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Whole Genome Sequencing of Candida glabrata for Detection of Markers of Antifungal Drug Resistance
Published on: December 28, 2017
A New Variant of Mutational and Polymorphic Signatures in the ERG11 Gene of Fluconazole-Resistant Candida albicans
Arome Solomon Odiba1,2, Olanrewaju Ayodeji Durojaye3,4,5, Ifeoma Maureen Ezeonu6
1Department of Biochemistry, Faculty of Biological Sciences, University of Nigeria, Nsukka, Enugu State, 410001, Nigeria.
Background:
Resistance to antifungal drugs for treating Candida infections remains a major concern globally despite the range of medications available. Most of these drugs target key proteins essential to the life cycle of the organism. An enzyme essential for fungal cell membrane integrity, lanosterol 14-α demethylase (CYP51), is encoded by the ERG11 gene in Candida species. This enzyme is the target of azole-based drugs. The organism has, however, devised molecular adaptations to evade the activity of these drugs.
Materials And Methods:
Classical methods were employed to characterize clinical isolates sampled from women and dogs of reproductive age. For fluconazole efficacy studies, CLSI guidelines on drug susceptibility testing were used. To understand the susceptibility pattern, various molecular and structural analytic approaches, including sequencing, in silico site-directed mutagenesis, and protein-ligand profiling, were applied to the ERG11 gene and CYP51 protein sequences. Several platforms, comprising Clustal Omega, Pymol plugin manager, Pymol molecular visualizer, Chimera-curated Dynameomics rotamer library, protein-ligand interaction profiler, Charmm36 force field, GROMACS, Geneious, and Mega7, were employed for this analysis.
Results:
The following Candida species distribution was obtained: 37.84% C. albicans, 8.12% C. glabrata, 10.81% C. krusei, 5.41% C. tropicalis, and 37.84% of other unidentified Candida species. Two codons in the nucleotide sequence of the wild-type (CTC and CCA) coding for LEU-370 and PRO-375, respectively, were mutated to L370S and P375H in the resistant strain. The mutation stabilized the protein at the expense of the heme moiety. We found that the susceptible isolate from dogs (Can-iso-029/dog) is closely related to the most resistant isolate from humans.
Conclusion:
Taken together, our results showed new mutations in the heme-binding pocket of caCYP51 that explain the resistance to fluconazole exhibited by the Candida isolates. So far, the L370S and P375H resistance-linked mutations have not been previously reported.
Insights
Antifungal resistance in Candida infections is a global concern. New mutations (L370S and P375H) in the CYP51 enzyme
Area of Science:
- Mycology
- Molecular Biology
- Drug Resistance
Background:
- Candida infections pose a significant global health challenge due to antifungal drug resistance.
- The lanosterol 14-α demethylase (CYP51) enzyme, encoded by the ERG11 gene, is a primary target for azole antifungal drugs.
- Candida species develop molecular adaptations to evade the efficacy of these essential antifungal medications.
Purpose of the Study:
- To investigate the molecular mechanisms underlying fluconazole resistance in clinical Candida isolates.
- To identify novel mutations in the ERG11 gene and CYP51 protein associated with antifungal drug resistance.
Main Methods:
- Characterization of clinical Candida isolates from humans and dogs using classical methods and CLSI guidelines for drug susceptibility testing.
- Application of molecular and structural analyses, including gene sequencing, in silico site-directed mutagenesis, and protein-ligand profiling of ERG11 and CYP51.
- Utilized various bioinformatics platforms such as Clustal Omega, Pymol, Chimera, Charmm36, GROMACS, Geneious, and Mega7 for sequence and structural analysis.
Main Results:
- Analysis revealed a distribution of Candida species including C. albicans (37.84%), C. glabrata (8.12%), C. krusei (10.81%), C. tropicalis (5.41%), and unidentified species (37.84%).
- Identified two novel mutations, L370S and P375H, in the ERG11 gene of resistant Candida strains, which alter the CYP51 protein structure.
- The identified mutations were found to stabilize the CYP51 protein, potentially impacting its interaction with the heme moiety and contributing to resistance. A susceptible dog isolate showed close genetic relation to a resistant human isolate.
Conclusions:
- New mutations, L370S and P375H, in the heme-binding pocket of Candida albicans CYP51 (caCYP51) confer resistance to fluconazole.
- These previously unreported mutations are critical in understanding and potentially overcoming fluconazole resistance in Candida infections.
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