Characterization of selected azole resistance-related genes by genome wide analysis and investigation of virulence in clinical Candida albicans isolates

• Main Author: Moron, Llewelyn S.
• Format: text
• Language: English
• Published: Animo Repository 2020
• Subjects: Candida albicans; Greater wax moth; Biology
• Online Access: https://animorepository.dlsu.edu.ph/etd_doctoral/1399; https://animorepository.dlsu.edu.ph/cgi/viewcontent.cgi?article=2424&context=etd_doctoral
• Institution: De La Salle University

Candida albicans is a diploid yeast and reported to be one of the most important human fungal pathogens, with wide range of infections from superficial symptoms to life-threatening disseminated candidiasis. This study aimed to investigate several mechanisms that might contribute to azole resistance in clinical C. albicans isolates and assess the virulence in vitro using the murine macrophage assay, and in vivo using the Galleria mellonella model. Comparative genome analyses among seven clinical C. albicans isolates and the reference strain C. albicans SC5314 revealed high number of structural variants (>76,000 single-nucleotide variations, >3,091 multi-nucleotide variations, >3,951 insertions, >5,336 deletions, and >412 replacements) and amino acid changes in the genomes (>46,302 non-exonic, >23,451 synonymous, and >16,354 nonsynonymous). Likewise, amino acid changes and novel genomic variations were detected in genes involved in the synthesis of ergosterol (amino acid changes A353T and A351I in ERG3 and amino acid changes V437I and E266D in ERG11) and transcriptional factors UPC2 (novel amino acid change N250S), TAC1 (novel amino acid changes L941P, I922L, T885K, and A430V), and MRR1 (novel amino acid changes S1037L, F1032L, N973K, G75R, P19L, and L592F). The amino acid change V437I in ERG11 and amino acid changes A353T and A351V in ERG3 are possible mechanisms of resistance to azoles. Gene expression studies of efflux pump genes (CDR1, CDR2, and MDR1) as a possible antifungal resistance mechanism were conducted. It was noted that fluconazole resistance was not due to such mechanism since no overexpression of the genes was identified (P > 0.05). In this study, we tried to construct double-knockout ERG11 mutants in clinical isolate CGP73 and SC5314 background to understand how this gene influences resistance and pathogenicity. Targeted gene deletion experiments using the SAT1 flipping strategy suggest the complexity of deleting this gene in our C. albicans due to the inability to construct homozygous mutants. We hypothesized that the accumulation of toxic sterols due to the conversion of tolerated sterols such as methylfecosterol to toxic sterol form by the ERG3 gene resulted to the inhibited growth of the mutants. Hence, no colonies were observed after second round of gene deletion transformations. Assessment of virulence in vitro using murine macrophages and in vivo in G. mellonella larvae indicated that the majority of the isolates were virulent. Optimization of infection load using G. mellonella-C. albicans infection model revealed that 2x105 yeast cells/larva was the suitable inoculum concentration in order to evaluate the pathogenicity of C. albicans. Using this fungal inoculum size, pathogenicity of the C. albicans clinical strains based on survival curves of infected waxworms displayed either higher (P < 0.0001) or equal virulence of clinical C. albicans compared to the drug susceptible strain SC5314 after six days post-infection. All clinical isolates regardless of azole-sensitivity were identified as pathogenic in vivo based on killing assays, fungal burden, and hemocyte counts. In addition, data on survival curves showed that in vitro resistance does not necessarily correlate with resistance in vivo. Studies on the efficacy of fluconazole at a concentration of 3.2 µg fluconazole/larva revealed that there was a protective effect against C. albicans infection in majority of the clinical isolates which were identified resistant in vitro. This study provides an exhaustive characterization of clinical C. albicans which provides information on the genetics of Candida and how this yeast interacts with the host. Keywords: Candida albicans, ERG11, Galleria mellonella, azole-resistance, efflux pumps