System. Appl. Microbiol. 23, 535-545 (2000) SYSTEMATIC AND © Urban & Fischer Verlag _htt-,--p:_Ilw_w_w_.ur_ba_nf_is_ch_er_.de--'.,/jo_u_rna_ls_/s_am ____________ APPLIED MICROBIOLOGY

Molecular Sequence Analyses of the Intergenic Spacer (lGS) Associated with rONA of the Two Varieties of the Pathogenic Yeast, Cryptococcus neoformans

MARA R. DIAZ1, TWN BOEKHOUT2, BART THEELEN2, and JACK W. FELL!

I Rosenstiel School of Marine and Atmospheric Science, University of Miami, Key Biscayne, Florida, USA 2 Centraalbureau voor Schimmelcultures, Yeast Division, Utrecht, The Netherlands

Received: November 2, 2000

Summary

The pathogen Crytococcus neoformans has been traditionally grouped in two varieties, C. neoformans var. neoformans (serotypes A, D and AD) and C. neoformans var. gattii (serotypes B and C). A recent taxonomic evaluation of C. neoformans var. neoformans described C. neoformans var. grubii as a new variety represented by serotype A isolates. Despite immunological, biochemical, ecological and molecu­lar differences the three varieties are classified within one species. We examined the genetic variability of one hundred and five clinical and environmental isolates that included all varieties and serotypes. Se­quence analysis of the intergenic spacer (IGS) associated with rDNA revealed significant differences in nucleotide composition between and within the varieties. Parsimony analysis showed five different geno­types representing distinct genetic lineages. Although there was a high degree of relatedness between serotype and genotype this relatedness was not exclusive as serotypes were not restricted to one particu­lar genotypic group. Serotyping and sequence analyses indicate that C. neoformans var. grubii (serotype A) should not be recognized as a separate variety. Based on this study we propose to accept two separate species, C. neoformans (serotypes A, D and AD) and C. bacillisporus (serotypes Band C synonymous with C. neoformans var. gattii).

Key words: C. neoformans var. neoformans - C. neoformans var. gattii - C. neoformans var. grubii - in­tergeneic spacer (IGS) - genotypes - nucleotide sequence analysis

Introduction

The basidiomycetous, encapsulated yeast, Cryptococ­cus neoformans (Sanfelice) Vuillemin has attracted the attention of the medical and epidemiological community as a pathogen in patients diagnosed with AIDS. Esti­mates indicate that up to 10% of AIDS patients will de­velop cryptococcosis in the United States with a higher worldwide percentage of infestation (LEVITZ 1999).

The anamorphic yeast, Cryptococcus neoformans was formerly described as a homogenous species, except for the existence of four different antigen types (serotypes A, B, C and D) in the polysaccharide capsule (EVANS, 1949 and 1950; WILSON et al., 1968). However, the discovery of the presence of two morphological sexual states result­ed in the description of two species in the teleomorphic states: Filobasidiella neoformans K won-Chung, with serotypes A, D or AD (KWON-CHUNG, 1975) and Filoba­sidiella bacillispora KWON-CHUNG, with serotypes B or C

(KWON-CHUNG 1976). Further studies on the taxonomy of Filobasidiella species and their anamorphs, led to the re-classification of F. bacillispora as F. neoformans var. bacillispora (KWON-CHUNG et al., 1982). In the anamor­phic state, the teleomorph F. neoformans is classified as C. neoformans var. neoformans and C. neoformans var. gattii Vanbreuseghem et Takashio (VANBREUSEGHEM and TAKASHIO, 1970). The variety gattii has also been de­scribed as Filobasidiella bacillispora KWON-CHUNG (KWON-CHUNG, 1976) and as Cryptococcus bacillisporus KWON-CHUNG et BENNETT (KWON-CHUNG et al., 1978). Differences between the antigenic properties of the polysaccharide structure of serotypes A and D, and B and C, led to the assumption that these serotypic groups are fundamentally different (CASADEVALL and PERFECT, 1998). The two varieties of C. neoformans are not recog­nized as different species, since they can mate with each

0723-2020/00/23/04-535 $ 15.00/0

536 M. R. DIAZ et al.

other. The intervariety matings have usually generated sterile progeny (KWON-CHUNG, 1998). The only success­ful mating that has been reported (serotype D and serotype B) produced only 30% viable basidiospores, of which opposite mating types: ex mating type and a mat­ing type were produced in a 1:1 ratio (KWON-CHUNG, 1998). Due to the low rate in viable spores, it was diffi­cult to determine whether these progeny were recombi­nants or carried a D dominant cytoplasmic factor, since all the progeny were classified as serotype D (KWON­CHUNG, 1998).

