In silico identification of Meyerozyma guilliermondii strain so potential virulence factors and pathogenicity verification of generated mutants in zebrafish model

Candidiasis, a fungal infection caused by Candida species, has been a global health concern that caused 40-60% mortality in the bloodstream infected patients. The virulence factors (VFs) in the most isolated C. albicans and other prominent Candida species, have been widely investigated. However, the reduction of antifungal drug susceptibility in non-prominent Candida spp., especially C. guilliermondii, is concerning. The roles of VFs in fungal pathogenicity in relevance to its structural data have been undermined for drug developments. The proper quantification on the fungal VFs was also lacking in the literature. A locally isolated Meyerozyma guilliermondii from spoiled orange (strain SO) that has been proven as a promising expression host for industrially important enzymes, exhibited ≥99% proteome similarity to other natural and clinical isolates but its VFs responsible for its pathogenicity towards zebrafish embryos remained unknown. Therefore, this research was aimed to comprehensively characterize and verify the VFs in M. guilliermondii strain SO using structure-guided mutants. A 19 out of 36 hits from 7 families [secreted aspartyl proteinase (Sap), agglutinin-like sequence, enolase, lipase, phytase, phospholipase, and heat-shock protein] detected using Hidden- Markov Model, exhibited the same conserved domain and stronger phylogenetic relationships with C. albicans SC5314. Next, the predicted and validated three-dimensional structures and their sequences were analyzed to verify the most potent VFs for fungal pathogenicity. The cell wall or cell membrane associated MgSap341 and extracellular MgSap1972 were targeted for deletion due to the predicted catalytic site enlargement, broader substrate specificity, and druggable active site clefts with the largest molecular surface area. Four general or VF-specific virulence assays were established to determine the pathogenicity of M. guilliermondii strain SO. The fungus demonstrated higher endoplasmic reticulum (ER), cell wall integrity but lower osmotic tolerances than Saccharomyces cerevisiae BY4742. Besides killing 75% more zebrafish embryos in vivo, it also produced 11× higher proteinase activity and 7.5× higher biofilm mass than S. cerevisiae. To determine the pathogenic roles of MgSap341 and MgSap1972, three mutants (ΔSAP341, ΔSAP1972 and ΔSAP1972 ΔSAP341) were constructed via homologous recombination strategy and they showed higher sensitivity towards osmotic, cell wall perturbing, and ER stresses than the wild type. MgSap1972 and MgSap341 contributed most to the reduction in biofilm mass (34.1%) and specific proteolytic activity (61.8%), respectively. All mutants showed virulence reduction (mortality and invasion rates) in the zebrafish embryos model: double mutant>single mutants>wild type. Thus, MgSap341 and MgSap1972 that were analyzed and targeted through computational approaches, indeed contributed to the fungal pathogenicity of M. guilliermondii strain SO. More potent VFs in M. guilliermondii were detected than the literature, signifying the proper and high coverage documentation strategy before the comprehensive, VF-specific in silico analyses were performed to examine the structurefunction relationship, subsequently bridging its potential pathogenic roles and antifungal drug development. The quantitative virulence assessments established allowed the comparison of fungal pathogenicity level at inter- and intra-species strata in diagnostic and pathology laboratories. Lastly, the establishment of FLP-SAT1 strategy allows other VFs’ pathogenic roles mining, providing insights into VF-specific antifungal drug screening and fungal pathogenic mechanisms.

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