Metadata analysis of the squalene epoxidase gene in dermatophytes.

Background: Squalene epoxidase gene is an azole antifungal target enzyme in the ergosterol biosynthesis pathway in fungi—the inhibition of enzyme aggregate squalene, a type of ergosterol derivative depletion that leads to fungal growth inhibition. Squalene epoxidase widely diverges in three fungal groups of dermatophytes Trichophyton, Microsporum and Epidermophyton. Methodology: The study has demonstrated a variety of squalene epoxidase genes in the dermatophyte genome. Squalene epoxidase gene was also studied for possible single nucleotide polymorphism (SNPs) in the Trichophyton group's homologs and found multiple SNP positions that induce non-synonymous mutation and change the sequence of amino acid into protein structure that can change phenylalanine to leucine. Results: Squalene epoxidase is widely present in dermatophytes. Mutation in the squalene epoxidase gene can mislead normal sterol formation in the fungal cell membrane that induces fungal resistance against several drugs, including azoles and polyenes. Squalene epoxidase gene carries 2 - 3 transcripts and 2 - 3 number of exons. Squalene epoxidase gene has FAD-dependent oxidoreductases and NADP binding domain conserved domains in fungal groups of dermatophytes. Conclusion: This study analyzed the abundance of the squalene epoxidase gene, suggesting that squalene epoxidase gene resistant mutants can occur naturally. Thus squalene epoxidase gene should be extensively studied in order to increase the potential of available antifungals.


Introduction
Dermatophytes are a very specialized group of keratinophilic fungi responsible for superficial mycoses of humans and animals 1 . Dermatophyte infections are a worldwide major health concern, specifically in an immunocompromised individual. They may pose severe risks. Dermatophytes are divided into three genera: Microsporum, Trichophyton and Epidermophyton, although the precise taxonomic associations of this fungal group are far from being fully determined. Regardless of the common occurrence of dermatophyte infections, little is known about these pathogens' molecular biology. Dermatophytes are slowgrowing as compare to other more significant fungal pathogens. They are not very well studied for their metabolic reactions and stress adaptive mechanisms. However, several natural mutants are investigated in clinical and agricultural practices 2 .
Squalene epoxidase is a monooxygenase that catalyzes the conversion of squalene to 2, 3oxidosqualene. It is a microsomal membraneassociated enzyme that functions as a significant regulator in the biosynthetic pathway of sterols. Ergosterol helps in the photo-oxidation of fungi. This enzyme needs oxygen, Nicotine amide, Adenine dinucleotide, Phosphate hydrogen and Flavin adenine dinucleotide for stimulation 3 . Previously, terbinafine resistant isolates have been reported in increasing number. Terbinafine inhibits the Squalene epoxidase (SE) enzyme and interferes with the ergosterol biosynthesis, an integral cell membrane constituent. Mutations have been identified within the squalene epoxidase gene like clinical terbinafine resistant isolates 4 .
Squalene epoxidase gene is involved in ergosterol biosynthesis, which converts squalene to lanosterol in sterol biosynthesis. This enzyme functions as a major regulator in the biosynthetic pathway to sterols 5 . Mutation in this gene causes resistance against fluconazole, ketoconazole, itraconazole, voriconazole and terbinafine drugs because of the modification of the target enzyme by mutation induces increased drug efflux, stress adaptation and overexpression of drug efflux, which degrade the drug effects 6 .
Squalene epoxidase is an enzyme involved in the early steps of ergosterol biosynthesis, is inhibited by allylamines such as terbinafine. This inhibition contributes to squalene accumulation that is toxic for fungi. Resistance is due to single point mutations in the gene encoding squalene epoxidase. Both mutations introduced missense replacements of amino acids (Leu393Phe in one case and Phe397Leu in the other), leading to 100 fold higher MIC 7 . Terbinafine resistant mutants of Trichophyton rubrum, Aspergillus nidulans and Aspergillus fumigatus strains were identified. This mutant carries a mutation in the squalene epoxidase gene that contributes to the replacement of leucine by phenylalanine in the mutant of Erg1 8 . ERG1 (squalene epoxidase) protein comprised of two flavin adenine dinucleotide domains (FAD) and one nucleotide-binding domain (NB). ERG1 required oxygen from energy molecules, NADPH and FAD. ERG1 protein alignment with other squalene epoxidase showed two strongly conserved FAD-binding domains, FAD1 and FAD 2 10 . Spontaneous PCR mutagenesis of the ERG1 gene identified one ERG1 allele that brings a mutation. It contributes to a single exchange of amino acids in the FAD1 domain near the ERG1 Nterminus 11 . Squalene epoxidase and related proteins were explored within acquired amino acid sequences of a FAD-binding domain. In the next step, FADbinding proteins were searched against a transmembrane domain NADB Squalene epoxidase contains a putative FAD-binding site and is a crucial enzyme in the sterol biosynthetic pathway. Putative transmembrane regions are found in the proteins C-terminus. For the detection of some unpredicted genes, we explored raw genomic sequences using NCBI nucleotide blast. Sequences of all known Squalene epoxidase genes were disguised from genomic sequences. All amino acid sequences of previously identified Squalene epoxidase genes were used as queries in nucleotide sequence blast.