The majority of infections of cryptococcosis are caused by C. neoformans var. neoformans (serotype A, ex mating type), which is found worldwide in pigeon exc­reta, and soils contaminated with bird droppings. Serotype A has been reported to occur frequently world­wide, whereas serotype D predominates in central Eu­rope (DROMER et aI., 1996; BARO et aI., 1999). In most cases, C. neoformans var. neoformans serotype A is the predominant variety in clinical isolates and the most prevalent variety in patients diagnosed with AIDS (RI­NALDI et aI., 1986). However, there have been few reports on the incidence of C. neoformans var. gattii infections in patients with AIDS (ROZENBAUM et aI., 1990; SPEED et aI., 1993). C. neoformans var. gattii (serotype B) is as­sociated with Eucalyptus camaldulensis (ELLIS and PFEIF­FER 1990) and Eucalyptus tereticornis (LICEA et aI., 1996). The first environmental isolation of C. neofor­mans var. gattii, serotype C was recently reported from almond trees (Terminalia capitata) in Colombia (CALLE­JAS et aI., 1998). Clinical isolates of gattii originated from tropical and subtropical regions (KWON-CHUNG and BEN­NETT, 1984; ELLIS, 1987; CURRIE et aI., 1990; GEZUELE et aI., 1993). The incidence of infestation by variety gattii is common in Australia and New Guinea, where 50-83% of the clinical isolates have been identified as this variety (ELLIS, 1987; CURRIE et aI., 1990 ). Only a few clinical cases of this variety have been reported in temperate zones (DROMER et aI., 1996). The route and source of in­fection of these temperate zone cases are unknown. They may represent reactivation of previous infections ac­quired in tropical zones or exposure to contaminated materials imported from tropical zones

Many studies focused on the molecular genetics of the varieties neoformans and gattii (MEYER et aI., 1993; BOEKHOUT et aI., 1997; Bertout et aI., 1999). Random amplified polymorphic DNA (RAPD) analyses, karyo­typing and fingerprinting demonstrate intraspecific ge­netic diversity between the two varieties (CASADEVALL and PERFECT, 1998). Despite differences in genetics, ecol­ogy, immunology and biochemistry these two varieties are usually classified as one species. A recent study based on karyotypes and RAPD analyses, however, suggests that the two varieties represent two different species (BOEKHOUT et aI., 1997). The purpose of this study was to elucidate the phylogenetic relationship and genetic di­versity between strains and serotypes of C. neoformans var. gattii and var. neoformans. We analyzed the nu­cleotide sequences of a -800 base region at the 5' end of the IGS region as a molecular tool to establish strain level

differentiation. The IGS has been recognized as a suitable molecular tool for population studies and strain level dif­ferentiation (POLANCO and PEREZ DE LA VEGA, 1994; FELL and BLATT, 1999; DIAZ and FELL, 2000). FELL and BLATT (1999) were able to identify different strains of Phaffia rhodozyma by comparing areas of insertions and dele­tions while DIAZ and FELL (2000) distinguished yeasts of the genus Mrakia by differences in nucleotide composi­tion in the IGS region.

Materials and Methods

Isolates A total of one hundred and five environmental and clinical

isolates of Cryptococcus neoformans var. gattii and C. neofor­mans var. neoformans were studied. The sources of isolation, serotypes, mating types and genotypes are listed in Table 1.

Serotyping Serotyping data was obtained from CBS collection and

BOEKHOUT et aI., (1997 and 2000).

peR amplification and cycle sequencing analysis Amplified products (-1.7 kb) from the intergenic spacer

