Abstract

Fifteen randomly selected microsatellites (simple sequence repeats; SSRs), from the H99 Cryptococcus neoformans var. grubii (serotype A) genome, were sequenced, characterized and applied to sequence 87 clinical and environmental C. neoformans var. grubii isolates from 12 different countries based on Multilocus Microsatellite Typing (MLMT). Among the 15 SSR loci, three (designated CNG1, CNG2 and CNG3) were polymorphic, while the remaining 12 SSR loci showed no variations. The specific PCR primers of the polymorphic microsatellites, i.e., CNG1, CNG2 and CNG3, amplified those loci only from strains of C. neoformans (C. neoformans var. grubii, C. neoformans var. neoformans and the AD hybrid) but not from Cryptococcus gattii, suggesting a species-specific association. The three polymorphic microsatellites are useful markers for strain genotyping, population genetic analysis, epidemiological studies, and may be helpful for the diagnosis of cryptococcosis due to C. neoformans.

Introduction

Members of the Cryptococcus species complex are the causal agents of cryptococcosis, a life-threatening human disease, affecting lungs, central nervous systems and skin 1. Of the two species and five serotypes that are currently recognized, Cryptococcus neoformans, is an opportunistic pathogen causing disease mainly in immunocompromised hosts. The 2 accepted varieties within this species are C. neoformans var. grubii (serotype A) 2, most predominant among AIDS patients 3, 4 and C. neoformans var. neoformans (serotype D). In addition, there is a hybrid of C. neoformans var. grubii and C. neoformans var. neoformans (serotype AD) 5. The second species, Cryptococcus gattii (serotypes B and C), may cause infections in immunocompetent and immunocompromised hosts 6.

To better understand the population genetic structure of the causative agents of cryptococcosis, several molecular approaches have been employed in strain typing and epidemiological studies. These methods have included multi-locus enzyme electrophoresis 7, DNA fingerprinting 8, PCR fingerprinting 9–12, restriction fragment length polymorphism (RFLP) analysis of the PLB113 and the URA5 genes 12, random amplified polymorphic DNA (RAPD) 9, 14–16, pulsed-field gel electrophoresis 17, amplified fragment length polymorphism (AFLP) 5, 18, sequencing of the URA5 gene 19, the internal transcribed spacer (ITS) region of rRNA gene cluster including the 5.8S gene 10, the intergenic spacer (IGS) region 20–22, mitochondrial DNA 23 and more recently multi locus sequence typing (MLST) 24. However, most of those techniques, with the exception of MLST, have serious limitations in their inter-laboratory reproducibility, and some are only useful in distinguishing varieties or major molecular types, but are unable to separate individual strains. The recently developed MLST approach 24 offers a powerful tool for strain typing with an excellent inter-laboratory reproducibility, and will most probably be used extensively in major reference laboratories in the near future. The MLST loci are highly variable in C. neoformans var. grubii molecular types VNI and VNII, for which they had been developed. However, when applied to other varieties, species or molecular types of the Cryptococcus species complex, they did not all amplify all molecular types or have been found to be variable as expected [Meyer, unpublished data]. That is reason there is still a need for additional markers which would increase the variation and allow, if possible, for the development of less expensive, highly discriminatory and reproducible strain typing methods. Microsatellites, also known as simple sequence repeats (SSRs) or short tandem repeats (STRs), are highly polymorphic and spread throughout all genomes, including humans, lower eukaryotes and fungi 25, 26.

The aim of the present studies was to identify, characterize and evaluate the genetic diversity of polymorphic microsatellite loci from a set of randomly chosen microsatellite loci isolated from the C. neoformans var. grubii genome, and discuss the usefulness of those microsatellites for strain typing within the Cryptococcus species complex.

Materials and methods

Strains used in the study

Eighty-seven clinical and environmental isolates of C. neoformans var. grubii from Belgium (2), Brazil (22), China (5), Chile (1), Costa Rica (12), Egypt (8), Italy (1), Japan (17), Taiwan (1), Thailand (6), USA (1), and Venezuela (11), maintained in the culture collection of the Medical Mycology Research Center, Chiba University, Japan, were retrospectively studied (Table 1). In addition the eight standard strains, representing each major molecular type of the Cryptococcus species complex, obtained from the Molecular Mycology Research Laboratory at Westmead Hospital, University of Sydney, Australia were included in the investigation. The latter consisted of: WM148 (C. neoformans var. grubbii, serotype A, VNI/AFLP1); WM626 (C. neoformans var. grubii, serotype A, VNII/AFLP1A); WM628 (AD hybrid, serotype AD, VNIII/AFLP2); WM629 (C. neoformans var. neoformans, serotype D, VNIV/AFLP3); WM179 (C. gattii, serotype B, VGI/AFLP4); WM178 (C. gattii, serotype B, VGII/AFLP6); WM161 (C. gattii, serotype B, VGIII/AFLP5); and WM779 (C. gattii, serotype C, VGIV/AFLP7) 5, 12. All strains were originally identified as C. neoformans using standard microbiological methods and growth on CGB media 27. The strains were grown on potato dextrose agar (PDA; Difco Laboratories) slants and incubated at 30°C for 48–72 h before DNA isolation.

Table 1

List of Cryptococcus neoformans var. grubii strains studied indicating their source, country of origin, isolation year, microsatellite and combined MLMT types.

