ORIGINAL RESEARCH published: 06 February 2018 doi: 10.3389/fmicb.2018.00086 Polymorphism in Mitochondrial Group I Introns among Cryptococcus neoformans and Cryptococcus gattii Genotypes and Its Association with Drug Susceptibility Felipe E. E. S. Gomes 1,2, Thales D. Arantes 1,2, José A. L. Fernandes 2, Leonardo C. Ferreira 1,2, Héctor Romero 3, Sandra M. G. Bosco 4, Maria T. B. Oliveira 5, Gilda M. B. Del Negro 6 and Raquel C. Theodoro 2* 1 Department of Biochemistry, Universidade Federal do Rio Grande do Norte, Natal, Brazil, 2 Institute of Tropical Medicine of Edited by: Rio Grande do Norte, Universidade Federal do Rio Grande do Norte, Natal, Brazil, 3 Laboratorio de Organizacion y Evolución Hector Mora Montes, del Genoma/Unidad de Genómica Evolutiva, Departamento de Ecología y Evolución, Facultad de Ciencias/CURE, Universidad de Guanajuato, Mexico Universidad de la República, Maldonado, Uruguay, 4 Department of Microbiology and Immunology, Institute of Biosciences, Reviewed by: Universidade Estadual Paulista Julio de Mesquita Filho, São Paulo, Brazil, 5 Department of Microbiology and Parasitology, Marilene Henning Vainstein, Universidade Federal do Rio Grande do Norte, Natal, Brazil, 6 Institute of Tropical Medicine of São Paulo, Universidade de Federal University of Rio Grande do São Paulo, São Paulo, Brazil Sul (UFRGS), Brazil Elizabeth R. Ballou, University of Birmingham, Cryptococcosis, one of the most important systemic mycosis in the world, is caused by United Kingdom different genotypes of Cryptococcus neoformans and Cryptococcus gattii, which differ in *Correspondence: their ecology, epidemiology, and antifungal susceptibility. Therefore, the search for new Raquel C. Theodoro [email protected] molecular markers for genotyping, pathogenicity and drug susceptibility is necessary. Group I introns fulfill the requisites for such task because (i) they are polymorphic Specialty section: sequences; (ii) their self-splicing is inhibited by some drugs; and (iii) their correct splicing This article was submitted to under parasitic conditions is indispensable for pathogen survival. Here, we investigated Fungi and Their Interactions, a section of the journal the presence of group I introns in the mitochondrial LSU rRNA gene in 77 Cryptococcus Frontiers in Microbiology isolates and its possible relation to drug susceptibility. Sequencing revealed two new Received: 23 October 2017 introns in the LSU rRNA gene. All the introns showed high sequence similarity to Accepted: 12 January 2018 Published: 06 February 2018 other mitochondrial introns from distinct fungi, supporting the hypothesis of an ancient Citation: non-allelic invasion. Intron presence was statistically associated with those genotypes Gomes FEES, Arantes TD, reported to be less pathogenic (p < 0.001). Further virulence assays are needed to Fernandes JAL, Ferreira LC, confirm this finding. In addition, in vitro antifungal tests indicated that the presence of LSU Romero H, Bosco SMG, Oliveira MTB, Del Negro GMB and Theodoro RC rRNA introns may influence the minimum inhibitory concentration (MIC) of amphotericin B (2018) Polymorphism in Mitochondrial and 5-fluorocytosine. These findings point to group I introns in the mitochondrial genome Group I Introns among Cryptococcus neoformans and Cryptococcus gattii of Cryptococcus as potential molecular markers for antifungal resistance, as well as Genotypes and Its Association with therapeutic targets. Drug Susceptibility. Front. Microbiol. 9:86. Keywords: group I introns, mtDNA, LSU, Cryptococcus genotypes, antifungal susceptibility, 5-fluorocytosine, doi: 10.3389/fmicb.2018.00086 homing endonuclease Frontiers in Microbiology | www.frontiersin.org 1 February 2018 | Volume 9 | Article 86 Gomes et al. Mitochondrial Group I Introns in Cryptococcus spp. INTRODUCTION involving sequencing (Meyer et al., 2009), multiplex PCR (Leal et al., 2008), AFLP (Boekhout et al., 2001) and RFLP (Meyer Cryptococcosis is a systemic mycosis that affects humans and et al., 2003). Therefore, new polymorphic DNA markers, with animals. Humans are often infected by inhalation of infective the potential for a practical and reproducible indication of propagules, which first colonize the lungs and subsequently drug resistance/susceptibility among Cryptococcus genotypes, invade the central nervous system (Chen et al., 2014). Currently, are important for clinical and epidemiological studies. Thus, two species are recognized as etiological agents of this disease: we evaluated the group I introns in the mitochondrial large Cryptococcus neoformans and Cryptococcus gattii (Kwon-chung subunit rRNA gene (LSU) as a possible candidate for this et al., 2014). However, a recent study using multi-loci analysis purpose. suggested the division of C. neoformans/C. gattii