(IGS) region between LrDNA and 5S genes were obtained by the polymerase chain reaction (PCR) using a large subunit rDNA (LrDNA) primer, Lrll (5' TTA CCA CAG GGA TAA CTG GC and a 5S rDNA primer, 5SR (5' GGA TCG GAC GGG GCA GGG TGC) . The reaction was carried out in micro­tubes at a final volume of 100 pI. The amplification reaction mixture contained 4 pi of template DNA; 10 mM Tris HCI (pH 9); 50 mM KCI; 0.1 % Triton X-lOO; 2 mM MgCI2; 100 nM each of Lr1l and 5SR; 2.5U of AmpliTaq DNA polymerase and 100 pM of each dNTP. This reaction mixture was incubated in a MJ Research PTC 100 thermal cycler. Reaction mixtures were denatured at 94°C for 2 min, followed by 40 cycles of denatu­ration at 94 °c, annealing for 1 min at 57°C and extension for 3 min at 72 0c. A final extension incubation of 7 min at 72 °c was included in the cycle. The amplification products were pu­rified using the QIAquick PCR Purification Kit (QIAGEN, Inc.). Amplicon synthesis was corroborated by agarose gel elec­trophoresis. The employed cycle sequence primers were the for­ward primer, IGlF, located at position number 3613 of the 3' end of LrDNA (5'CAG ACG ACT TGA ATG GGA ACG) and the reverse primer IG2R (5 'ATG CAT AGA AAG CTG TTG G) located at position no. 791 of the IGS1 region. The sequences were obtained with a U-COR Automated Sequencer using a standard protocol. Alignments were made with MegAlign (DNA Star) and visually corrected. Phylogenetic trees were computed with PAUP*4.0 using parsimony analysis (heuristic search, stepwise addition, random addition sequence, nearest neighbor interchange, 100 maximum trees). The type strains of Mrakia nivalis (CBS 5266), Mrakia frigida (CBS 5270) Mrakia stokesii (CBS 5917) and Mrakia gelida (CBS 5272) were select­ed as outgroups for the tree construction.

Results

IGS sequence analysis

The IGS sequence of -800 bases, initiating at the 3' end of the LrDNA of the two varieties of C. neoformans were analyzed. Our sequence data show that this region,

Molecular Sequence Analyses of the Intergenic Spacer (IGS) 537

located between the 285- ike (LrDNA) and the 55 genes, initiates at base - 94 of the sequence for C. neoformans var. gattii and C. neoformans var. neoformans. A highly conserved region was observed at the 5' end of the aligned sequence, which represents the 3'end of the 285 rDNA. Beyond nucleotide position number 145, diver­gence in sequences was noticeable among the strains of C. neoformans var. gattii and C. neoformans var. neofor­mans. Divergence in sequences was also observed in dif­ferent sub-groups within the two varieties. All the se­quences showed short tandem repeat motifs of GG, GA and CT at different locations of the IG5. The most com­mon motifs were: GGGGGACTT and GAGA (Table 2). Variations in both repeats were observed at various loca­tions (Table 2). Two consecutive stretches of CAGTCT separated by CAT were present in C. neoformans var. neoformans (positions: 259-264, 268-273) as well as a TG rich area with a consensus of TGTGCTGTG (posi­tion: 203-211). An area characterized by the presence of

Cryptococcus neoformans var. neoformans

233

Cryptococcus neoformans var. gattii

-5 changes

161

185

CBS 5266 CBS 5270

CBS 5917 CBS 5272

Genotype 1A

Genotype 1B

Genotype 1C

67

32

3g~~mB 18~~ 1~~~ ~

CBS 1933 A CBS 4572 A CBS 4868 A RV 53794 D CBS 7779 A CBS 879 A CBS 880 A CBS 886 A CBS 887 A CBS 889 A CBS916A CBS 5756 A RV 52733 D