IFM strain number Source of isolation Country of origin Year of isolation CNG1 allele type (repeat number) CNG2 allele type (repeat number) CNG3 allele type (repeat number) MLMT type 
48812 CSF of HIV (+) patient Belgium nd 4 (13) 3 (10) 2 (7) 22 
48813 CSF of HIV (+) patient Belgium nd 4 (13) 3 (10) 2 (7) 22 
5874 Unknown Brazil 1988 2 (11) 4 (11) 6 (12) 13 
5876 Unknown Brazil 1988 2 (11) 4 (11) 6 (12) 13 
5877 Unknown Brazil 1997 2 (11) 4 (11) 6 (12) 13 
5888 Unknown Brazil 1997 2 (11) 2 (9) 4 (10) 
47274 Unknown Brazil 1998 2 (11) 5 (12) 6 (12) 14 
48224 CSF of HIV (+) patient Brazil 1997 2 (11) 2 (9) 6 (12) 
48225 CSF of HIV (+) patient Brazil 1997 2 (11) 2 (9) 6 (12) 
48229 CSF of HIV (+) patient Brazil 1997 2 (11) 3 (10) 6 (12) 11 
48231 CSF of HIV (+) patient Brazil 1997 2 (11) 2 (9) 6 (12) 
48235 Blood of HIV (+) patient Brazil 1997 4 (13) 2 (9) 6 (12) 21 
48254 CSF of HIV (+) patient Brazil 1997 4 (13) 2 (9) 2 (7) 19 
48255 CSF of HIV (+) patient Brazil 1997 1 (9) 2 (9) 2 (7) 
48256 CSF of HIV (+) patient Brazil 1997 4 (13) 2 (9) 1 (5) 18 
48257 Bronchoalveolar lavage Brazil 1997 2 (11) 4 (11) 3 (9) 12 
48259 CSF of HIV (+) patient Brazil 1997 4 (13) 4 (11) 1 (5) 25 
48260 CSF of HIV (+) patient Brazil 1997 4 (13) 4 (11) 4 (10) 27 
48261 HIV (+) patient Brazil 1997 4 (13) 4 (11) 4 (10) 27 
48293 CSF of HIV (+) patient Brazil 1999 3 (12) 2 (9) 2 (7) 15 
48294 CSF of HIV (+) patient Brazil 1997 4 (13) 4 (11) 4 (10) 27 
48295 CSF of HIV (+) patient Brazil 1997 4 (13) 2 (9) 6 (12) 21 
48296 CSF of HIV (+) patient Brazil 1997 4 (13) 2 (9) 4 (10) 20 
48304 Sputum of HIV(+) patient Brazil 1997 4 (13) 4 (11) 6 (12) 28 
48279 CSF of HIV (+) patient Chile 1999 4 (13) 4 (11) 2 (7) 26 
45718 Unknown China 1990 3 (12) 5 (12) 2 (7) 17 
45728 Unknown China 1990 4 (13) 5 (12) 2 (7) 29 
45729 Pigeon dropping China 1990 3 (12) 4 (11) 2 (7) 16 
45732 Pigeon dropping China 1990 3 (12) 5 (12) 2 (7) 17 
45735 Pigeon dropping China 1990 3 (12) 5 (12) 2 (7) 17 
46551 Unknown Costa Rica 1994 3 (12) 5 (12) 2 (7) 17 
46553 Unknown Costa Rica 1994 2 (11) 4 (11) 6 (12) 13 
46563 Unknown Costa Rica 1994 3 (12) 3 (10) 2 (7) 16 
46664 Unknown Costa Rica 1994 1 (9) 3 (10) 2 (7) 
46665 Unknown Costa Rica 1994 1 (9) 3 (10) 2 (7) 
46670 Unknown Costa Rica 1994 1 (9) 3 (10) 2 (7) 
46706 Pigeon dropping Costa Rica 1994 1 (9) 4 (11) 1 (5) 
46713 Pigeon dropping Costa Rica 1994 2 (11) 4 (11) 6 (12) 13 
46716 Pigeon dropping Costa Rica 1994 2 (11) 4 (11) 6 (12) 13 
46728 Pigeon dropping Costa Rica 1994 2 (11) 3 (10) 2 (7) 
46736 Pigeon dropping Costa Rica 1994 2 (11) 4 (11) 6 (12) 13 
46760 Pigeon dropping Costa Rica 1994 2 (11) 4 (11) 6 (12) 13 
52965 CSF of HIV (+) patient Egypt nd 4 (13) 3 (10) 2 (7) 22 
52966 CSF of HIV (+) patient Egypt nd 4 (13) 3 (10) 2 (7) 22 
52967 CSF of HIV (+) patient Egypt nd 4 (13) 3 (10) 2 (7) 22 
52968 CSF of HIV (+) patient Egypt nd 4 (13) 3 (10) 2 (7) 22 
52969 CSF of HIV (+) patient Egypt nd 4 (13) 3 (10) 2 (7) 22 
52970 CSF of HIV (+) patient Egypt nd 4 (13) 3 (10) 2 (7) 22 
52971 CSF of HIV (+) patient Egypt nd 4 (13) 3 (10) 2 (7) 22 
52972 CSF of HIV (+) patient Egypt nd 4 (13) 3 (10) 2 (7) 22 
48810 CSF of HIV (+) patient Italy before 1997 4 (13) 3 (10) 2 (7) 22 
5807 Unknown Japan before 1999 3 (12) 5 (12) 2 (7) 17 
5808 Unknown Japan before 2000 3 (12) 5 (12) 2 (7) 17 
5814 Unknown Japan before 2001 3 (12) 5 (12) 2 (7) 17 
8569 Unknown Japan nd 3 (12) 5 (12) 2 (7) 17 
9738 Unknown Japan nd 4 (13) 5 (12) 2 (7) 29 
10177 Unknown Japan nd 3 (12) 5 (12) 2 (7) 17 
