Cryptococcus neoformans and Cryptococcus gattii: speciation in progress Marjan Bovers bovers_V4.indd 1 25-10-2007 10:14:00 Promotor: Prof. dr. I. M. Hoepelman Co-promotor: Dr. T. Boekhout Commissie: Dr. F. E. J. Coenjaerts Prof. dr. P. W. Crous Prof. dr. R. F. Hoekstra Prof. dr. J. A.G. van Strijp Prof. dr. H. A. B. Wösten Paranimfen: Eiko Kuramae Ruben IJpelaar Cryptococcus neoformans and Cryptococcus gattii: speciation in progress Proefschrift Universiteit Utrecht, ISBN: 978-90-393-46884 Copyright © 2007 by Marjan Bovers, All rights reserved Foto voorkant: Artist impression of Cryptococcus neoformans and Cryptococcus gattii by Eiko E. Kuramae Lay-out en drukwerk: Ponsen en Looijen Het onderzoek dat beschreven wordt in dit proefschrift is uitgevoerd op het Centraalbureau voor Schimmelcultures (Utrecht) en is financieel ondersteund door het Odo van Vloten fonds. De uitgave van dit proefschrift werd mede mogelijk gemaakt door financiële steun van de Eijkman Graduate School en de J.E. Juriaanse stichting. bovers_V4.indd 2 25-10-2007 10:14:01 Cryptococcus neoformans and Cryptococcus gattii: speciation in progress Cryptococcus neoformans en Cryptococcus gattii: voortdurende soortvorming (met een samenvatting in het Nederlands) Proefschrift ter verkrijging van de graad van doctor aan de Universiteit Utrecht op gezag van de rector magnificus, prof. dr. J. C. Stoof, ingevolge het besluit van het college voor promoties in het openbaar te verdedigen op donderdag 29 november 2007 des middags te 12.45 uur door Marjan Bovers geboren op 7 september 1979, te Eindhoven bovers_V4.indd 3 25-10-2007 10:14:01 Promotor: Prof. dr. I. M. Hoepelman Co-promotor: Dr. T. Boekhout bovers_V4.indd 4 25-10-2007 10:14:01 Contents Chapter 1 Introduction 7 Chapter 2 Six monophyletic lineages identified within Cryptococcus 29 neoformans and Cryptococcus gattii by multi-locus sequence typing Chapter 3 The mitochondrial genome of Cryptococcus gattii shows 59 evidence of recombination Chapter 4 Unique hybrids between fungal pathogens Cryptococcus 83 neoformans and Cryptococcus gattii Chapter 5 Promiscuous mating of Cryptococcus neoformans and 101 Cryptococcus gattii: discovery of a novel AB hybrid Chapter 6 Identification of genotypically diverse Cryptococcus 117 neoformans and Cryptococcus gattii isolates using Luminex xMAP technology Chapter 7 General discussion and Summary 139 Samenvatting 157 Dankwoord 167 Curriculum Vitae and List of publications 173 bovers_V4.indd 5 25-10-2007 10:14:01 bovers_V4.indd 6 25-10-2007 10:14:02 Chapter 1 Introduction bovers_V4.indd 7 25-10-2007 10:14:02 bovers_V4.indd 8 25-10-2007 10:14:02 Chapter 1: Introduction Taxonomic history 1 The science of Cryptococcus neoformans and Cryptococcus gattii started in 1894, when the Italian researcher Sanfelice first isolated Saccharomyces neoformans from peach juice (Sanfelice, 1894). In the same year Saccharomyces hominis was observed in a human infection (Busse, 1894; Buschke, 1895) and two years later an encapsulated bacilliform yeast, which was named Saccharomyces subcutaneous tumefaciens, was isolated from a young and healthy man (Curtis, 1896). Several of these cultures were re-examined by another researcher, but as the ascospore characteristics of the genus Saccharomyces were not present, these isolates were placed in the genus Cryptococcus (Vuillemin, 1901). Cryptococcus hominis was later described as a synonym to Cryptocococus neoformans (Lodder and Kreger-van Rij, 1952). In 1970, an atypical strain of C. neoformans was isolated from a leukemic patient and this strain was described as C. neoformans var. gattii (Vanbreuseghem and Takashio, 1970). The teleomorph of C. neoformans was discovered when two serotype D isolates were mated and it was named Filobasidiella neoformans (Kwon-Chung, 1975). However, when serotype B and C isolates were mated, a teleomorph that clearly differed from F. neoformans was observed. This resulted in the description of Filobasidiella bacillispora (Kwon-Chung, 1976b). In addition, several physiological assays showed that C. neoformans serotype B and C isolates differed from C. neoformans serotype A and D isolates, which resulted in the description of Cryptococcus bacillisporus for serotype B and C isolates (Kwon- Chung et al., 1978). Although previous mating experiments with C. neoformans var. gattii had been unsuccessful (Kwon-Chung et al., 1978), in 1982, viable basidiospores were produced when C. neoformans var. gattii was mated with C. bacillisporus and C. neoformans (Schmeding et al., 1981; Kwon-Chung et al., 1982a). Furthermore, mating of C. bacillisporus with C. neoformans resulted in the formation of viable basidiospores (Schmeding et al., 1981; Kwon-Chung et al., 1982a). Kwon-Chung et al. (1982a) therefore proposed to treat the two taxa as varieties of C. neoformans, namely C. neoformans var. neoformans and C. neoformans var. gattii. In 1999, based on phenotypic and genotypic data, Franzot et al. (1999) proposed to install a third variety, namely C. neoformans var. grubii, corresponding to serotype A. Despite the results of earlier mating experiments, C. gattii has recently been described as a separate species, because of the increasing amount of data suggesting the distinctiveness of C. neoformans and C. neoformans var. gattii (Kwon-Chung et al., 2002). Currently, two species are recognized within the C. neoformans – C. gattii species complex. These species are the anamorphic yeast C. neoformans (Sanfelice) (Vuillemin, 1901) with its teleomorph Filobasidiella neoformans (Kwon-Chung) (Kwon-Chung, 1975) and the anamorphic yeast C. gattii (Vanbreuseghem and Takashio) (Kwon- Chung et al., 2002) with its teleomorph Filobasidiella bacillispora (Kwon-Chung, 1976b). 