Squalene epoxidase protein prediction
The number of genes contained FAD and NABD binding domain, which was similar in all species of dermatophytes. These domains have an alphabeta-alpha configuration. NAD binding involves numerous hydrogen and van der Waals contacts.

Transcript Exon and intron analysis
Several exons and introns of different species of dermatophytes were obtained from NCBI. A single gene can produce multiple different RNAs (transcripts). Similarly, the numbers of transcripts were also obtained from the CBI database (https://www.ncbi.nlm.nih.gov/).

Squalene epoxidase gene Phylogenetic Analysis
A sequence alignment was done with ClustalX. Squalene epoxidase genes were determined by a conserved domain database. Phylogenetic analysis was made in MEGA6. Phylogenetic analysis was done using maximum possibility and neighbourhood joining method.

Single nucleotide polymorphism
Single nucleotide polymorphism (SNPs) analysis was found in the Squalene epoxidase gene of the Trichophyton group of dermatophytes. SNPs were obtained from NCBI and compared with the reported.

Bioinformatics survey of Squalene epoxidase gene
The squalene epoxidase gene was analyzed using different bioinformatics tools. The comparison was made based on percentage similarity, exon count, conserved domains and query cover. Sequences were aligned, and phylogenetic trees were constructed. The squalene epoxidase gene in dermatophytes has an average sequence length of 489 amino acids.
Squalene epoxidase protein prediction NAD binding involves numerous hydrogen-bonds and van der Waals contacts, particularly H-bonding of residues in turn between the first strand and the subsequent helix of the Rossmann-fold topology. Characteristically, this turn exhibits a consensus binding pattern. The first 2 glycines participate in NAD(P)-binding, and the third facilitate close packing of the helix to the beta-strand (https://go.usa.gov/xARNY).

Prediction of conserved domains of Squalene epoxidase
In this study, we used genomic sequences of all available dermatophyte species. The first step is the identification of all Squalene epoxidase genes within their genomes. Squalene epoxidase consists of two types of domain. FAD and NADB. The identified genes were containing FAD and NABD binding domain. The E value of 1e-135 was used. Query cover ranges from 95% to 99%. The length of the amino acid sequence was 489 bp. Percentage similarity was very low such as 45.34% for Trichophyton rubrum. Trichophyton rubrum has the highest percentage similarity to other Trichophyton species, as shown in table 1.

Single nucleotide polymorphisms (SNPs) in Squalene epoxidase gene
Single nucleotide polymorphisms (SNPs) have been identified in the squalene epoxidase gene of the Trichophyton group. Resistance in the gene encoding squalene epoxidase is attributable to point mutations.
The mutation was observed in Trichophyton interdigital and Trichophyton rubrum isolates. Trichophyton interdigitale nucleotide substitution in the Squalene epoxidase gene was CTC, which was substituted into TTC at 1184. Similarly, another substitution was found at position 1185, leading to the replacement of TTC by CTC.
For Trichophyton rubrum, a substitution occurred at position 1380, leading to the replacement of CAT by TAT.
Another substitution was at position 1175, leading to the replacement of TTC by TTA in the squalene epoxidase gene, as shown in table 5. Single-nucleotide polymorphism (SNP) is a substitution of a single nucleotide at a specific genome position. Single nucleotide polymorphisms occur within coding sequences of genes, non-coding regions of genes, or in the intergenic regions between genes. SNPs in the coding regions are of two kinds synonymous and non-synonymous SNPs. Synonymous SNPs do not affect protein or DNA sequence, while nonsynonymous SNPs change the amino acid sequence of protein or DNA 18 . SNPs have been identified in squalene epoxidase homologs of Trichophyton group 19 . Nucleotide substitution within the squalene epoxidase gene was observed in Trichophyton interdigitale all isolates that substitutes TTC with CTC at 1185 position 20 . In Trichophyton rubrum, nucleotide substitution occurred at 1380, leading to the replacement of CAT with TAT 8 . Another substitution occurred at 1175 position leading to the replacement of TTC by TTA that was near related to previous study 21 in which the nucleotide and amino acid substitution in the squalene epoxidase gene varies between homologs of the Trichophyton group.

Conclusion
Squalene epoxidase is an antifungal targeted key enzyme in the ergosterol biosynthesis pathway of several fungal groups. This inhibition of the gene contributes to squalene aggregation, ergosterol depletion and the inhibition of fungal growth. Studies have demonstrated a variety of squalene epoxidase genes in a major group of dermatophytes. Squalene epoxidase has widely diverged in three fungal groups of dermatophytes Trichophyton, Epidermophyton and Microsporum. Mutation in the squalene epoxidase gene can mislead normal sterol formation in the fungal cell membrane that induces fungal resistance against several drugs, including azoles and polyenes. Squalene epoxidase gene carries 2 -3 transcripts and 2 -3 number of exons in dermatophytes. Squalene epoxidase gene has FAD-dependent oxidoreductases and NADP binding conserved domains similar in three major fungal groups of dermatophytes. Squalene epoxidase gene was also studied for possible single nucleotide polymorphism (SNPs) in the Trichophyton group's homologs and found multiple SNP positions on 1184, 1185 1380, 1175 and 1368 nucleotide positions of TTC→CTC. This position was the cause of nonsynonymous mutation, which can change the sequence of amino acid into a protein structure that can change phenylalanine to leucine.

Conflicts of Interest
None.