10201 Unknown Japan nd 4 (13) 5 (12) 2 (7) 29 
10271 Unknown Japan nd 3 (12) 5 (12) 2 (7) 17 
45923 Pigeon dropping Japan 1991 3 (12) 5 (12) 2 (7) 17 
45933 Pigeon dropping Japan 1991 3 (12) 5 (12) 2 (7) 17 
45981 Pigeon dropping Japan 1991 3 (12) 5 (12) 2 (7) 17 
45985 Pigeon dropping Japan 1991 3 (12) 5 (12) 2 (7) 17 
46641 PCNB of lung Japan before 1994 3 (12) 5 (12) 2 (7) 17 
46652 Sputum Japan before 1995 3 (12) 5 (12) 2 (7) 17 
46653 CSF Japan before 1996 3 (12) 5 (12) 2 (7) 17 
46660 Lung tissue Japan before 1997 3 (12) 5 (12) 2 (7) 17 
46661 PCNB of lung Japan before 1998 3 (12) 5 (12) 2 (7) 17 
45705 CSF of HIV (+) patient Taiwan nd 3 (12) 5 (12) 6 (12) 17 
46501 HIV (+) patient Thailand 1995 2 (11) 2 (9) 5 (11) 
46502 HIV (+) patient Thailand 1995 2 (11) 3 (10) 5 (11) 10 
46503 HIV (+) patient Thailand 1995 2 (11) 3 (10) 5 (11) 10 
46504 HIV (+) patient Thailand 1995 2 (11) 1 (7) 5 (11) 
46515 HIV (+) patient Thailand 1995 2 (11) 1 (7) 5 (11) 
46531 HIV (+) patient Thailand 1995 2 (11) 2 (9) 5 (11) 
40216 CSF, NCCLS USA nd 1 (9) 4 (11) 6 (12) 
48263 CSF of HIV (+) patient Venezuela 1999 4 (13) 3 (10) 6 (12) 24 
48264 CSF of HIV (+) patient Venezuela 1999 4 (13) 5 (12) 6 (12) 30 
48265 CSF of HIV (+) patient Venezuela 1999 4 (13) 3 (10) 2 (7) 22 
48274 CSF of HIV (+) patient Venezuela 1999 4 (13) 3 (10) 2 (7) 22 
48282 HIV (+) patient Venezuela 1999 4 (13) 4 (11) 1 (5) 25 
48283 HIV (+) patient Venezuela 1999 4 (13) 3 (10) 2 (7) 22 
48284 HIV (+) patient Venezuela 1999 4 (13) 3 (10) 6 (12) 24 
48286 HIV (+) patient Venezuela 1999 4 (13) 3 (10) 2 (7) 22 
48287 HIV (+) patient Venezuela 1999 4 (13) 3 (10) 2 (7) 22 
482300 Blood of renal TB patient Venezuela 1999 4 (13) 3 (10) 3 (9) 23 
482301 CSF of HIV (+) patient Venezuela 1999 4 (13) 4 (11) 1 (5) 25 
IFM strain number Source of isolation Country of origin Year of isolation CNG1 allele type (repeat number) CNG2 allele type (repeat number) CNG3 allele type (repeat number) MLMT type 
48812 CSF of HIV (+) patient Belgium nd 4 (13) 3 (10) 2 (7) 22 
48813 CSF of HIV (+) patient Belgium nd 4 (13) 3 (10) 2 (7) 22 
5874 Unknown Brazil 1988 2 (11) 4 (11) 6 (12) 13 
5876 Unknown Brazil 1988 2 (11) 4 (11) 6 (12) 13 
5877 Unknown Brazil 1997 2 (11) 4 (11) 6 (12) 13 
5888 Unknown Brazil 1997 2 (11) 2 (9) 4 (10) 
47274 Unknown Brazil 1998 2 (11) 5 (12) 6 (12) 14 
48224 CSF of HIV (+) patient Brazil 1997 2 (11) 2 (9) 6 (12) 
48225 CSF of HIV (+) patient Brazil 1997 2 (11) 2 (9) 6 (12) 
48229 CSF of HIV (+) patient Brazil 1997 2 (11) 3 (10) 6 (12) 11 
48231 CSF of HIV (+) patient Brazil 1997 2 (11) 2 (9) 6 (12) 
48235 Blood of HIV (+) patient Brazil 1997 4 (13) 2 (9) 6 (12) 21 
48254 CSF of HIV (+) patient Brazil 1997 4 (13) 2 (9) 2 (7) 19 
48255 CSF of HIV (+) patient Brazil 1997 1 (9) 2 (9) 2 (7) 
48256 CSF of HIV (+) patient Brazil 1997 4 (13) 2 (9) 1 (5) 18 
48257 Bronchoalveolar lavage Brazil 1997 2 (11) 4 (11) 3 (9) 12 
48259 CSF of HIV (+) patient Brazil 1997 4 (13) 4 (11) 1 (5) 25 
48260 CSF of HIV (+) patient Brazil 1997 4 (13) 4 (11) 4 (10) 27 
48261 HIV (+) patient Brazil 1997 4 (13) 4 (11) 4 (10) 27 
48293 CSF of HIV (+) patient Brazil 1999 3 (12) 2 (9) 2 (7) 15 
48294 CSF of HIV (+) patient Brazil 1997 4 (13) 4 (11) 4 (10) 27 
48295 CSF of HIV (+) patient Brazil 1997 4 (13) 2 (9) 6 (12) 21 
48296 CSF of HIV (+) patient Brazil 1997 4 (13) 2 (9) 4 (10) 20 
48304 Sputum of HIV(+) patient Brazil 1997 4 (13) 4 (11) 6 (12) 28 
48279 CSF of HIV (+) patient Chile 1999 4 (13) 4 (11) 2 (7) 26 
45718 Unknown China 1990 3 (12) 5 (12) 2 (7) 17 