6 ~~~~~~ RV 66025 A RV 58145 A

2~~%!~~~~ RV 64610 A RV 59379 A CBS 7812A 1~M1446A

AVB2A AVB12 A AVB10A AVB5A RV 61756 AD AVB3A AVB7A

1 t~~~~1 A RV 55451 A RV 59351 A RV 59369 A

4 ~~af~10A AVBOA AVB11 A

LJ2AVB13 A 5~ RV46115A

'-"- RV 58146 A CBS 6886 D

Genotype 2A CBS 7822 D AVB6D

jo!eBBS 8888 D

!2CCBSS 41~~ ~ CBS 6900 D CBS 6901 D CBS 7000 D

Genotype 2B 2CBS 7824 D CBS 6995 D CBS 7825 AD

19~~~~~%tD CBS 6885 D

1BA1 D CBS 950 AD BA3AD CBS 132 D CBS 5728 D

Genotype 2C g~~ ~~ A

,--_....;4,;;.8_-f1 ~~S5m~ ~ 8~~~~.pD 8~~~mB CBS 7814 D CBS 131 AD

3 ~~S6~~~J' D

I CCBS 1930 B 35 BS 6956 B

I IMH 1658 B Genotype 3

96

Genotype 4

CBS 7750 B a CBS 5757 B fl. CBS 6992 B Id CBS 6998 B

1 8~~ ~?~~ ~ CBS919B C£1-. fjf90 B 56AB

2 55A B 48AB ,!!. 59A B

1 C%~A6~89B RV 5265 B

11g~~g~g~ Genotype 5 26 CBS 6955 C

NIH 178C CBS 6994C NIH 139C

Fig. 1. Genotypes of Cryptococcus neoformans based on parsimony analysis of -800 nucleotides in the 5' region of the intergenic spacer rDNA. The phylogenetic tree was computed with PAUP"4 (heuristic search, stepwise addition, random addition sequence, nearest neighbor interchange, 100 maximum trees). Numbers refer to base substitution.

538 M. R. DIAZ et al.

Table 1. List of experimental strains.

Strain Source of isolation Serotype Mating type Genotype EMBL#

C. neoformans var. neoformans

CBS 131 Institut Pasteur, Paris, France AD 2c A]300916 CBS 132 Fermenting fruit juice D a 2c A]300905 CBS 464 Laboratoire de Parasitologie, Paris,France A 2c A]300908 CBS 879 Ulcerated cheek A a la A]300848 CBS 880 Unknown A a la A]300849 CBS 882 Nasal tumor of horse, type strain of Torula nasalis D a 2a A]300890 CBS 886 Unknown A a la A]300850 CBS 887 Unknown A a la A]300851 CBS 888 Unknown D a 2a A]300891 CBS 889 Unknown A la A]300852 CBS 916 Unknown A a la A]300853 CBS 918 Dead white mouse D a 2c A]300917 CBS 939 Unknown D 2c A]300911 CBS 950 Tumor AD 2c A]300903 CBS 1143 Cerebrospinal fluid A a la A]300841 CBS 1144 Cerebrospinal fluid A a la A]300840 CBS 1584 Unknown A 2c A]300910 CBS 1931 Soil A a la A]300842 CBS 1932 Soil A la A]300843 CBS 1933 Mastitic cow, USA A la A]300839 CBS 4194 Spleen, Germany D a 2a A]300892 CBS 4572 Cerebrospinal fluid A a la A]300844 CBS 4868 Sputum, The Netherlands A la A]300845 CBS 5467 Milk from mastitic cow, Switzerland D a 2c A]300912 CBS 5474 Mastitic cow D 2c A]300914 CBS 5728 Nonmeningitic cellulitis & osteomyelitis, USA D a 2c A]300906 CBS 5756 Unknown A a la A]300854 CBS 6885 Lesion on bone of Man D a 2c A]300901 CBS 6886 Dropping of pigeon D a 2a A]300886 CBS 6900 Genetic offspring of CBS 6885 x CBS 7000 D a 2b A]300893 CBS 6901 Genetic offspring of CBS 6885 x CBS 7000 D a 2b A]300894 CBS 6961 Man, Oklahoma, USA A a la A]300877 CBS 6995 Cerebrospinal fluid fluid, Illinois, USA D a 2b A]300897 CBS 7000 Pigeon droppings, Denmark D a 2b A]300895 CBS 7779 Urease negative isolate from AIDS patient, Argentina A ? la A]300847 CBS 7812 Cerebrospinal fluid A la A]300864 CBS 7814 Air, Belgium D a 2c A]300915 CBS 7815 Pigeon droppings, Czechoslovakia D a 2c A]300913 CBS 7816 Cuckoo dropping, Thailand D a,a 2b A]300900 CBS 7822 Genetic offspring of CBS 6886 x CBS 7814 D a 2a A]300887 CBS 7824 Unknown D a 2b A]300896 CBS 7825 Unknown AD ? 2b A]300898 CBS 7826 Unknown AD 2b A]300899 RV 46115 Plants, India A 1b A]300884 RV 52733 Pigeon droppings, Belgium D la A]300856 RV 52755 Cerebrospinal fluid, Belgium D 2c A]300909 RV 53794 Canary bird droppings, Belgium D 1a A]300846 RV 55446 House dust, Zaire A la A]300865 RV 55447 Air inside house, Zaire A la A]300855 RV 55451 Cockroach, Zaire A Ib A]300878 RV 58145 Wood, Zaire A la A]300859 RV 58146 Wood, Zaire A lc A]300885 RV 59351 Parrot droppping, Belgium A Ib A]300880 RV 59369 Parrot droppping, Belgium A Ib A]300879 RV 59379 Air in Zoo, Belgium A la A]300863 RV 60074 Skin, cryptococcosis, Belgium A 1a A]300857 RV 61756 Man, Belgium (visited Zaire) AD la A]300872 RV 61790 Man, Belgium A la A]300860 RV 62210 Cerebrospinal fluid, Belgium fluid from AIDS patient, A 1b A]300881

Belgium

Molecular Sequence Analyses of the Intergenic Spacer (IGS) 539

Table 1. (Continued).

