Introns do grupo I no LSU rRNA mitocondrial de Cryptococcus neoformans e Cryptococcus gattii e a sua relação com genótipos e susceptibilidade a antifúngicos

Abstract

Cryptococcosis, caused by the fungal species Cryptococcus neoformans or Cryptococcus gattii, is one of the most important systemic and/or opportunistic diseases in the world. Each species has four genotypes, usually accessed by PCR-RFLP of the URA5 gene, which present differences in their ecology, epidemiology, geographical distribution and antifungal susceptibility. Easier accessible molecular markers are attractive for rapid recognition of genotypes or relevant characteristics such as virulence and antifungal susceptibility. In this way, group I autocatalytic introns in the mitochondrial LSU rRNA were evaluated as potential molecular marker for the genotypes of C. neoformans and C. gatti, as well as their relationship to antifungal susceptibility. Seventy-seven Brazilian isolates were used, most of the genotype VNI (39 strains) followed by 20 VGII, 5 VNIV, 4 VNII, 3 VNIII, 2 VGI, 2 VGIII and 2 VGIV. The introns Cne.mL2449 and Cne.mL2504 were amplified in a single PCR with complementary primers to the flanking region of the introns LSU rRNA gene. PCR products showed a significant polymorphism between C. neoformans and C. gattii genotypes. Sequencing of the PCR products indicated that some strains had none, one, two, three or four introns followed. This new two introns, not previously described in the mitochondrial genome of Cryptococcus, were named Cne.mL2439 and Cne.mL2584 in C. neoformans and Cga.mL2439 and Cga.mL2584 in C. gattii. Cne.mL2439/Cga.mL2439 introns were classified as belonging to IB2, whereas Cne.mL2584/Cga.mL2584, as belonging IA1 subclass. Interestingly, genotypes with some intronless strains, VNI, VGII, VGI and VNIV, are those known to be more virulent and less susceptible to antifungal agents. Here, we observed that those intronless isolates had significant higher MICs values for 5-flucytosine. The findings suggest that these elements can be used as potential molecular markers for antifungal resistance. Finally, phylogenetic analyzes suggested high sequence similarity between the introns Cne.mL2449, Cne.mL2504, Cne.mL2439/Cga.mL2439 and Cne.mL2584/Cga.mL2584 with other mitochondrial introns present in the genes COX1, COX2, COX3, NAD5, ATP9, COB, LSU of fungi supporting the “introns early” hypothesis, as well as its dispersion to heterologous sites by reverse splicing.