45728 Unknown China 1990 4 (13) 5 (12) 2 (7) 29 
45729 Pigeon dropping China 1990 3 (12) 4 (11) 2 (7) 16 
45732 Pigeon dropping China 1990 3 (12) 5 (12) 2 (7) 17 
45735 Pigeon dropping China 1990 3 (12) 5 (12) 2 (7) 17 
46551 Unknown Costa Rica 1994 3 (12) 5 (12) 2 (7) 17 
46553 Unknown Costa Rica 1994 2 (11) 4 (11) 6 (12) 13 
46563 Unknown Costa Rica 1994 3 (12) 3 (10) 2 (7) 16 
46664 Unknown Costa Rica 1994 1 (9) 3 (10) 2 (7) 
46665 Unknown Costa Rica 1994 1 (9) 3 (10) 2 (7) 
46670 Unknown Costa Rica 1994 1 (9) 3 (10) 2 (7) 
46706 Pigeon dropping Costa Rica 1994 1 (9) 4 (11) 1 (5) 
46713 Pigeon dropping Costa Rica 1994 2 (11) 4 (11) 6 (12) 13 
46716 Pigeon dropping Costa Rica 1994 2 (11) 4 (11) 6 (12) 13 
46728 Pigeon dropping Costa Rica 1994 2 (11) 3 (10) 2 (7) 
46736 Pigeon dropping Costa Rica 1994 2 (11) 4 (11) 6 (12) 13 
46760 Pigeon dropping Costa Rica 1994 2 (11) 4 (11) 6 (12) 13 
52965 CSF of HIV (+) patient Egypt nd 4 (13) 3 (10) 2 (7) 22 
52966 CSF of HIV (+) patient Egypt nd 4 (13) 3 (10) 2 (7) 22 
52967 CSF of HIV (+) patient Egypt nd 4 (13) 3 (10) 2 (7) 22 
52968 CSF of HIV (+) patient Egypt nd 4 (13) 3 (10) 2 (7) 22 
52969 CSF of HIV (+) patient Egypt nd 4 (13) 3 (10) 2 (7) 22 
52970 CSF of HIV (+) patient Egypt nd 4 (13) 3 (10) 2 (7) 22 
52971 CSF of HIV (+) patient Egypt nd 4 (13) 3 (10) 2 (7) 22 
52972 CSF of HIV (+) patient Egypt nd 4 (13) 3 (10) 2 (7) 22 
48810 CSF of HIV (+) patient Italy before 1997 4 (13) 3 (10) 2 (7) 22 
5807 Unknown Japan before 1999 3 (12) 5 (12) 2 (7) 17 
5808 Unknown Japan before 2000 3 (12) 5 (12) 2 (7) 17 
5814 Unknown Japan before 2001 3 (12) 5 (12) 2 (7) 17 
8569 Unknown Japan nd 3 (12) 5 (12) 2 (7) 17 
9738 Unknown Japan nd 4 (13) 5 (12) 2 (7) 29 
10177 Unknown Japan nd 3 (12) 5 (12) 2 (7) 17 
10201 Unknown Japan nd 4 (13) 5 (12) 2 (7) 29 
10271 Unknown Japan nd 3 (12) 5 (12) 2 (7) 17 
45923 Pigeon dropping Japan 1991 3 (12) 5 (12) 2 (7) 17 
45933 Pigeon dropping Japan 1991 3 (12) 5 (12) 2 (7) 17 
45981 Pigeon dropping Japan 1991 3 (12) 5 (12) 2 (7) 17 
45985 Pigeon dropping Japan 1991 3 (12) 5 (12) 2 (7) 17 
46641 PCNB of lung Japan before 1994 3 (12) 5 (12) 2 (7) 17 
46652 Sputum Japan before 1995 3 (12) 5 (12) 2 (7) 17 
46653 CSF Japan before 1996 3 (12) 5 (12) 2 (7) 17 
46660 Lung tissue Japan before 1997 3 (12) 5 (12) 2 (7) 17 
46661 PCNB of lung Japan before 1998 3 (12) 5 (12) 2 (7) 17 
45705 CSF of HIV (+) patient Taiwan nd 3 (12) 5 (12) 6 (12) 17 
46501 HIV (+) patient Thailand 1995 2 (11) 2 (9) 5 (11) 
46502 HIV (+) patient Thailand 1995 2 (11) 3 (10) 5 (11) 10 
46503 HIV (+) patient Thailand 1995 2 (11) 3 (10) 5 (11) 10 
46504 HIV (+) patient Thailand 1995 2 (11) 1 (7) 5 (11) 
46515 HIV (+) patient Thailand 1995 2 (11) 1 (7) 5 (11) 
46531 HIV (+) patient Thailand 1995 2 (11) 2 (9) 5 (11) 
40216 CSF, NCCLS USA nd 1 (9) 4 (11) 6 (12) 
48263 CSF of HIV (+) patient Venezuela 1999 4 (13) 3 (10) 6 (12) 24 
48264 CSF of HIV (+) patient Venezuela 1999 4 (13) 5 (12) 6 (12) 30 
48265 CSF of HIV (+) patient Venezuela 1999 4 (13) 3 (10) 2 (7) 22 
48274 CSF of HIV (+) patient Venezuela 1999 4 (13) 3 (10) 2 (7) 22 
48282 HIV (+) patient Venezuela 1999 4 (13) 4 (11) 1 (5) 25 
48283 HIV (+) patient Venezuela 1999 4 (13) 3 (10) 2 (7) 22 
48284 HIV (+) patient Venezuela 1999 4 (13) 3 (10) 6 (12) 24 
48286 HIV (+) patient Venezuela 1999 4 (13) 3 (10) 2 (7) 22 
48287 HIV (+) patient Venezuela 1999 4 (13) 3 (10) 2 (7) 22 
482300 Blood of renal TB patient Venezuela 1999 4 (13) 3 (10) 3 (9) 23 
482301 CSF of HIV (+) patient Venezuela 1999 4 (13) 4 (11) 1 (5) 25 

