Cryptococcus (C.) neoformans, a fatal pathogenic yeast, was previously divided into two varieties comprising C. neoformans var. neoformans, which is the opportunistic agent of cryptococcosis in immunodepressed hosts, and C. neoformans var. gattii, which is a probable cause of cryptococcosis in immunocompetent hosts.1,2 Recently, C. neoformans var. gattii has been defined as a separate species, C. gattii, due to the divergence of ecological, biochemical, and molecular characteristics.3,4 The lastest related study even suggested C. gattii deserved to have varieties because of genetic diversity.5 Currently, taxonomic studies3–5 described C. neoformans into C. species complex contains two sibling taxa, C. neoformans and C. gattii, both of which are basidiomycetous yeasts and major fungal pathogens of humans and other mammals. Two varieties of C. neoformans are recognized: C. neoformans var. grubii (serotype A),6 which is distributed worldwide, and C. neoformans var. neoformans (serotypes D and AD), which occurs mainly in Europe and South America.1,2,7 In contrast to C. neoformans, C. gattii is divided into two serotypes: serotype B and serotype C, which has been regarded as a tropical and subtropical pathogenic organism until a recent outbreak of cryptococcosis caused by C. gattii in Vancouver Island, Canada, where temperate region is.8
In recent years, several molecular studies, including electrophoretic karyotyping (EK),9 polymerase chain reaction (PCR) fingerprinting,10 restriction fragment length polymorphism (RFLP),11 amplified fragment length polymorphism (AFLP),12 rDNA sequencing,13,14 multilocus sequence typing (MLST) analysis15,16 and multilocus microsatellite typing (MLMT)17,18 greatly improved our understanding of morphologic and genetic diversity of C. species complex isolates. Among these molecular techniques, PCR fingerprinting has being the most prevalent molecular type approach of C. species complex worldwide, and the Cryptococcal Working Group I (Genotyping of C. neoformans and C. gattii) of the International Society for Human and Animal Mycology (ISHAM) recommended MLST as the preferred method of choice for future strain typing in light of its high discriminatory power as well as reproducibility between different laboratories.19 For example, PCR fingerprint patterns based on M13 microsatellite DNA10 grouped all globally obtained C. neoformans species complex isolates into eight distinct molecular types: VNI and VNII (C. neoformans var. grubii, serotype A); VNIV (C. neoformans var. neoformans, serotype D); VNIII (hybrid, serotype AD); and VGI, VGII,VGIII and VGIV (C. gattii, serotypes B and C). Additionally, molecular epidemiology focused on cryptococcal isolates infections in China suggested that molecular type VNI isolates accounted for the vast majority (72.4–89.6%) of cryptococcosis, but C. gattii isolates (molecular type VGI and VGII) was only responsible for a small part (7.8–11.4%) of cryptococcosis in China.20,21 However, Liao and his team isolated a strain S8012 with unique phenotype of C. gattii from cerebrospinal fluid (CSF) of a 43-year-old male patient in the Shanghai Changzheng Hospital and described as C. neoformans var. shanghaiensis in 1980.22 An array of strategies had been used to analyze the strain S8012, including direct microscopic examination (i.e., light microscopy and electron microscopy ) and animal studies since 1980s.23,24 Several morphological differences was observed to occur among strain S8012 and classical C. gattii strains.22–25 Based on those differences, Liao and his colleagues described S8012 as a new variety of Cryptococcus species complex, namely C. neoformans var. shanghaiensis22 in spite of our following study reclassified strain S8012 as a C. gattii strain.26
Actually, different genetic background of C. gattii isolates appears to distinct virulence and antifugal susceptibilities,27–29 and it is highly significant for the treatment of proper cryptococcosis treatments. Nevertheless, the reports on exact genetic background and polymorphism of C. gattii isolates from China are few. Hence, PCR fingerprint pattern and MLST were used to evaluate the genetic background and polymorphism of clinical C. gattii isolates represented by strain S8012 from China.
METHODS
Strains
Fifteen C. species complex strains were studied in this study. The reference strains were used to determinate the molecular type, the others were obtained from the Chinese Cultural Collection Commission for Microbiology, Nanjing, China. The reference strains information of serotype, isolation, genotype and accession number were obtained from the database of the ATCC, BCCM or CBS collection and presented in Table 1.
