identified in M. guilliermondii strain SO genome dataset and subsequently analyzed using in silico approaches according to the presence of domain SAP, phospholipase C (PLC) and phospholipase D (PLD), similar to those in C. albicans.

Results and discussion

Genomic features of M. guilliermondii strain SO

The draft genome of M. guilliermondii strain SO was generated using Illumina HiSeq 4000 system with a total throughput of 10.6 Mbp. Across the whole genome, the structure of 142 tRNA and 3 rRNA (8S; 18S; 28S) were detected by HMM model. The dataset comprising of 69 contigs which 52% of it were larger than 1 kbp and the largest contigs racked up to 1.34 Mbp. The final assembled contigs were merged into 51 scaffolds with GC content of 43.72%. About 5,372 CDS were predicted according to fungal-specific intron organization in GeneMark-ES algorithm (Table 1), covering 78.1% of the entire genome.

Table 1 Assembly statistics of M. guilliermondii strain SO genome features demonstrated from de novo Velvet program

The annotation of genes was identified up to 99% homolog in public databases, and 88.5% of the sequences was classified in GO categories (Additional file 1: Fig. 1). Concurrently, 3,495 domains in protein families (pfam) database locally aligned to 4,597 (85.9%) protein sequences of M. guilliermondii strain SO (Additional file 1: Fig. 2). In addition, a total of 966 predicted proteins were assigned to 851 EC number and subsequently mapped to 134 KEGG pathway maps as per updated on April 2021.

Approximately, 1,918 (35.8%) of protein queries in M. guilliermondii strain SO dataset were assigned to the KEGG network-based system. Furthermore, KofamKOALA was performed to complement the analysis and identified 64.4% genes subset to 502 modules that further linked to 6 major classes (metabolism pathway, genetic information processing, environmental information processing, cellular processes, organismal systems, and human diseases) and secondarily, associated with 53 KEGG networks (Fig. 1).

Fig. 1
figure 1

Tabulation of M. guilliermondii strain SO proteins into 53 interconnected KEGG pathway. The stacked on the column base indicates solely hits of annotation to the respected pathway maps

The initial verification of the genome set examined using BUSCO Assessment succeeded to inspect 97% of the assembly completeness subjected to the catalog of conserved single-copy orthologs from Ascomycota dataset in OrthoDB. From the assessment of predicted gene models, all 1,315 BUSCO orthologs are accounted within the predicted gene sets, albeit a few of those orthologs are fragmented. Gene prediction analysis was carried out adequately as most predicted single-copy orthologs were presented in the genome (Additional file 1: Table 1).

Prospective virulence factor in putative aspartic proteases encoding gene

Proteolytic enzymes or also defined as proteases, peptidases and proteinases catalyze the breakdown of peptide bonds in proteins through hydrolysis reaction. Aspartic proteases (EC 3.4.23.24) classified under the group of enzymes candidapepsin possess virulence determinants through the degradation of subendothelial extracellular matrix, albumin, immunoglobulin G, hemoglobin, fibronectin and laminin in the host components [39]. Specifically named as eukaryotic aspartyl protease, this domain architecture can be found in 11,794 sequences in the Ensembl public database (verified in April 2021). These catalytic residues are α/β monomer that is composed of bilobed shape, positioned within the hallmark motif Asp-Thr/Ser-Gly to the active site based on the InterPro HMM database. The eight genes of aspartic proteases were identified in M. guilliermondii strain SO with encoded ID’s; 1971_t, 1972_t, 4064_t, 4978_t, 4979_t, 4980_t, 70_t and 999_t (Additional file 1: Table 2). From the eukaryotic protease homolog, the closest identity to M. guilliermondii strain SO profile besides the other strains of M. guilliermondii are the proteases from K. phafii, S. cerevisiae and C. albicans with the E-value 6.1e−65, 7.5e−120 and 2.4e−107, respectively (Table 2).