into seven Group I introns are structural sequences capable of catalyzing different species (Hagen et al., 2015). Nevertheless, for clinical their own splicing from precursor RNA (Lambowitz and Caprara, practicality, the names C. neoformans and C. gattii species 1999; Haugen et al., 2005). Some of them are mobile elements due complexes are used. to the presence of homing endonucleases, which trigger a DSB Various molecular techniques have already been applied to (Double Strand Break), usually in an allelic site, that is followed the epidemiological study of cryptococcosis (Brandt et al., 1995; by homologous recombination repair (Belfort and Roberts, 1997; Yamamoto et al., 1995; Boekhout et al., 2001; Meyer et al., 2003, Stoddard, 2011). These introns are also important drug targets 2009; Leal et al., 2008), resulting in the recognition of eight because once the splicing is inhibited, the precursor RNA remains genotypes: VNI (serotype A), VNII (serotype A), VNIII (hybrid non-functional. In Candida albicans, for example, the nuclear AD), VNIV (serotype D) for C. neoformans, and VGI, VGII, group I intron in the Ca.LSU rRNA gene is related to increased VGIII, VGIV (serotypes B and C) for C. gattii. susceptibility to pentamidine (Miletti and Leibowitz, 2000; These genotypes differ in many aspects. Ecologically, Zhang et al., 2002) and bleomycin (Jayaguru and Raghunathan, C. neoformans is known to be associated with feces of pigeons 2007), since these drugs selectively inhibit group I intron (Columba livia), and other birds, whereas C. gattii is frequently self-splicing. isolated from vegetal material, such as Eucalyptus trees (Sorrell, The search for new therapeutic targets is urgent due to 2001; Gullo et al., 2013). Epidemiologically, for many years the limited availability of antifungal agents in the face of the infections caused by C. neoformans were considered the major increasing occurrence of opportunistic fungal infections and cause of morbidity and mortality in immunosuppressed patients drug resistant strains (Dismukes, 2000; Singh, 2001; Cowen (mainly those with AIDS), while C. gattii usually infects et al., 2014). In addition, the antifungals commonly used immunocompetent patients (Sorrell, 2001; Iqbal et al., 2010; in cryptococcosis treatment, such as amphotericin B, can D’Souza et al., 2011). Although all the C. gattii genotypes are able cause serious toxic side effects in patients, usually requiring to cause disease as primary pathogens, VGI and VGII were shown hospitalization (Laniado-Laborín and Cabrales-Vargas, 2009). In to affect immunocompetent individuals more frequently than this sense, group I introns may be considered a safe therapeutic VGIII and VGIV (Farrer et al., 2015). Some genotype-associated target because they are absent in the human genome (Disney clinical differences have also been observed; for example, VGI et al., 2001). infection usually affects the central nervous system, presenting The present study was designed to investigate the occurrence cryptococcomas and simultaneous lung lesions, whereas VGII and variability of mitochondrial LSU rRNA introns in C. generally causes pulmonary disease. These differences require neoformans and C. gattii, and their possible relationship to refined therapeutic recommendations specific for the causative genotype and antifungal susceptibility. Two group I introns C. gattii genotype (Chen et al., 2014). The genotypes also show (Cne.mL2449 and Cne.mL2504) in the mitochondrial LSU rRNA geographic variation; VGII and VNI are frequently found in of Cryptococcus have already been described in a C. neoformans the Americas, VGIV predominates in the southern countries of var. neoformans (VNIII genotype) isolate (Cannone et al., 2002; Africa, and VGI and VNIV occur in Europe (Matos et al., 2012; Litter et al., 2005). In this work we also propose the nomenclature Chen et al., 2014; Kwon-chung et al., 2014). Finally, antifungal and structure for two newly found LSU rRNA group I introns responses may also vary. In some studies, C. gattii disease has in C. neoformans and C. gattii. Our main findings indicate that been reported to present longer clinical courses (delayed clinical the LSU rRNA intronless genotypes are those reported to be the and mycological cure) than C. neoformans (Sorrell, 2001). most virulent ones (especially VGII, VGI, and VNI) and also that Additionally, the molecular type VGII is less susceptible to in vitro antifungal tests indicate a relationship between intron antifungal drugs (especially azoles), followed by VGI, VNI, and absence and high MIC (minimum inhibitory concentration) VNIV (Chong et al., 2010; Hagen et al., 2010; Iqbal et al., 2010; values for 5-fluorocytosine. Trilles et al., 2012). Thus, the Cryptococcus species, genotype