Extraction of DNA

Genomic DNA was extracted as described previously 28. In brief, 3 to 4 loops of yeast cells from PDA slants were suspended in 200 µl of TE-buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) in a 1.5 ml microcentrifuge tube. Guanidine thiocyanate solution (6 M in 50 mM Tris, pH 8.3), 250 µl, and 450 µl of phenol (buffered with Tris-HCl, pH 8.0), were added to the washed yeast cells in the microcentrifuge tube and boiled for 15 min. Then 250 µl of chloroform-isoamylalcohol (24:1) was added and the aqueous phase was separated by centrifugation at 14,000 g, mixed with an equal amount of isopropanol and 1/10 volume of 3M ammonium acetate, and placed at −20°C for 1 h. Samples were centrifuged again at 14,000 g for 20 min, and resulting nucleic acid pellets were washed with ice-cold 70% ethanol, dried, and resuspended in TE-buffer at a DNA concentration of 5 µg/ml.

Serotype determination

The serotype was determined using the slide agglutination test (Cryptocheck Iatron RM 304-K kit; Mitsubishi Kagaku Iatron, Inc., Tokyo, Japan) according to the manufacturer's protocol.

Microsatellite selection and PCR amplification

The H99 C. neoformans var. grubii (serotype A) genome (http://cneo.genetics.duke.edu, Duke Institute for Genome Science and Policy, USA), was initially searched for SSR motifs longer than 10 base-pairs using the computer program DNASIS version 2.2 (Hitachi software Co. Ltd., Japan). From those identified, 15 microsatellites were then randomly selected for further studies. PCR primers flanking each of those microsatellites were designed using the Genetyx program v. 11.2.2 (Genetyx corp., Tokyo, Japan) and are listed in Table 2. The microsatellites were amplified using ‘Ready-To-Go PCR Beads’ (Amersham Pharmacia Co. Piscataway, NJ, USA), a set of primers at final concentrations of 1 µM (see Table 2), and 25 pg of genomic DNA, in a volume of 25 µl using a GeneAmp 9600 thermocycler (Perkin-Elmer Inc., USA). The PCR conditions were as follows; initial denaturation at 94°C for 4 min, followed by 25 cycles of denaturing at 94°C for 1 min, annealing at 57°C for 30 sec, and extension at 72°C for 30 sec, followed by a final extension at 72°C for 5 min. All reaction products were checked by 1.5% agarose gel electrophoresis in 1×TBE buffer (45 mM Tris-borate, 1 mM EDTA, pH 8.0) at 80 V for 90 min, followed by staining in 0.5 µg/ml ethidium bromide solution and visualization by UV transillumination.

Table 2

List of polymorphic SSR loci, PCR primers, SSR repeat unit, SSR type, DDBJ accession number, number of sequence alleles and discriminatory power (D).

SSR locus PCR primer name and sequence SSR repeat motive SSR allele type Number of repeats DDBJ accession number Number of alleles Discriminatory power (D) 
CNG1 CNG1F: 5′-CACTTATGGTCTCAGAGGTA-′3 TA AB200387 0.698 
 CNG1R: 5′-AACTTGGCTCGCTGCATCGT-′3    AB200881   
   11 AB200386   
AB200880        
AB200882        
   12 AB200379   
AB200380AB200883        
   13 AB200383   
     AB200384AB200388   
        
CNG2 CNG2F: 5′-CCGGAGAATGAGATTGTCGT-′3 GA AB200898 0.800 
 CNG2R: 5′-TTATCGACGGCCATAGCTTC-′3  AB200895AB200889   
   10 AB200884AB200888AB200893AB200897   
   11 AB200894   
   12 AB200885AB200887   
        
CNG3 CNG3F: 5′-AGATACTACCCGCAAACGTC-′3 CAT AB249641 0.633 
 CNG3R: 5′-TCCCAGTCCTATTCCTCACT-′3  AB249639   
   AB249640   
   10 AB249638   
   11 AB249642   
   12 AB249643   
SSR locus PCR primer name and sequence SSR repeat motive SSR allele type Number of repeats DDBJ accession number Number of alleles Discriminatory power (D) 
CNG1 CNG1F: 5′-CACTTATGGTCTCAGAGGTA-′3 TA AB200387 0.698 
 CNG1R: 5′-AACTTGGCTCGCTGCATCGT-′3    AB200881   
   11 AB200386   
AB200880        
AB200882        
   12 AB200379   
AB200380AB200883        
   13 AB200383   
     AB200384AB200388   
        
CNG2 CNG2F: 5′-CCGGAGAATGAGATTGTCGT-′3 GA AB200898 0.800 
 CNG2R: 5′-TTATCGACGGCCATAGCTTC-′3  AB200895AB200889   
   10 AB200884AB200888AB200893AB200897   
   11 AB200894   
   12 AB200885AB200887   
        
CNG3 CNG3F: 5′-AGATACTACCCGCAAACGTC-′3 CAT AB249641 0.633 
 CNG3R: 5′-TCCCAGTCCTATTCCTCACT-′3  AB249639   
   AB249640   
   10 AB249638   
   11 AB249642   
   12 AB249643   

Polyacrylamide gel analysis

One µl of the PCR product was loaded on a 22.5 cm long polyacrylamide/urea gel (8% polyacrylamide in 7 M urea and 10×TBE buffer). The gels were run in 1×TBE buffer at 1,500 V for 45 min at 51°C on a LI-COR IR2 automated DNA sequencer (LI-COR Biosciences, USA). LI-COR Molecular size markers were loaded every 8 to 10 lanes. The 50-350 bp LI-COR sizing standard was loaded for the polymorphic SSR locus CNG1, while the 50-700 bp LI-COR sizing standard was used for the polymorphic loci CNG2 and CNG3. Products of the CNG1 locus were labeled with IRD 700 dye and the CNG2 and CNG3 loci products were labeled with the IRD 800 dye (LI-COR) using the respective M13 tailed labeled primers. Signals were automatically read and the data were stored and analyzed with the Saga software (generation 2, LI-COR Biosciences, USA) using the automated lane finding and loci location options.