Table 2 HMM homologs hits on M. guilliermondii strain SO eukaryotic aspartyl protease profiles

Overall, aspartic proteases have limited homology except for the active site region approximately at residue 78 to 271. This profile is involved in aspartic-type endopeptidase for proteolysis activity. Figure 2 shows eight genes of strain SO slightly diversed between one another and dispersed to the same branch of M. guilliermondii ATCC 6260, C. albicans, K. phaffii and S. cerevisiae separately. It could be defined that the gene 70_t, 1971_t and 1972_t were prospective to be the virulence-associated protease based on 37.45% to 43.19% identical to the pathogenic yeast C. albicans with 99% query cover in BLAST analysis. But, as compared to Scheffersomyces stipitis which converged from the same taxon of C. albicans, it can be classified as non-pathogenic species [40]. Whereas, the enzymatic activity from 999_t gene which was similar to the non-pathogenic species, S. cerevisiae and K. phaffii might exert less toxicity. Proteases genes denoted as 4064_t, 4978_t, 4979_t, and 4980_t were in parallel to the protease motif of Candida tenuis and its reference genome, M. guilliermondii ATCC 6260. The quality of consensus and conservation region generated by BioEdit also exhibited the data proficiency.

Fig. 2
figure 2

Profiling of putative aspartic proteases encoding gene according to public databases to signify virulence factors as indicated pathogenic in C. albicans. a The consensus region of aspartic proteases from HMM hits coordinate represented using BioEdit and b the phylogenetic tree of M. guilliermondii strain SO from HMM protease model. Three genes of M. guilliermondii strain SO (1971_t, 1972_t and 70_t) shared connected elements with C. albicans

Prospective virulence factor in putative phospholipases encoding gene

The construction of HMM analyses was based on the curated architecture of the phospholipase domain which was annotated to the domain of lysophospholipase catalytic (family PLA2_B; PF01735.19), phosphoinositide phospholipase C (family PI-PLC-X; PF00388.20), phosphatidylinositol-specific phospholipase C, Y domain (family PI-PLC-Y; PF00387.20), phospholipase D (family PLDc; PF00614.23) and PLD-like domain (family PLDc_2; PF13091.7). Pfam database had discovered six genes of M. guilliermondii strain SO denoted to the enzyme belonging to this superfamily (Additional file 1: Table 3). The family of hydrolases consisting of lysophospholipase (phospholipase B) and cytosolic phospholipase A2 (included the domain type PLA2_B) were found in the M. guilliermondii strain SO proteome at the gene ID 1966_t and 913_t. However, based on prior study, the production of esterase enzymes carried virulence properties in M. guilliermondii [36]. Thus, the family of phospholipase B, which is not classified as a group of esterase enzyme, was withdrawn from further investigation. Nevertheless, both PLC and PLD are rationally considered to play roles in pathogenicity because the mechanism is contributed to Candida virulence factors [40]. These putative lytic genes possibly become a specific target site for downstream analysis in facilitating gene modification.

Phospholipase C

Phospholipase C (PLC) mediates in assigning signal transduction intracellularly and intercellularly by cleaving phospholipids in eukaryotic organisms. The family member of this enzyme has been characterized into thirteen different isoform and contributes to specific cellular function [41]. The gene that encodes phosphatidylinositol-specific phospholipase C (PI-PLC) in S. cerevisiae is PLC1 which is also an ortholog to the gene CAPLC1 in C. albicans delta-form isoenzyme and consists of the associated of catalytic domain X and Y [42]. Nevertheless, the phospholipase C identified in C. albicans does not contain N-terminal signal peptides which probably secrete intracellular phospholipases and are unidentical to the other classes of phospholipases [43].

PLC breaks the glycerophosphate bond at position before phosphate in glycerophospholipid molecule to produce diacylglycerol and a phosphate-containing head group. Thirteen isoenzymes are categorized into six subfamilies (β, γ, δ, ε, ζ and η) according to their structures [44]. The secretion of phosphoinositide phospholipase C (EC 3.1.4.11) constitutes an inositol signaling pathway and is involved in the invasion and penetration of host barrier cells [45]. These phosphatidylinositol-specific phospholipase C proteins contain highly conserved X and Y catalytic domains coordinated at residue 499 to 644 and 703 to 818, respectively. The order of these domains is (–NH2–X–Y–COOH–), nevertheless, the spacing may fluctuate according to species. This domain architecture is identified in 1,140 sequences in the Ensembl database (as per analyzed in April 2021) and is responsible for lipid metabolic process, signal transduction and intracellular signal transduction.