Strain Source of isolation Serotype Mating type Genotype EMBL#

RV 62692 Skin cryptococcosis, Belgium D 2c AJ300918 RV 64610 AIDS patient, Rwanda A 1a AJ300862 RV 65662 Man, Portugal visited Venezuela) A 1a AJ300861 RV 66025 Cryptococcoma, Belgium A 1a AJ300858 AVBO AIDS patient, The Netherlands A 1b AJ300883 AVB1 AIDS patient, The Netherlands A Ib AJ300882 AVB2 AIDS patient, The Netherlands A 1a AJ300867 AVB3 AIDS patient, The Netherlands A 1a AJ300873 AVB4 AIDS patient, The Netherlands A 1a AJ300875 AVB5 AIDS patient, The Netherlands A 1a AJ300871 AVB6 AIDS patient, Rotterdam D 2a AJ300888 AVB7 AIDS patient, The Netherlands A 1a AJ300874 AVB10 AIDS patient, The Netherlands A 1a AJ300870 AVB11 AIDS patient, The Netherlands A 1b AJ300869 AVB12 AIDS patient, The Netherlands A 1a AJ300868 AVB13 AIDS patient, The Netherlands A 1b AJ300876 BA1 AIDS patient, Bordeaux D 2c AJ300902 BA3 AIDS patient, Bordeaux AD 2c AJ300904 BA4 AIDS patient, Bordeaux AD 2c AJ300907 BD2 AIDS patient, France A 1a AJ300866 J9 AIDS patient, New York, USA D 2a AJ300889

C. neoformans var. gattii

CBS 919 Meningoencephalic lesion, type strain B 4 AJ300928 Torulopsis neoformans

CBS 1930 Sick goat, Aruba B 3 AJ300919 CBS 5757 Unknown B a 4 AJ300924 CBS 5758 Unknown C a 5 AJ300929 CBS 6290 Man, Congo B 4 AJ300930 CBS 6289 Subculture of type strain RV 20186 B a 4 AJ300937 CBS 6955 Spinal fluid, type strain of Filobasidiella bacillispora, C a 5 AJ300940

California CBS 6956 Sputum, Washington B a 3 AJ300920 CBS 6992 Man B a 4 AJ300923 CBS 6994 Cerebrospinal fluid, New Jersey C a 5 AJ300942 CBS 6996 Man B a 5 AJ300939 CBS 6998 Cerebrospinal fluid, Thailand B a 4 AJ300925 CBS 7229 Meningitis, type strain C. neoformans var B ? 4 AJ300926

shanghaiensis, China CBS 7748 Air in hollow, Eucalyptus camaldulensis, Australia B 4 A]300927 CBS 7750 Bark debris of E. camaldulensis, California B 3 AJ300922 RV 5265 Cerebrospinal fluid, Zaire B 4 A]300938 NIH 139 Patient, USA, LA C 5 A]300943 NIH 178 Patient, USA, Long Beach C 5 AJ300941 IMH 1658 Nest of Wasp, Uruguay B 3 A]300921 48A Lung of a goat, Spain B 4 A]300934 52A Brain of a goat, Spain B 4 A]300931 55A Lung of a goat, Spain B 4 AJ300933 56A Gut of a goat, Spain B 4 AJ300932 59A Lung of a goat, Spain B 4 AJ300935 60A Lung of a goat, Spain B 4 AJ300936

CBS, Centraalbureau voor Schimmelcultures; RV, Institute of Tropical Medicine; NIH, National Institutes of Health; EMBL#: Euro-pean Molecular Biology Laboratory accession number. The rest of the isolates were provided by individual researchers from Ameri-ca and Europe.

540 M. R. DIAZ et al.

T repeats, ranging from 5-8 bp (starting at position 153) was found to be unique to C. neoformans var. gattii. All sequences in this study have been deposited in EMBL. For accession numbers see Table 1.

