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0INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" _25-26 SEPTEMBER, 2024_
FUTURE PERSPECTIVE OF METABOLOMIC STUDY IN DERMATOPHYTE CULTURES
1Saumya Chaturvedi, 2Khushboo Arya, 3Sana Akhtar Usmani, 4Shikha Chandra, 5Deeksha
Jattan, 6Nitin Bhardwaj, 7Ashutosh Singh
1Research Scholar- Department of Biochemistry, University of Lucknow, Lucknow, Uttar Pradesh, India 226024, 2Research Scholar- Department of Biochemistry, University of Lucknow,
Lucknow, Uttar Pradesh, India 226024, 3Research Scholar- Department of Biochemistry, University of Lucknow, Lucknow, Uttar Pradesh, India 226024, ^Research Scholar- Department of Biochemistry, University of Lucknow, Lucknow, Uttar Pradesh, India 226024, 5Research Scholar- Department of Biochemistry, University of Lucknow, Lucknow, Uttar Pradesh, India 226024, 6Department of Zoology and Environmental Science, Gurukula Kangri (Deemed University), Haridwar, 249404, Uttarakhand, India, 7Assistant Professor - Department of Biochemistry, University of Lucknow, Lucknow, Uttar Pradesh, India 226024. https://doi.org/10.5281/zenodo.13832344
Abstract: Superficial fungal infections affect one quarter of the world's total population and are the 5th biggest cause of death in humans. Dermatophytes are a group of fungi responsible for causing superficial fungal infection and many known drugs have become resistant towards different strains of dermatophyte. It has become very important to develop some newer treatment strategies to eradicate this infection. Metabolic profiling of strains of dermatophytes can be one such strategy that can help in solving this problem. In this article, we have discussed the importance of metabolomics studies and a strategy to obtain metabolic profiles of dermatophytes. Further, we emphasize that the obtained data can be used to treat, explore pathogenic pathways and identify novel drug targets to treat contagious infection caused by dermatophytes.
Keywords: Dematophyte, Trichophyton, Metabolomics, Mass spectrometry
Introduction
Dermatophytes are a group of human pathogenic fungi responsible for causing Athlete's Foot (Tinea Pedis), Toenail Fungal Infections (Tinea Unguium or Onychomycosis), Crotch Itch (Tinea Cruris), Ringworm (Tinea Corporis). It includes genera like Trichophyton. It can infect skin, nail, groin, scalp, beard area. Immunocompromised individuals are at higher risk of developing infection due to Trichophyton. Alkaline pH, sweating, humidity, high temperature promotes the infection [5]. It shows great contagious potential, spreading through contact with the infected individual. Key factor of its pathogenicity is the keratinolytic activity which proceeds by the colonization of dermatophyte in the keratinized tissues where it hydrolyzes the disulfide bonds of keratin by the process of sulfitolysis and further degrading keratin into smaller peptide and amino acid (using exopeptidases and endopeptidase) fulfilling its metabolic need.
According to report published by GAFFI, fungal infection is the 5th biggest cause of deaths annually and around 50% of HIV patients have died because of fungal infection. It is estimated that globally 20% to 25% of people are affected by superficial fungal infection of skin, hair and nails making it fourth most common cause of human diseases [7]. 10% of population world-wide is affected with onychomycosis alone, caused by Trichophyton rubrum. According to data published in 2022 it has been found that 10% students of urban primary school in Kolkata have
INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" _25-26 SEPTEMBER, 2024_
prevalence of tinea capitis, representing 90% of total tinea capitis cases [7]. The highest prevalence rate of 75.9% to 77.5% for Trichophyton species is shown by T. mentagrophytes followed by T. rubrum [9].
Metabolites are product of metabolic pathways, and the scientific study of chemical processes involving these metabolites is known as 'metabolomics'. Metabolomics is a very sensitive technique because every minute changes can result in concentration change or absence or presence of metabolites. Metabolomics can help in identifying metabolites being produced by the organism in different physiological conditions. Metabolomic profiling of Trichophyton species can help us in understanding host-pathogen interaction, metabolites being produced during the infection can be identified and pathways involved in the synthesis of pathogenic compounds can be studied, promoting better understanding of infection mechanism.
Further, metabolomic profiling can help in identification of disease specific biomarkers, which can help in early diagnosis and treatment of fungal infection. Additionally, it can help in identification of new drug target for the antibiotic-resistant species such as T. indotineae and can help in planning newer treatment strategies for such strains.