The genes denoted as 3887_t in M. guilliermondii strain SO proteome was verified by hmmscan to annotate PLC-1 enzyme. The specific hits for gene 3887_t (length size: 2.8 kbp) showed a high confident association to be inferred as PLC domain X. The MSA of this gene to the PI-PLC gene of C. albicans P37005 (Genbank: KGQ83690.1) had shown 42% identity that covered 88% at 0.0e+00 E-value. The ORF of this gene mapped to the conserved domain comprised of PI-PLC, EF-hand and C2 superfamilies region (Additional file 1: Fig. 3). The alignment results also showed high identity of the gene to the pathogenic C. albicans and contained complete structure of domain X and Y in phosphatidylinositol-specific phospholipase C.

Phmmer and hmmscan were used as homolog finder to identify the motif pattern of PLC in 3887_t gene against reference proteome database and pfam database, respectively, with default E-value cutoff, 0.01 and BLOSUM62 scoring matrix substitution. From the HMM searching algorithm, PLC that targeted on Hyphopichia burtonii, Scheffersomyces stipites and Suhomyces tanzawaensis were the most significant to M. guilliermondii strain SO according to the E-value. Notably, the coverage hits of these two domains (PI-PLC-X and PI-PLC-Y) consensus motif was also not established consistently in the conserved region, supported clearly by incongruent quality, conservation and consensus (Fig. 3). The phylogenetic tree specifying the relatedness of PLC enzyme in M. guilliermondii strain SO in comparison with S. cerevisiae had shown two lineages that split taxon from ancestral node, as well as the latter species grouped together to the other non-pathogenic yeast, K. phaffi. The constitution of PLC properties in M. guilliermondii strain SO and C. albicans might need to be considered as toxic to the host as attributed in the topography scale that corresponded to share similar bases which might impute to have virulence factors (Fig. 3).

Fig. 3
figure 3

Profiling of putative phospholipase C encoding gene according to public databases to signify virulence factors as indicated pathogenic in C. albicans. a The consensus region of phospholipase C from HMM hits coordinate represented using BioEdit and b the phylogenetic tree of M. guilliermondii strain SO from HMM phospholipase C model

Phospholipase D

Phospholipase D (PLD) hydrolyzes the phosphodiester bond of glycerolipid phosphatidylcholine, yielding phosphatidic acid and free choline. The enzymes are important in modifying membrane lipids in eukaryotes and the production of phosphatidic acid signifies the signal of gene activity [46]. Two isoforms of PLD; PLD1 and PLD2 have been reported [47]. PLD1 (EC 3.1.4.4) stimulates the morphogenic transition from yeast to hyphal growth and plays the role as a modifier of membrane structure and function [48]. These phosphatidylcholine-hydrolyzing PLD isoforms possess a homolog of phosphatidylserine synthase and cardiolipin synthase, which apparently displayed the occurrence of two motifs containing well-conserved lysine, histidine and/or asparagine residues, and this may subsequently contribute to the active site aspartic acid as in sequence feature (Additional file 1: Fig. 4).

The proteomic data of M. guilliermondii strain SO had distinguished the gene with sequence ID 5149_t annotated as PLD1 supported by the output analysis from hmmscan. The following verification was using the same platform as PLC, and recognized the molecular structure of PLD identified by the motif of catalytic sequence; HxKxxxxD (HKD), represented by the abbreviations of amino acids histidine (H), lysine (K), aspartic acid (D), while x represents non-conservative amino acids [49].

Verification on public databases using BLASTX had found the gene of 5149_t, length size 5.2 kbp nucleotide, hit specifically on PLD in C. albicans P34048 (GenBank: KGU31558.1) that covered 69% of its gene with 55% identity and zero E-value. This gene was also mapped into conserved protein domain family PLN02866, phospholipase D, that would eventually construct the tertiary structure of this protein and perform the enzyme function. According to the constructed ORF, 5149_t gene fits the specification for toxicity analysis as it scores 1590 bits to C. albicans. The catalytic PLD motif was identified as “HEKLCVID” in M. guilliermondii strain SO and this is parallel with the subject strain of C. albicans, “HEKLCIID.” (Table 3).