Phylogenetic analysis

The phylogenetic tree obtained by parsimony analysis of partial sequences of the IGS region is shown in Fig. 1. This tree comprises five main groups, or clusters, in which C. neoformans var. neoformans and C. neofor­mans var. gattii represent two distinct phylogenetic groups differing by over 200 nucleotides. The upper clus­ter, representing C. neoformans var. neoformans, segre­gates into two main groups consisting of genotype 1 (serotypes A, D and one AD) and genotype 2 (serotypes A, D and AD). Sequence data demonstrate that geno­types 1 and 2 differ by over 80 nucleotides. These two genotypes segregate into three individual sub groups: genotypes 1 a, 1 band 1 c (genotype 1) and genotypes 2a, 2b and 2c (genotype 2). The other main phylogenetic group is represented by C. neoformans var. gattii. This group comprises 3 genotypes: genotype 3 (serotype B), genotype 4 (serotype B) and genotype 5 (serotypes C and B). Genotypes 4 and 5 differ by over 40 nucleotides whereas genotype 3 differs by over 60 nucleotides from genotypes 4 and 5. Overall, most of the nucleotide differ­ences between the genotypes 1 and 2 appear as scattered nucleotide variations and/or deletions/insertions areas

throughout the sequence alignment. The most pro­nounced divergence was observed at location 325-400, where genotype 2 differed from genotype 1 by 29 nu­cleotide substitutions and 19 deletionslinsertions. The se­quences of genotype 3 were characterized by the presence of various nucleotides segments of consecutive nucleotide substitutions and insertions. For example, at location 428-454 the divergence of genotype 3 from genotypes 4 and 5 involved 15 nucleotide substitutions and 6 dele­tions/insertions.

serotype and genotype

The serotype classification for each individual isolate is indicated in Table 1. A serological comparison among the clinical and environmental isolates showed the fol­lowing distribution: serotype A, 43.8%; serotype D, 25.7%; serotype AD, 6.7%; serotype B, 19% and sero­type C, 4.8%. Isolates from AIDS patients were serotype A, except for BA1, AvB6 and J9, which were serotype D and BA3 and BA4, which were serotype AD. Serotype A isolates from AIDS patients represented 33 % of the total number of isolates with serotype A. The serotype inci­dence was as follows: genotype 1 (serotype A, 93.6%; serotype D, 4.3% and serotype AD, 2.1 %.), genotype 2 (serotype A, 12.1 %; serotype D, 66.7% and serotype AD; 21.2%), genotype 3 (serotype B, 100%), genotype 4 (serotype B, 100%) and genotype 5 (serotype C, 83% and serotype B, 16%).

Table 2. Repeats in the IGS region of the two varieties of Cryptococcus neoformans. Dashes represent the same nucleotide as found in the consensus.

Variety Genotype

neoformans and gatti 1,2,3,4,5 neoformans and gattii 1,2,3,4,5 neoformans 1,2 gattii 3,4,5

neoformans and gattii 1,2,3,4,5 neoformans 1 gattii 3,4,5 neoformans 2 neoformans 2 neoformans 2 gattii 3,4,5 neoformans 2 neoformans 1 neoformans 2 gattii 3,4,5 neoformans 1 gattii 3,4,5

neoformans 1,2 neoformans 1,2 gattii 3,4,5 gattii 3,4,5

Location

Consensus:

423-431 576-585 507-515 507-515

Consensus:

128-132 198-201 202-205 485-488 608-611 198-201 198-201 202-205 202-205 485-488 485-488 608-611 608-611

Consensus:

259-264 268-273 259-264 268-273

GGGGGACTT

-TTATG-GG

GAGA

deletion AG-­-T-T AT-T --TA -CGG A--G --A-

CAGTCT

-CA--G ACT-TA

Molecular Sequence Analyses of the Intergenic Spacer (IGS) 541

There is no conclusive correlation between genotype and serotype. The distribution and common occurrence of serotypes A, D and AD among the two main groups of C. neoformans var. neoformans indicate that these sero­types are not restricted to a single genotype (Figure 1). This is based on the distribution of serotypes D or AD within genotype 1 (serotype D: RV 53794 and RV 52733, and serotype AD: RV 61756) and serotypes A or AD within genotype 2 (serotype A: CBS 464 and CBS 1584, and serotype AD: BA3, Ba4, CBS 131, CBS 950, CBS 7825 and CBS 7826). A similar lack of correlation was found among serotypes Band C within genotypes of C. neoformans var. gattii. Within this variety, CBS 6996 (serotype B) resided in genotype 5, which is mostly repre­sented by serotype C strains. The phylogenetic tree also shows the segregation of C. neoformans var. gattii strains (serotype B) into two distinct groups: genotypes 3 and 4.

