Polymorphism in Mitochondrial Group I Introns among Cryptococcus neoformans and Cryptococcus gattii Genotypes and Its Association with Drug Susceptibility.

Felipe E E S Gomes,Thales D Arantes,Gilda M B Del Negro,Leonardo C Ferreira,Maria T B Oliveira,Sandra M G Bosco,Raquel C Theodoro,Héctor Romero,José A L Fernandes

doi:10.3389/fmicb.2018.00086

Abstract

Cryptococcosis, one of the most important systemic mycosis in the world, is caused by different genotypes of Cryptococcus neoformans and Cryptococcus gattii, which differ in their ecology, epidemiology, and antifungal susceptibility. Therefore, the search for new molecular markers for genotyping, pathogenicity and drug susceptibility is necessary. Group I introns fulfill the requisites for such task because (i) they are polymorphic sequences; (ii) their self-splicing is inhibited by some drugs; and (iii) their correct splicing under parasitic conditions is indispensable for pathogen survival. Here, we investigated the presence of group I introns in the mitochondrial LSU rRNA gene in 77 Cryptococcus isolates and its possible relation to drug susceptibility. Sequencing revealed two new introns in the LSU rRNA gene. All the introns showed high sequence similarity to other mitochondrial introns from distinct fungi, supporting the hypothesis of an ancient non-allelic invasion. Intron presence was statistically associated with those genotypes reported to be less pathogenic (p < 0.001). Further virulence assays are needed to confirm this finding. In addition, in vitro antifungal tests indicated that the presence of LSU rRNA introns may influence the minimum inhibitory concentration (MIC) of amphotericin B and 5-fluorocytosine. These findings point to group I introns in the mitochondrial genome of Cryptococcus as potential molecular markers for antifungal resistance, as well as therapeutic targets.

Highlights

Cryptococcosis is a systemic mycosis that affects humans and animals
We studied the potential of group I introns in the mitochondrial large subunit rRNA gene (LSU) rRNA of C. neoformans and C. gattii as a drug susceptibility indicator since in other fungi, their presence is related to attenuated virulence and antifungal susceptibility (Mercure et al, 1993; Miletti and Leibowitz, 2000; Jayaguru and Raghunathan, 2007)
Experimental data associating virulence profiles for Cryptococcus isolates with and without introns in their mitochondrial genome are necessary to confirm this hypothesis. Reinforcing these previous observations, we found a strong association between intronless strains and high minimum inhibitory concentration (MIC) values for the 5-fluorocytosine drug, which is corroborated by other studies in C. albicans

Summary

Introduction

Cryptococcosis is a systemic mycosis that affects humans and animals. A recent study using multi-loci analysis suggested the division of C. neoformans/C. gattii into seven different species (Hagen et al, 2015). For clinical practicality, the names C. neoformans and C. gattii species complexes are used. Various molecular techniques have already been applied to the epidemiological study of cryptococcosis (Brandt et al, 1995; Yamamoto et al, 1995; Boekhout et al, 2001; Meyer et al, 2003, 2009; Leal et al, 2008), resulting in the recognition of eight genotypes: VNI (serotype A), VNII (serotype A), VNIII (hybrid AD), VNIV (serotype D) for C. neoformans, and VGI, VGII, VGIII, VGIV (serotypes B and C) for C. gattii

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