Table 3 HMM homologs hits on M. guilliermondii strain SO phospholipase C

The analysis of phmmer showed that the homolog of PLD genes from subject species hit perfectly to the target gene, 5149_t M. guilliermondii strain SO with E-value 0.0e+00, concluding high identity motif for this domain family (Table 4) where 550 catalytically significant residues of this domain architecture were detected to conserve at reading frame 5’ to 3’ nucleotide position 757 to 1094 in HMM execution against Ensembl database. Similar to the phylogenetic tree constructed for PLC, PLD enzyme also clustered in one clade indicating that homologous structure of these proteins was inherited with minimum evolution (Fig. 4). From the analysis, it is fair to conclude that in silico findings showed close relation of virulence factor from PLD enzyme in M. guilliermondii and C. albicans. Hence, the suppression of this gene in M. guilliermondii strain SO may alleviate or revert the pathogenic effects for further development as a host.

Table 4 HMM homologs hits on M. guilliermondii strain SO phospholipase D
Fig. 4
figure 4

Profiling of putative phospholipase D encoding gene according to public databases to signify virulence factors as indicated pathogenic in C. albicans. a The consensus region of phospholipase D from HMM hits coordinate represented using BioEdit and b the phylogenetic tree of M. guilliermondii strain SO from HMM phospholipase D model

Regardless of how the identification of virulence factors was conveyed from this analysis, there is still a requirement to evaluate M. guilliermondii strain SO through in vitro analysis. Assessment on quantifying the level of expression is recommended to certify the level of pathogenicity or toxicity corresponding to the enzymatic activity of those genes. In order to accelerate the research, one of the advancements in quantifying the transcription of those above-mentioned virulence factors is using NanoString technology which detects the target genes digitally in the samples of input material besides microarray analysis. Nevertheless, for the development of strain SO to host a heterologous expression system, the corresponding virulence factor genes are proposed to be knocked out to repress or inhibit the operational function of these enzymes through CRISPR-Cas9 advancement technology. The method is currently having a wide range of potential, yet precise and versatile in genomic editing by removing, adding or altering the virulence domain section of DNA sequence, subsequently, qualifying GRAS standard.

Conclusion

Complete information on the genome M. guilliermondii strain SO may be used optimally to complement this recently developed expression system and supports the future genetic manipulation for the recombinant protein production industry. Prior to the commercialization of this methanol-free expression host, the previously available draft genome of M. guilliermondii strain ATCC 6260 should not be solely relied. The proteomic data of M. guilliermondii strain SO were deployed to comprehend the fundamental knowledge regarding virulence factors in this species. In silico toxicology analysis in identifying the presence of potential virulence factors; phospholipases and proteases protein would specify the potential traits according to the genome annotations, subsequently predicting the adverse effects implicated by the fungal pathogenicity. Resemblance molecular features of virulence factors in the most clinically isolated C. albicans were identified in M. guilliermondii strain SO proteome. The recognition algorithm programs delivered by the hidden Markov model had shown the properties of those selected virulence determinants with low E-value to C. albicans, indicating that these factors might elicit virulence for candidiasis infection and toxicity as a yeast expression host. The finding supported by the construction of neighbor-joining (NJ) phylogenetic tree was confirmed with 1000 bootstrapping.

Materials and methods

Yeast isolation and DNA extraction

The sample of wild-type yeast cell, M. guilliermondii strain SO was obtained from a previous study [18]. The cryopreserved cells in 30% (v/v) glycerol were streaked on YPD agar (Millipore Sigma, USA), prepared from (in w/v): 1% yeast extract, 2% peptone, 2% dextrose and 2% bacteriological agar, then incubated at 30 °C for 3 days. A single colony of the yeast cell revived on the plate was cultivated in YPD medium broth containing (in w/v): 1% yeast extract, 2% peptone and 2% dextrose, and was grown overnight in an incubator shaker at 30 °C, 250 rpm. The genomic DNA extraction was carried out using Wizard Genomic DNA Purification Kit (Promega, USA). A 600 µl of 0.5 M phosphate buffer, pH 7.5 (dipotassium phosphate and monopotassium phosphate) was added to the pellet cells from 10 mL culture and mixed gently with 50 U/g of Zymolyase®-100 T enzymes (Nacalai Tesque, Japan) and 1% 14 mM β-mercaptoethanol (v/v) before incubating for 2 h at 37 °C. The next procedure followed the protocol supplied by the manufacturer. The final step is rehydration of DNA; the samples, however, were eluted with 10 mM Tris–Cl pH 8.5 without EDTA to avoid under-tagmentation by enzymatic inhibitors. Quantification using NanoDrop One Microvolume UV–Vis Spectrophotometers (Thermo Fisher Scientific, USA) determined the concentration of starting material contains 93.6 ng/µl in 24.52 µg total mass of DNA with purity A260/280 at 1.9. The specificity assessment of genomic DNA was precisely measured using Qubit 4 Fluorometer (Thermo Fisher Scientific, USA) at 93.0 ng/µl and visualized the nucleic acid fragments in agarose gel electrophoresis (Additional file 1: Fig. 5).