Isolates from AIDS patients grouped into two differ­ent genotypic groups, genotypes 1 and 2. Seventy five percent of these isolates belonged to genotype 1, whereas 25% belonged to genotype 2. The higher incidence of AIDS isolates occurred in genotype la, followed by geno­type 1 b, genotype 2c and genotype 2a. The distribution of AIDS isolates among the genotypes is shown in Table 3.

Geographic distribution

There is a geographic substructure among strains from C. neoformans var. gattii. Genotype 5 and genotype 3 consist of strains originated from the Americas, except CBS 5758 (unknown origin and source of isolation). These two genotypes, with serotypes Band C differed from genotype 4, which contained a group of serotype B strains isolated from Australia/Asia/Africa and a sub­group of six strains isolated from goats in Extremadura, Spain. These six strains were found in goats from five herds infected by five outbreaks of cryptococcosis during 1990 to 1994 (BARO et al., 1998). The IGS genetic pro­files of these isolates proved to be independent of the site of isolation in the body. Also, their homogeneous genetic pattern appears to suggest that clonal expansion oc­curred in this geographic site of Northern Spain. Another strain, isolated from a sick goat in the Americas (Aruba), did not follow the same genotypic pattern as the Spanish

isolates but belonged to genotype 3, rather than genotype 4. In contrast to the apparent correlation between geo­graphic distribution and serotyping (genotypes 3 and 4) in var. gattii, there is no correlation among genotypes 1 and 2, as their geographic distributions appear to be more heterogeneous. Genotype 1 isolates originated from locations as diverse as Africa, Europe, Asia, South Amer­ica and United States, whereas genotype 2 originated from Europe, Asia and United States. Sequence data of isolates from AIDS patients did not show any geographic distribution. Genotype 1 contained isolates from AIDS patients in The Netherlands, France, Belgium and Rwan­da, whereas genotype 2 contained isolates from AIDS patients in New York, The Netherlands and France (Table 3).

Discussion

Genetic Diversity

The genetic variability of 105 isolates, representing C. neoformans var. neoformans and C. neoformans var. gattii was examined (Table 1). The sequence divergence in the IGS region indicated the presence of distinct genet­ic lineages within the varieties (Figure 1). The most ex­tensive sequence divergence was found between the two varieties. Based on these differences we define five geno­typic lineages, which are grouped into two main phyloge­netic clusters: Cluster 1): C. neoformans var. neoformans (genotypes 1 and 2) and cluster 2): C. neoformans var. gattii (genotypes 3, 4 and 5). The phylogenetic tree in Figure 1 indicates genotypes 1 and 2 (serotypes A, D, AD) are genetically distinct lineages from genotypes 3, 4 and 5 (serotypes B and C). Previous studies on the molec­ular phylogeny of C. neoformans indicated that the two serotypes of C. neoformans var. neoformans (serotypes A and D) are phylogenetically distinct. In contrast, the serotypes of C. neoformans var. gattii possessed nearly identical genetic profiles (MEYER et al., 1993). Using DNA fingerprinting with hybridization probes as single primers, MEYER et al. (1993), identified three distinct banding patterns associated with serotypes A, D or B/C. The nearly indistinguishable genetic banding pattern as­sociated with strains of serotype Band C along with the

Table 3. Distribution of genotype and origins of C. neoformans var. neoformans strains isolated from AIDS patients.

Genotype Total Number % Origin

Genotype 1 n = 15 75 Argentina, Rwanda, France, Belgium & The Netherlands genotype la n = 10 50 Argentina, Rwanda & France, genotype Ib n=5 25 Belgium & The Netherlands genotype lc n=O 0

Genotype 2 n=5 25 United States, The Netherlands & France genotype 2a 11=2 10 United States, The Netherlands genotype 2c n=3 15 France genotype 2b n=O 0