Genomic DNA extraction
DNA extractions were carried out according to the procedures described.30 The concentration and quality of the purified DNA was evaluated both by 1% agarose gel electrophoresis and by spectrophotometry (Biophotometer, Germany).
PCR fingerprint analyses
A primer of the minisatellite-specific core sequence of the wild-type phage M13 was used as a single primer, and the PCR condition was in accordance with that originally described by Meyer et al.10 Amplification reactions were performed with Taq DNA MasterMix (TIANGEN, China) with a total volume of 50 μl and 20 ng of DNA as template. Amplification products were separated by gel electrophoresis on 1.4% agarose gels in 1×TAE buffer at 110 V for 1 hour. DNA bands of each fingerprinting pattern were defined manually by comparison to the reference strains of the eight major molecular types.
rDNA sequencing
The ITS region including 5.8S rDNA cluster was amplified using the ITS5 and ITS4 primer set.13 The IGS1 locus was amplified using the primer pair IGS1F (5′-2;-ATCCTTTGCAGACGACTTGA-3′-2;) and IGS1R (5′-2;- GTGATCAGTGCATTGCATGA-3′-2;).14 All amplification reactions were performed with Taq DNA MasterMix (TIANGEN) with a total volume of 50 μl and 20 ng of genomic DNA as template. PCR products were directly sequenced on an ABI 3730 automated DNA sequencer twice in opposite directions.
Multilocus analysis
The sequences were aligned using ClustalX version 1.83 software (http://bips.u-strasbg.fr/fr/Documentation/ClustalX). The phylogenetic tree from the combined DNA sequences of the ITS, IGS1, RPB1, RPB2, LAC1, and TEF1 genes was inferred using neighbor-joining by MEGA3.1 software. In addition, two C. neoformans type strains (CBS10085, B-3501) were included as outgroup. The accession number for the six loci was listed in Table 2.
Phylogenetic analysis
The sequences were aligned with ClustalX version 1.83. The phylogenetic tree was constructed by MEGA software version 3.1 (http://megasoftware.net) with the neighbour joining (NJ) or minimum evolution method based on the nucleic acid sequences. Bootstrap analysis with 1000 replications was done to test the robustness of the internal branches.
RESULTS
PCR fingerprint pattern analyses
PCR fingerprint analysis of strain S8012 showed that S8012 belonged to VGI genotype PCR fingerprint pattern, and C. neoformans var. shanghaiensis showed no significant morphologic differences with C. gattii type strains on PCR fingerprint pattern which showed in Figure 1.
rDNA sequence analyses
Phylogenetic tree using minimum evolution method from the combined DNA sequences of the ITS and IGS1 regions, showed a phylogenetic relatedness of strain S8012 between the different molecular types strains of C. species complex (Figure 2).
MLST analyses
The phylogenetic tree from the combined DNA sequences of the ITS, IGS1, RPB1, RPB2, CNLAC1, and TEF1 genes was NJ analyzed using MEGA3.1 software. In addition, outgroup sequences were included. Two C. neoformans isolates (CBS10085, B-3501) representing the two varieties of C. neoformans were included as outgroup for C. gattii data set. Phylogenetic result suggested strain S8012 was grouped into the cluster of C. gattii environmental isolates originated from Eucalyptus camaldulensis trees in Australia (Figure 3).