Whole genome sequencing (WGS) analysis

The purified genomic DNA of M. guilliermondii strain SO was sent offsite to Codon Genomics Sdn. Bhd. in Selangor, Malaysia, for second-generation sequencing using Illumina instruments. The sequencing of M. guilliermondii strain SO genome was performed approximately at 146 × coverage accumulating 20.588 million raw reads containing 3.088 Gb of data from both paired-end (short insert; 300 bp) and mate-pair (long insert; 5 Kbp) library construction. The quality of data throughput was verified using FASTQC [50] and FASTX-Toolkit [51] before downstream analysis with 91% reads longer than 140 bp retained after filtering quality (Additional file 1: Fig. 6, 7). The de novo assembly was computed by Velvet assembler [52] using 99 k-mer length (Additional file 1: Fig. 8). The scaffolding and gap-filling assemblies were carried out by SSPACE and Gapfiller, respectively, for further improvement in the genome arrangement. Structural prediction of tRNA and rRNA were identified by tRNAscan-SE v1.3.1 [53] and rRNAmmer v1.2 [54], respectively. HMM spotter model was applied to detect rRNA (8S; 18S; 28S) genes across the whole genome. While covariance model search was applied to differentiate pseudogenes from true RNA where the score of covels exceeds 20.0 bit. Homology search algorithm, BLASTX [55] annotated the gene function from public protein databases, RefSeq National Center for Biotechnology Information (NCBI) reference sequence and Swiss-Prot (SP) curated database at default cutoff E-value (database downloaded at 05th Jan 2018). A gene prediction program, GeneMark-ES [56] employed hidden state Markov model (HSMM) approaches to predict the region of noncoding and coding (CDS) genes. The domains of retrieved protein were identified from protein family (pfam) database and subsequently interconnected to Gene Ontology (GO), KEGG Orthology (KO) and Enzyme Commission (EC) knowledge bases (Additional file 1: Fig. 9). The initial affirmation of the genome set was examined using BUSCO Assessment against the catalog of 1,315 conserved single-copy orthologs from Ascomycota dataset in OrthoDB. Concomitantly, in order to synchronize the annotation, GhostKOALA was generated to infer its molecular functional cluster according to KEGG ORTHOLOGY (KO), collaterally, reconstruct KEGG maps, BRITE hierarchies and KEGG modules. KofamKOALA was assigned to annotate based on hmmsearch and provided the identification of strain SO genes. The completeness of genome assembly was quantified using Benchmarking Universal Single-Copy Orthologs (BUSCO) software [57]. Assessment of genome assembly with a technical measure of N50 only inspected the quality of the assembly; however, did not assess assembly completeness in terms of gene content. On the other hand, BUSCO provides measures for quantitative assessment of genome assembly completeness based on evolutionarily informed expectations of gene content from near-universal single-copy orthologs selected from OrthoDB. The assembled draft genome of strain SO was subjected to completeness assessment in BUSCO using the Ascomycota dataset which comprised 1,315 conserved single-copy orthologs. Furthermore, the full repertoire of peptide sequences (≥ 33 aa) from predicted genes in FASTA file format was verified by BUSCO to ensure the completeness of gene set.

The probabilities of candida-like virulence factors to be identified in M. guilliermondii strain SO genome dataset were analyzed according to the presence of aspartic proteases, phospholipase C and phospholipase D. The reference genome data of M. guilliermondii, strain ATCC6260 were included for interspecies comparison. P. pastoris or its binomial name K. phaffi and S. cerevisiae were used as a GRAS control model [58] while Candida albicans was used as a reference for pathogenic model for yeast [59].