DNA sequencing and BlastN analysis

The amplified PCR products were purified using the PCR product pre-sequencing kit (ExoSAP-IT; USB Corp., Cleveland, OH, USA), and the DNA sequences were determined by an automatic sequencer (ABI PRISM™ 3100; PE Applied Biosystems, Tokyo, Japan) using the big dye terminator cycle sequencing kit with the protocol recommended by the manufacturer. BlastN analysis against the genome sequence databases was performed to identify the respective genes or localizing the sequences in the genome.

Calculation of the discriminatory power (D) for the polymorphic microsatellites

The ability of the microsatellites to discriminate between isolates was assessed by using the Simpson's index of diversity formulated by Hunter & Gaston 29: 

formula
where N is the total number of strains in the sample population, S is the total number of types described, and nj is the number of strains belonging to the j type.

Results

Serotype determination

All 87 isolates were determined to be serotype A using the Iatron serotyping kit.

Degree of polymorphism of microsatellite loci as determined by polyacrylamide gel analysis

The PCR products of all 15 randomly selected microsatellite loci were checked for their degree of polymorphism within the studied set of globally obtained cryptococcal isolates using polyacryamide gel electrophoresis. Out of the 15 microsatellite loci tested, three revealed a high degree of polymorphism and were named; CNG1, CNG2, and CNG3 (Fig. 1.). The remaining 12 microsatellie loci showed no variation in their PCR product lengths (data not shown). For the locus CNG1, the amplified PCR products ranged from 256 and 294 bp (Fig. 1A), while for the locus CNG2, the products were from 408 to 438 bp (Fig. 1B) and for CNG3, the products ranged from 577 to 676 bp were amplified (Fig. 1C).

Fig. 1

Selected microsatellite loci separated by polyacrylamide gel electrophoresis after PCR amplification of the loci CNG1 (‘TA’ repeat), CNG2 (‘GA’ repeat) and CNG3 (‘CAT’ repeat) from randomly selected isolates, showing polymorphisms between the studied strains. Lanes 1–48 are global selections of C. neoformans var. grubii strains; Lanes 49–52 are the four molecular standard strains (WM 148=VNI, WM 626=VNII, WM 628=VNIII and WM 629=VNIV); M=marker lanes, the 50–350 bp size marker was used for the CNG1 locus, and the 50–700 bp size marker was used for the loci CNG2 and CNG3.

Fig. 1

Selected microsatellite loci separated by polyacrylamide gel electrophoresis after PCR amplification of the loci CNG1 (‘TA’ repeat), CNG2 (‘GA’ repeat) and CNG3 (‘CAT’ repeat) from randomly selected isolates, showing polymorphisms between the studied strains. Lanes 1–48 are global selections of C. neoformans var. grubii strains; Lanes 49–52 are the four molecular standard strains (WM 148=VNI, WM 626=VNII, WM 628=VNIII and WM 629=VNIV); M=marker lanes, the 50–350 bp size marker was used for the CNG1 locus, and the 50–700 bp size marker was used for the loci CNG2 and CNG3.

Species and variety specificity of the polymorphic microsatellite loci

The primers for the locus CNG1 amplified PCR products from the C. neoformans reference strains of serotype A (VNI and VNII), serotype AD (VNIII) and D (VNIV) (Fig. 2, upper panel), but did not amplify any C. gattii standard reference strains. The primers for the locus CNG2 amplified PCR products in the reference strains of C. neoformansvar. grubii (VNI and VNII) and the serotypes AD hybrid strains (VNIII), but did not amplify either the reference strains of C. neoformans var. neoformans, serotype D (VNIV), or C. gattii (Fig. 2, middle panel). Finally the primers for the locus CNG3 amplified PCR products in the C. neoformans var. grubii reference standard strains WM148 (serotype A, VNI) and WM628 (serotype AD), but did not amplify the C. neoformans var. grubii standard strain WM626 (serotype A, VNII), the C. neoformans var. neoformans reference standard strain (serotype D, VNIV) or any reference standard strains of C. gattii (Fig. 2, lower panel).

Fig. 2

PCR amplification of polymorphic microsatellite loci from the eight major molecular types of the Cryptococcus species complex, C. neoformans var. grubii WM148 VNI/AFLP1 and WM626 (serotype A), C. neoformans var. neoformans WM628 VNIII/AFLP2 (serotype AD), and WM629 VNIV/AFLP3 (serotype D), WM179 VGI/AFLP4, WM178 VGII/AFLP6 and WM161 VGIII/AFLP5 (serotype B), and WM779 VGIV/AFLP7 (serotype C). CNG1, CNG2 and CNG3 correspond to the polymorphic microsatellite loci with ‘TA’, ‘GA’ and ‘CAT’ repeat motives, respectively.

Fig. 2

PCR amplification of polymorphic microsatellite loci from the eight major molecular types of the Cryptococcus species complex, C. neoformans var. grubii WM148 VNI/AFLP1 and WM626 (serotype A), C. neoformans var. neoformans WM628 VNIII/AFLP2 (serotype AD), and WM629 VNIV/AFLP3 (serotype D), WM179 VGI/AFLP4, WM178 VGII/AFLP6 and WM161 VGIII/AFLP5 (serotype B), and WM779 VGIV/AFLP7 (serotype C). CNG1, CNG2 and CNG3 correspond to the polymorphic microsatellite loci with ‘TA’, ‘GA’ and ‘CAT’ repeat motives, respectively.