DISCUSSION
C. neoformans and C. gattii differ from each other not only in host range and geographic distribution, but in clinical manifestations.1 Although both species infect the central nervous system, patients infected with C. gattii seem to maintain symptoms longer before presentation, and therapy is often required for a longer period of time.1,31,32 In the past, C. gattii has often been associated with Eucalyptus trees in tropical and subtropical climates, causing life-threatening infection in otherwise healthy hosts and to a lesser extent in immunocompromised hosts at low incidences.1,2,33,34 However, the recent outbreak of C. gattii infections occurred on Vancouver Island, Canada, which has expanded the range of this yeast to temperate regions.8,29,35 In PCR fingerprint genotyping pattern, C. gattii isolates can be grouped into four discrete molecular types,10 VGI, VGII, VGIII and VGIV, which appear to have distinct biogeoclimatic distribution zones. For instance, most clinical isolates from Australia and eucalyptus-associated C. gattii isolates belong to molecular type VGI; VGII isolates have been found in domestic animals in the Northern Australia, Canada and Pacific Northwest region of the United States.8,34,35 Remarkably, recent molecular epidemiology focused on cryptococcosis in China suggested that molecular type VGI strains accounted for the vast majority (88.9–91.7%) of C. gattii infections in Chinese cryptococcosis.20,21 It has a significant difference from the recent Pacific Northwest C. gattii outbreak infections in Canada and the United States because VGII strains accounted for approximately 95%.8 Undoubtedly, understanding the genetic background and polymorphism of Chinese clinical isolates of C. gattii is crucial to the development of proper cryptococcosis treatments. In our opinion, strain S8012 is the representative molecular type VGI strain of C. gattii isolated from China. Actually, Liao and his colleagues isolated this strain with a peculiar morphological appearance was isolated from CSF of a male patient in Shanghai Changzheng Hospital in 1980. Direct smear examination of the CSF stained with Indian ink showed some special forms of S8012: bacilliform, needle, gourd, shuttle forms, about 3.33–8.33 μm × 7.33–13.32 μm.22 The capsule is about 23.31–24.98 μm wide, and they were single and double walled, contained 1–7 glistening granules.22 There were several differences between S8012 and the classical C. gattii strain. For instance, 5.13% difference of genomic G+C contents between strain S8012 of C. neoformans var. shanghaiensis and type strain RV20186 of C. gattii was shown in the following study.23 Therefore, Liao and his colleagues22 once proposed C. neoformans var. shanghaiensis as a new variety of C. neoformans in 1980s. Certainly, our subsequent research solved the controversies about the taxonomic status of C. neoformans var. shanghaiensis,26 and the results showed that strain S8012 belonged to the ITS subtype 3 and IGS subtype 4a genotype strain,13,14 which are the molecular types belong to C. gattii. In the current study, phylogenetic analysis based on ITS and IGS1 sequence also showed that strain S8012 fell into the C. gattii group, and the PCR fingerprinting analyses data showed strain S8012 fell into VGI molecular type cluster, which is in agreement with the report by Bovers et al16 that the strain S8012 (CBS7229) belongs to C. gattii group. From the gradual changes of strain S8012’s taxonomic status, we can draw the following conclusions that it was not proper to identify the taxonomic status of C. gattii isolates merely by morphology analysis nowadays because C. gattii expresses highly heterogeneous not only on the genetics, but also on the phenotype; and clinical C. gattii isolates originated from China exists highly polymorphism on the phenotype and genetic background.
Additionally, since C. gattii was firstly isolated from Eucalyptus camaldulensis in 1989,34 this species was soon reported by the isolation from other trees such as Eucalyptus tereticornis, Eucalyptus microcorys and Syncarpia glomulifera, etc.36 Actually, large numbers of Eucalyptus camaldulensis were immigrated in the China mainland from Australia, including Shanghai, Zhejiang, Guangdong, Guangxi, Jiangxi and Yunnan provinces since 1900s.37 In 1999, Li et al38 once attempted to isolate C. gattii strains from Eucalyptus camaldulensis in Jiangxi province where located in southeast China with subtropical climate, but no C. gattii strain was isolated from the 819 sampled Eucalyptus camaldulensis including flowers, buds and leaves, etc. So explored the potential genetic relationship among the molecular type VGI C. gattii strains from other country with China has important scientific significance. In this study, we analyzed 6 Loci sequence of eighteen VGI C. gattii strains using MEGA3.1 programme. Interestingly, the phylogenetic tree suggested strain S8012 was clear to the C. gattii VGI strains isolated from Eucalyptus camaldulensis trees in Australia on genetic background. Despite that this interesting genetic background of Chinese C. gattii isolates needs further analysis in times to prove, C. gattii strains isolated from China, especially molecular type VGI strains, may be immigrated in China mainland with the Australia native Eucalyptus camaldulensis trees transplanted in China.
In summary, the molecular analysis of Chinese clinical C. gattii strain S8012 suggested that clinical C. gattii strains from China existed highly heterogeneous on the phenotype and genetics, and the molecular type VGI strains was possible to be immigrated in China mainland with the Australia native Eucalyptus camaldulensis trees transplanted in China before hundreds of years.
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