Identification of virulence factors in M. guilliermondii strain SO

The proteomic profiles of M. guilliermondii strain SO were conferred initially to alternative yeast nuclear code (known as translation table 12) [60] and the regions that mapped significantly to virulence factor enzymes of aspartic proteases and phospholipases, then retrieved it in FASTA format. Subsequently, the annotated profiles were proceeded to multiple sequence alignments (MSA) using MUSCLE build-in provided by MEGAX platform to generate highly conserved polymorphic sequences. Meanwhile for the genes without consensus profiles, reverse PSI_BLAST approach was applied to BLASTX with a default cutoff value lower than 0.01 to ensure those determinant genes were accurately selected for toxicity analysis. The output with hits to the domain of selected virulence factors was pooled and interpreted the adequacy by implying the bit-score, the query coverage, E-value and percentage of identity. The alignment of M. guilliermondii strain SO genes that matched the query enzyme was visually checked according to the score colors. Furthermore, the architecture of the hit subject was verified whether the interval hits performed within specific or non-specific boundaries of superfamily domain, which defined the structural homology of the gene.

Hidden Markov model analysis

The identification of those selected genes in M. guilliermondii strain SO were analyzed in silico using hidden Markov model (HMM) approach based on the proteome database gathered from prior analysis. Profile HMMs implemented in HMMER was used to perform the interactive searches against the queries from M. guilliermondii strain SO proteome database and next, proposed the probabilistic models to predict the similar homologs on selected reference databases. In this study, hmmsearch was used to configure the similarity of sequences that matched a multiple sequence alignment of domain proteases. While phmmer and hmmscan were executed to a single query protein like phospholipase C and phospholipase D. For the identification of match features, hmmscan utilized the HMM database from pfam with default cutoff parameter. The sequence database ensembled genomes (v.44) was selected as a target to correspond to this analysis. Phylogenetic tree was constructed inferring the Neighbor-Joining (NJ) method [61] to compute the distance matrix of protease enzymes among these species. The predicted virulence factor genes in M. guilliermondii strain SO were executed using HMMER and phmmer algorithms to configure the pathogenic domain in other species. Henceforth, pfam database was used as a reference to annotate those domains and subsequently visualized the output analysis in BioEdit and MegaX.

Availability of data and materials

A whole genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession VMSI00000000 and Sequence Read Archive (SRA) under BioProject accession number PRJNA547962. The version described in this paper is the second version, VMSI01000000 (https://www.ncbi.nlm.nih.gov/nuccore/VMSI00000000).

Abbreviations

AOX:

Alcohol oxidase

CDS:

Coding region sequences

EC:

Enzyme commission

E-value:

Expected value

FLD:

Formaldehyde dehydrogenase

GC content:

Guanine–cytosine content

GO:

Gene ontology

GRAS:

Generally recognized as safe

HMM:

Hidden Markov model

KEGG:

Kyoto encyclopedia of genes and genomes

Kbp:

Kilobase pair

Mbp:

Megabase pair

mRNA:

Messenger RNA

MSA:

Multiple sequence alignment

nm:

Nanometer

pfam:

Protein families

PLC:

Phospholipase C

PLD:

Phospholipase D

rRNA:

Ribosomal ribonucleic acid

SAP:

Aspartic proteases

tRNA:

Transfer ribonucleic acid

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Acknowledgements

This study was supported by Graduate Research Fellowship (GS46691) and Geran Inisiatif Putra Siswazah from Universiti Putra Malaysia (GP- IPS/2016/9513300).

Funding

This work, including the efforts of Siti Nurbaya Oslan, was funded by Geran Inisiatif Putra Siswazah from Universiti Putra Malaysia (GP- IPS/2016/9513300).

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The authors confirm their contribution to the paper as follows: RAZ, SS, ABS, AA, RFRK and SNO contributed to conception or design of the work; RAZ collected the data; RAZ and RFRK analyzed and interpreted the data; RAZ, RFRK and SNO drafted the article; RAZ, SS, ABS, RFRK and SNO critically revised the article; RAZ, SS, ABS, RFRK and SNO provided final approval of the version to be published. All authors read and approved the final manuscript.

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Correspondence to Siti Nurbaya Oslan.

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Zainudin, R.A., Sabri, S., Salleh, A.B. et al. In silico identification of prospective virulence factors associated with candidiasis in Meyerozyma guilliermondii strain SO from genome dataset. Egypt J Med Hum Genet 24, 6 (2023). https://doi.org/10.1186/s43042-023-00384-3

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Keywords

  • Virulence factors
  • Whole genome sequencing
  • Yeast expression system
  • Meyerozyma guilliermondii