9 bovers_V4.indd 9 25-10-2007 10:14:03 Chapter 1: Introduction Within C. neoformans two varieties have been described, namely var. grubii and var. neoformans (Franzot et al., 1999). Both C. neoformans and C. gattii belong to the Filobasidiella clade of the Tremellales (Basidiomycota, Hymenomycetes) (Scorzetti et al., 2002). Species that are closely related to C. neoformans and C. gattii are Cryptococcus amylolentus, Tsuchiyaea wingfieldii and Filobasidiella depauperata (Fell et al., 1992; Mitchell et al., 1992, Guého et al., 1993; Kwon-Chung et al., 1995; Fell et al., 2000; Scorzetti et al., 2002). Genotypes and serotypes Cryptococcus neoformans and C. gattii can be distinguished by serotype (A, D and AD for the former, B and C for the latter) (Kwon-Chung et al., 1982a; Kwon-Chung et al., 2002), karyotype (Wickes et al., 1994; Boekhout et al., 1997), RAPD pattern (Ruma et al., 1996; Boekhout et al., 1997; Ellis et al., 2000), PCR fingerprint (Meyer et al., 1993), RFLP pattern (Meyer et al., 2003; Latouche et al., 2003), AFLP fingerprint (Boekhout et al., 2001) and DNA sequence (Diaz et al., 2000; Xu et al., 2000b; Sugita et al., 2001; Chaturvedi et al., 2002; Biswas et al., 2003; Katsu et al., 2004; Butler and Poulter, 2005; Diaz et al., 2005; Bovers et al., 2007a; Bovers et al., 2007b). Several molecular methods showed that two genotypic groups can be recognized within C. neoformans (Meyer et al., 1993; Ruma et al., 1996; Diaz et al., 2000; Ellis et al., 2000; Xu et al., 2000b; Boekhout et al., 2001; Sugita et al., 2001; Chaturvedi et al., 2002; Biswas et al., 2003; Latouche et al., 2003, Meyer et al., 2003; Katsu et al., 2004; Butler and Poulter, 2005; Diaz et al., 2005; Bovers et al., 2007a) and these groups have been described as separate varieties, namely var. grubii (serotype A) and var. neoformans (serotype D) (Franzot et al., 1999). However, although several molecular methods distinguished four genotypic groups within C. gattii (Ruma et al., 1996; Ellis et al., 2000; Chaturvedi et al., 2002; Biswas et al., 2003; Latouche et al., 2003; Meyer et al., 2003; Butler and Poulter, 2005; Diaz et al., 2005; Fraser et al., 2005; Bovers et al., 2007a), these groups have not been described as distinct taxa. Interestingly, serotypes B and C do not correspond to a specific group, but occur in all genotypic groups C. gattii (Meyer et al., 2003). Epidemiology and ecology Cryptococcus neoformans and C. gattii differ in epidemiology and in their ecological niche. Cryptococcus neoformans var. grubii occurs throughout the world and causes 99% of the cryptococcal infections in AIDS patients (Mitchell and Perfect, 1995). Cryptococcus neoformans var. neoformans is also found worldwide, but it occurs most commonly in Europe, where 20% of the reported infections are caused by var. neoformans (Kwon- Chung and Bennett, 1984; Dromer et al., 1996, Tortorano et al., 1997, Tintelnot et al., 10 bovers_V4.indd 10 25-10-2007 10:14:03 Chapter 1: Introduction 2004; Viviani et al., 2006). Cryptococcus neoformans var. neoformans is able to infect AIDS patients (Dromer et al., 1996; Tortorano et al., 1997). Among patients diagnosed with 1 cryptococcosis, elderly people as well as people that receive corticosteroid therapy have an increased risk of infection with C. neoformans var. neoformans (Dromer et al., 1996). In contrast to C. neoformans, C. gattii mainly infects otherwise healthy individuals (Rozenbaum and Goncalves, 1994; Speed and Dunt, 1995; Mitchell et al., 1995; Chen et al., 2000) and occurs predominantly in (sub)tropical areas (Kwon- Chung and Bennett, 1984). Cryptococcus gattii has, however, been isolated in Europe (Montagna et al., 1997; Baró et al., 1998; Velegraki et al., 2001; Colom et al., 2005) and in a temperate climate zone in Colombia (Escandón et al., 2006). Furthermore, C. gattii is responsible for the ongoing outbreak of cryptococcosis on Vancouver Island, Canada (Stephen et al., 2002; Hoang et al., 2004; Kidd et al., 2004), and has been detected in other areas of the Pacific Northwest, USA, although recent data suggests that C. gattii has not permanently colonized those areas (MacDougall et al., 2007). Cryptococcus neoformans is often isolated from avian excreta, mainly from pigeon excreta (Emmons, 1955; Casadevall and Perfect, 1998). In addition, it has been isolated from soil (Emmons, 1951) and decaying wood (Lazéra et al., 1996). Cryptococcus gattii has been isolated from several tree species since the first finding of C.