Molecular profiling and phylogenetic analysis of the erg11 gene of fluconazole resistant strains of candida species isolated from humans and dogs of reproductive age

Abstract

The clinical resistance of Candida species to antifungal medications, particularly fluconazole, is rising. A major mechanism responsible for this resistance is the alteration in the nucleotide sequence of the gene that codes for the ERG11 protein, a key enzyme in the ergosterol synthesis pathway, which remains the target of fluconazole. This study investigated the distribution of different species of Candida in human and dog vaginal swabs, the susceptibility of the Candida species to fluconazole, the profile of the ERG11 gene, and the phylogenetic relationship of the Candida species based on the nucleotide sequence of the ERG11 gene. A total of 57 human samples and 7 dog samples were screened for the presence of Candida species. Twenty-eight (28) of the human samples were positive (+ve) to yeast growth. A total of 37 Candida isolates were obtained from the 28 human specimens that were positive to yeast growth. Of the 37 isolates, 13 (35%) were C. albicans, 4 (9%) C. glabrata, 4 (9%) C. krusei, 2 (6%) C. tropicalis, and 14 (38%) other Candida species. Of the 28 human specimens that were positive to Candida growth, 21 had single species, 5 had two different species and 2 had three different species. Four different species of Candida, including C. albicans, C. tropicalis, C. krusei, and C. glabrata were identified. The antifungal susceptibility test revealed that 33 (89.2%) of the Candida species were susceptible (≥19mm) to 25 μg fluconazole. The most fluconazole-resistant isolate was C. glabrata, while the most fluconazole-susceptible isolate was a C. albicans. Both isolates were obtained from humans. The four (4) isolates whose ERG11 genes were sequenced, were the most resistant, C. glabrata, the susceptible dose-dependent, C. albicans, the most susceptible, C. albicans and the dog isolate, C. krusei. The nucleotide sequence lengths of the ERG11 gene of these isolates varied from 1431 bases (Can Iso-001) to 1668 bases (Can Iso-029). Similarly, the molecular weight varied from 56183.10Da (Can Iso-001) to 65139.25Da (Can Iso-029), while the isoelectric point varied from 8.88 pI (Can Iso-029) to 9.60 pI (Can Iso-001). The predicted half-life (t1/2) of these proteins in mammalian cells was 100 hours, and the instability coefficients were 35.70, 36.98, 44.50 and 37.69, for Can Iso-001, Can Iso-017, Can Iso-028 and Can Iso-029, respectively. The grand average of hydrophobicity (GRAVY) of these Candida ERG11 proteins were 0.195, 0.478, 0.195 and 0.576, respectively. The four Candida ERG11 proteins were predicted to be localized in the plasma membrane. The first 12 amino acids in the multiple sequence alignment (MSA) make-up the first major conserved domain, while the second major conserved regions are in positions 331-337, 332-338, 330-336 and 330-336, for Can Iso-001, Can Iso-017, Can Iso-028 and Can Iso-029, respectively. The total residues of alpha helix, beta pleated sheets and turns for these proteins varied. The most fluconazole-resistant isolate (Can Iso-001) had the highest percentage of α-helix in its ERG11 protein, while the most fluconazole-susceptible isolate (Can Iso-028) had the lowest percentage of α-helix in its ERG11 protein. There was a low-level similarity between the Can Iso-017 and Can Iso-028 ERG11 tertiary structural models. The Tertiary protein structure of the Can Iso-001 ERG11 was not similar to the other isolates. Can Iso-001 ERG11 protein had no single fluconazole-binding site, while Can Iso-017, Can Iso-028 and Can Iso-029 ERG11 proteins had unique binding sites to which the drug can effectively bind. Similarly, Can Iso-001 ERG11 protein possesses 10 antigenicity sites, while the Can Iso-017, Can Iso-028 and Can Iso-029 ERG11 proteins, possess 16, 21 and 15 antigenicity sites, respectively. The amino acids; 2-8: ETVIDGI was identified as a disease-causing region common to all, in addition to other disease-causing regions that were peculiar to each of the isolates. The phylogenetic analysis based on the ERG11 gene showed that, the four isolates were closely related. However, the most resistant isolate (Can Iso-001) and the dog isolate (Can Iso-029), seemed to have originated from a common ancestor, implying an even closer evolutionary relatedness.