Repeat number analysis of the polymorphic microsatellite loci

PCR products amplified from all 15 microsatellite loci have been sequenced. Variable repeat numbers were revealed by polyacrylamide gel electrophoresis for the three identified polymorphic microsatellite loci among the studied strains (Table 2, Fig. 3). The repeat number of the ‘TA’ motif of the microsatellite locus CNG1 varied between 9 to 13, resulting in 4 microsatellite allele types (Table 2). The repeat number of the ‘GA’ motif of the locus CNG2 varied between 7 to 12, resulting in 5 microsatellite allele types (Table 2). Finally the repeat number of the ‘CAT’ motif of the locus CNG3 varied between 5 to 12, resulting in 6 microsatellite allele types (Table 2). Representative examples of the variable motif sequences were deposited to DDBJ (Table 2). The SSR types identified by sequencing did not correspond to the alleles obtained after polyacrylamide gel electrophoresis. The observed discrepancy between the alleles detected by polyarcylamide gel electrophoresis and those determined by sequencing are due to the fact that the size variation detected by polyacrylamide gel electrophoresis was the result of a combination of the variation of the repeat number and the variation in the flanking regions (see Fig. 3 for examples of the CNG1 locus, results for the loci CNG2 and CNG3 are not shown). The other 12 microsatellite loci showed no variation in their repeat numbers.

Fig. 3

Alignment of DNA sequences obtained using primers specific for the CNG1 locus, illustrating the variation found in the repeat number of the microsatellite and in its flanking region.

Fig. 3

Alignment of DNA sequences obtained using primers specific for the CNG1 locus, illustrating the variation found in the repeat number of the microsatellite and in its flanking region.

Genome location of the polymorphic microsatellite loci

The obtained sequences were BlastN searched against the H99 genome to determine the location of the amplified microsatellites. The ‘TA’ motif region of the SSR locus CNG1 was found to be located in the intron of the 1,544 bp functional gene, (181.mo7938) glutamate carboxypeptidase (CNA01640), on chromosome 1. The ‘GA’ motif region of CNG2 was located at 230 bp upstream from the 5′ ATG initiation codon of gene (181.m08568), corresponding to an expressed protein of unknown function on chromosome 1. The ‘CAT’ motif region of CNG3 was located at around 122,4411th base from the top of chromosome 3 — piece 24.

Discriminatory power of the polymorphic microsatellite loci

The highest level of discriminatory power (D) was observed for the microsatellite locus CNG2 repeat motifs (D=0.800). Discriminatory powers for CNG1 and CNG3 were D=0.698, and D=0.633, respectively (Table 2). When taking the combined MLMT SSR profile of the three polymorphic loci into account the discriminatory power increased to D=0.992.

Global distribution of the MLMT types

Thirty different combined Multilocus Microsatellite Typing (MLMT) types were globally found in the 87 C. neoformans var. grubii strains studied. The highest genetic variation was detected in South and Latin American strains, which had 27 of the 30 MLMT types found. Genotype 22, a combination of CNG1 type 4 (13 repeats), CNG2 type 3 (10 repeats) and CNG3 type 2 (7 repeats), was found globally with the exception of Asia. MLMT type 17, a combination of CNG1 type 3 (12 repeats), CNG2 type 5 (12 repeats) and CNG3 type 2 (7 repeats), was found in Asia and Latin America and MLST types 5, 7, 10 and 29 were specific to Asia (Figs. 4 and 5).

Fig. 4

Geographical distribution of the 86 Cryptococcus neoformans var. grubii strains studied with their MLMT type, made up of the combination of the three polymorphic microsatellite alleles. In brackets is the number of strains given per MLMT locus. Showing the largest genetic variation is present in South and Latin America.

Fig. 4

Geographical distribution of the 86 Cryptococcus neoformans var. grubii strains studied with their MLMT type, made up of the combination of the three polymorphic microsatellite alleles. In brackets is the number of strains given per MLMT locus. Showing the largest genetic variation is present in South and Latin America.

Fig. 5

Global distribution of the established MLMT types, made up of the three polymorphic microsatellite loci, showing that the most common MLMT type is type 22, which is present in all continents except Asia. In addition Asia has four unique MLMT types, 5, 7, 10 and 29. The highest genetic variation among the tested strains, as indicated by the highest number of different MLMT types, is located in the Americas.

Fig. 5

Global distribution of the established MLMT types, made up of the three polymorphic microsatellite loci, showing that the most common MLMT type is type 22, which is present in all continents except Asia. In addition Asia has four unique MLMT types, 5, 7, 10 and 29. The highest genetic variation among the tested strains, as indicated by the highest number of different MLMT types, is located in the Americas.