For example, lipid metabolites play a key role in the life processes of fungi playing such as spore germination, hyphae formation, virulence, cell division and apoptosis. Proper regulation of lipid homeostasis is therefore very important for the survival of pathogenic fungi like Trichophyton. Moreover, inhibiting lipid synthesis results in significant killing of T. tonsurans and T. interdigitale [1]. In order to understand the mechanism of action of such inhibitions in detail, and to identify novel converging pathways that could be used to target these pathogenic fungi, a detailed metabolomics study is much demanded. Metabolic profiling of different species of Trichophyton such as T. rubrum, T. tonsurans, T. interdigitale will allow us to map different metabolites present in these strains and identify major and unique structures present in different strains. Metabolomics of transition of spore to hyphae form will allow us to identify any differences that occur during the establishment of fungal infection, within the fungi itself. Metabolic profiling of drug treatment conditions should help us identify the metabolite changes that result in overall reduced fungal growth. This should be helpful in deciphering the mechanism of action of drugs inhibiting Trichophyton growth.
Methodology
Metabolite extraction: Cells are disrupted mechanically by adding cold methanol-water in pellet followed by bead beater or sonication, and extracted with solvents such as methanol, chloroform, water etc it will also help in disrupting the cell. Choice of extraction sample depends on various parameters. After phase separation, solvent is dried and metabolites are later reconstituted in suitable solvent system [6,8]. Samples may be derivatized post-extraction using reagents such as methoxyaminehydrochloride in pyridine (for oximation of carbonyl group) or N-methyl-N-(trimethylsilyl)trifluroacetamide (MSTFA) it silylate hydroxyl and amine group [3].
Mass spectrometry: While gas chromatography (GC) is more suitable for volatile metabolites, liquid chromatography (LC) is preferred for non-volatile metabolites. Often, a few classes of metabolites can be separated on GC after derivatization steps. Post-separation, the identity and amount of the metabolite can be determined using a mass spectrometer (MS) as a detector. MS will identify metabolites on the basis of their mass-charge ratio and by comparing
INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" _25-26 SEPTEMBER, 2024_
obtained mass spectra against NIST and Fiehn libraries or available external standards. Retention index is also used for identification. Both internal and external standard methods may be used for the quantification of the metabolite signal [3].
Statistical methods: MS often requires preprocessing of obtained data which includes noise removal, deconvolution etc. Retention time needs to be aligned across the sample according to the obtained peak of the chromatogram, followed by normalization which corrects the variation. Correlation can be calculated using Pearson's rank. Since area of the peak is proportional to the concentration of metabolite, hence peak integration is used for quantification of metabolite concentration. After preprocessing further statistical analysis like t-test, ANOVA, PCA, DA, etc. may be performed. Data
Results and Discussion
Metabolomics of dermatophytes can give us significant insights into the pathogenesis mechanism and can help us in planning newer treatment strategies. In 2021, Ciesielska et al. analysed metabolome of Trichophyton and Microsporum using LC-MS. Here, they could identify 62 compounds in T. rubrum and 58 compounds inM. canis. Specific changes in growth conditions can result in the production of different metabolites. For example, in case of T. rubrum, L-alanine, kynurenic acid and cysteine, and in the case of M. canis, cysteine and riboflavin are produced. Metabolites play different roles, and metabolomics provides different information about their contribution in fungal pathogenesis mechanisms. For example, the production of higher concentration of kynurenic acid indicates that up to some level immune cells of the host is being suppressed. This is probably because kynurenic acid inhibits Th17 lymphocyte differentiation which is responsible for the release of IL-17 as an antifungal response. Higher concentration of L-alanine also indicates some role in pathogenesis which requires further studies. Cysteine which would have been produced after keratin degradation also plays important role in pathogenesis, as it gets oxidised into cysteine sulphuric acid by cysteine dioxygenase and act as a precursor for taurine or pyruvate and sulphate which again causes keratin degradation. Increased concentration of riboflavin is also very important for virulence as deletion of gene riboB, responsible for its synthesis have shown loss of virulence in some fungal species [2]. However, other than this single, the dermatophyte metabolomics field is largely unexplored.
Conclusion
Metabolomics of dermatophytes should provide some insights about the pathogenesis and homeostasis, biomarkers for strain/species identification making the diagnosis easy. Specific species could be identified at early stage before the infection gets aggressive. Profiling of metabolite will help in exploring the factors that are responsible for different level of virulence in different species of dermatophytes; we can identify the factors responsible for drug resistance and will help in exploring the pathways involved in spore to hyphae transition. A dedicated metabolomics of dermatophytes can help in developing new treatment strategies and can help in identification of pathways that can be targeted.
Acknowledgements: AS thanks support from ICMR (No.52/08/2019-B10/BMS), DST-PURSE program (SR/PURSE Phase 2/29(C)), UP Higher Education (No. 10/2021/281/-4-Sattar-2021-04(2)/2021 and No. 39/2024/242/Sattar-4-2024-001-4(33)/2023), DBT
INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS"
_25-26 SEPTEMBER, 2024_
(BT/PR38505/MED/29/1513/2020) and DST (CRG/2022/001047) and the University of
Lucknow.
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