Discussion

Although it has been shown that MLST is a powerful and useful technique in molecular sub-typing of C. neoformans var. grubii24, MLST loci used in previous studies could be amplified from all eight major molecular types of the Crytococcus species complex. Therefore, it was necessary to develop further loci or other techniques. Microsatellites are known for their high levels of polymorphism compared with those of other molecular markers 25, 26. Those markers can be combined in a multilocus microsatellite typing (MLMT) scheme, which indexes repeat variation within microsatellite sequences. The ideal MLMT scheme should fulfill the following two criteria; (1) it should amplify and type the same loci from all studied strains using the same set of primers, and (2) the loci should contain sufficient sequence diversity to produce a discriminatory typing scheme. In the present paper, microsatellite polymorphisms within the species C. neoformans have been identified as allelic length differences, which are due to the different numbers of repeat units present in the alleles detected by PCR amplification and sequencing. Three of the 15 loci studied were polymorphic, i.e., CNG1, CNG2 and CNG3. Polyacrylamide gel electrophoresis revealed 13 different alleles for those three polymorphic loci (Fig. 1). However, DNA sequencing identified only 4, 5 or 6 alleles for CNG1, CNG2, and CNG3, respectively. Sequencing revealed that those discrepancies in the allele number identified by these two different techniques were due to the fact that the polymorphism detected by polyacrylamide gel electrophoresis are the result of the combination of the variation found in the actual repeat number, as well as the sequence variation found in the flanking regions (see e.g., CNG1 in Fig. 3). These results confirm previous reports of similar findings in the genus Neurospora30. Since the present study was directed towards the identification and characterization of microsatellite repeats, we subsequently restricted our analysis to the actual repeat number of the microsatellites. For the microsatellite locus CNG1 the most frequent allele type was No. 4, with 13 repeats of the ‘TA’ motive (frequency 41.4%). For the SSR locus CNG2, the most frequent allele type was No. 3, with 10 repeats of the ‘GA’ motive (frequencies of 31.0%), and for CNG3 the most frequent allele type was No. 2, with 7 repeats of the “CAT” motive (frequency 55.2%). No correlations between the observed MLMT type and the source of the clinical isolates were found. Although the sample numbers of C. neoformans var. grubii strains from some countries were small, our results show that the MLMT type is more or less restricted to certain geographic regions (Fig. 5). MLMT type number 22 was found in Europe and around the Mediterranean Sea (Italy and Egypt), as well as in Venezuela (Fig. 4) indicating a link between the old and the new world. These findings suggest that cryptococcal strains that are present today in Africa and Latin America might have been introduced during colonization 31. MLMT type 29 is found only in China and Japan. Similarly MLMT type 17 is found in China, Japan and Taiwan, reflecting the historical link between those countries (Fig. 5). Costa Rica, Brazil, Chile, Thailand, the USA and Venezuela harbor their own unique MLMT type(s). Three (5, 7, and 10) out of the four (5, 7, 10, 29) unique Asian MLMT genotypes are specific to Thailand (Figs. 4 and 5), which suggests a unique situation of Thailand within Asia. The highest genotype diversity was observed in South and Latin American countries, such as Brazil (15 different MLMT types), Costa Rica (5 different MLMT types) and Venezuela (different MLMT types) (Fig. 5), which confirmed the variation described earlier in a number of South and Latin American countries 9, 12, 18, 31, 32. Our finding indicated that the three microsatellite loci, individually or in combination, could be useful markers in the prediction of the geographical origin of C. neoformans strains. Further globally obtained strains and a larger number of polymorphic microsatellites need to be studied before a final judgment of the uniqueness of certain MLMT genotypes can be made.

Based on the investigation of standard strains of the eight major molecular types of the Cryptococcus species complex 12, the polymorphic microsatellite locus CNG1 showed a high specificity to C. neoformans, serotypes A, D and AD, while the microsatellite loci CNG2 and microsatellite CNG3 showed high specificity to only C. neoformans, serotypes A, and AD (Fig. 2), suggesting that the microsatellite specific primers developed in this study could, in addition to strain typing, also be used for the quick identification of the species C. neoformans present in clinical specimens. In addition the VNIII standard AD hybrid strain amplified two different alleles of 256 and 264 bps at the CNG1 locus, reflecting the diploid nature of this strain (Fig. 1, CNG1 panel, lane 51) 33, 34.

Since microsatellites have considerable length variations in various organisms, they have been successfully applied for strain typing, population structure analyses, and epidemiological studies of several fungi, including Aspergillus35, 36, Candida37, 38, Saccharomyces39, Penicillium40 and Coccidioides41. To our knowledge this is the first report of an application of microsatellite loci for strain typing within the Cryptococcus species complex. Although the sample number of C. neoformans strains typed herein was small, our study showed that the three polymorphic microsatellites described in this study, in combination with other microsatellites currently studied (Karaoglu & Meyer, unpublished data) could form the basis for the development of a multiplex typing system for the members of the Cryptococcus species complex. MLMT typing as applied herein could form an alternative to MLST typing or could extend the number of loci used in a combined MLMT/MLST typing system to study the spread of strains around the globe or to trace outbreak strains such as the ones which are currently causing the cryptococcosis outbreak on Vancouver Island, Canada 42. If the number of the repeats and the variations in the flanking regions were taken into account, the markers could be separated on a capillary electrophoresis system. This in turn would allow for automatic detection and analysis, eliminating the sequencing step and drastically reducing the costs and turnaround time as has been done for Aspergillus fumigatus36. The prospective MLMT typing system should include further microsatellite loci described from the Cryptococcus species complex 43, 44 or obtained from the five cryptococcal genomes currently being sequenced.

The final aims of such a typing system are (i) to standardize microsatellite typing for the Cryptococcus species complex, including the primers, the separation technique and the MLMT nomenclature used, and (ii) to establish a public database that would make cryptococcal microsatellite allele data available worldwide.

Acknowledgements

This work was supported by a grant from The Ministry of Education, Culture, Sports, Science and Technology of Japan, provided to H.A. and an NH&MRC project grant #9937187, Canberra, Australia to W.M. The authors are also grateful to Dr Eric R. Dabbs, School of Molecular and Cell Biology, University of the Witwatersrand, South Africa, and Dr Wael S. El-Sayed, Central Research Institute of Electric Power Industry, Biotechnology Sector, Abiko-shi, Japan, for their critical comments of the manuscript. In addition we would like to thank Nathalie van de Wiele Luciana Trilles for computer support. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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