542 M. R. DIAZ et al.

similiarity coefficient values (S ranging from 0.72 and 0.86), revealed that e. neoformans var. gattii strains (serotypes Band C) are nearly identical. These findings, however might be influenced by factors as diverse as the number of isolates studied, source and origin of the iso­lates and primer selection. For example, different primers have been found to yield different levels of genetic het­erogeneity when using the DNA fingerprinting technique (VAN BELKUM et al., 1993). In contrast to MEYER et al. (1993), our IGS sequence data revealed genotypic differ­ences between the serotypes of e. neoformans var. neo­formans and the presence of genotypic diversity among serotypes Band e. For example, serotype C strains in genotype 5 differed by approximately 4% and 8% from serotype B strains in genotype 4 and serotype B strains in genotype 3, respectively. Similar findings on the genetic variability of e. neoformans var. gattii (serotypes Band C) were reported by CASADEVALL and FAN (1992), who documented a 9% sequence divergence in the URA5 genes between e. neoformans var. gattii strains. Also, VARMA and KWON-CHUNG (1992) found that e. neofor­mans var. gattii isolates (serotypes Band C) displayed a greater genetic diversity than e. neoformans var. neofor­mans isolates (serotypes A and D).

Genetic divergence of up to 6% between serotypes A and D has been suggested by URA5 gene sequences and DNA fingerprinting analysis from geographically related clinical strains of e. neoformans var. neoformans (CASADEVALL et al., 1992). Most of the base pair substitu­tions were observed in the third codon or within introns (CASADEVALL et al., 1992). Another study that focused on specific gene sequences showed that the topoisomerase I gene of serotypes A and D strains differs by 3.6% (CASA­DEVALL and PERFECT, 1998). Our study is in agreement with the above reports as we estimate nearly 8% of ge­netic divergence between serotypes A and D. A recent work, based on URA5 sequences and DNA fingerprint patterns of e. neoformans var. neoformans (serotypes A and D) recognized e. neoformans var. grubii as a new va­rietal status for serotype A isolates of e. neoformans var. neoformans (FRANZOT et al., 1999). Despite fundamental genotypic differences between serotypes A and D, our se­quence data show serotype and genotype relatedness is not exclusive since serotypes A, D and AD occur in two different genotypic groups. Thus, the recognition of a new e. neoformans variety classification based mainly on the serological characterization of the isolate should be re-considered. Our IGS data suggest the presence of two separate species, each representing two or three dif­ferent genotypes. Whether these genotypes represent va­rieties within each species remains unclear. Studies that combined molecular techniques such as karyotypes, RAPD analyses, PFGE, serotype and killer toxin analysis also recognized the two varieties as two different species (BOEKHOUT et al., 1997). Hybridization studies focused on DNA base composition and sequence homology demonstrated that both varieties are genetically related by 55 to 63% (AULAKH et al., 1981). Furthermore, recent studies showed that the IGS sequence identity between e. neoformans varieties (serotype B and D) was only 78.5%

(FAN et al., 1995). The reported identity and DNA-DNA re-association values suggest that there is a considerable genetic divergence between these two taxa. FAN et al., (1995) also noticed extensive size variation (100 to 300 nucleotides) in the amplified segments of the IGS of the two varieties. Although, we documented size variation ranging between 10 to 30 nucleotides (e. neoformans var. neoformans: 894-917 bp and e. neoformans var. gattii: 883-886 bp), our length variation differs from oth­ers since our sequences are based on partial regions of the IGS as opposed to complete IGS sequences. Also, the length variability in our IGS partial sequences does not appear to relate to the numbers of repeats as those re­ported for e. neoformans and Phaffia (FAN et al., 1995; FELL and BLATT, 1999). These ribosomal DNA repeat size polymorph isms appear to be common in fungi and other eukaryotes (JEMTLAND et aI., 1986; FAN et aI., 1995). Their size variation has been linked to crossing over and gene conversion events (JEMTLAND et aI., 1986; LUSCHNIG et aI., 1993). In this study, the occurrence of the repeats GGGGGACTT and GAGA were found in both varieties, with the exception of CAGTCT, which was only present in variety neoformans. Whether these repeats playa role in the regulation of transcription or in rRNA processing in e. neoformans (ApPELS et al., 1986; POLANCO and PEREZ DE LA VEGA, 1994) needs to be elucidated.

Contrary to the above findings on the genetic differ­ences in the IGS region between varieties, similar rDNA sequences have been reported in the 16S rDNA and 26S rDNA (FAN et al., 1994). As few as 2 and 10 nucleotide differences were documented for the two varieties in the 16S rDNA and 26S rDNA, respectively. These similari­ties can be expected since both regions are highly con­served. In this regard, the non coding IGS region is a more powerful tool to distinguish between closely related strains (FELL and BLATT, 1999, DIAZ and FELL, 2000) . Se­quences of the IGS region have also been shown to vary considerable among taxa and have been found to be species specific (DELCAsso-TREMOUSAYGUE et al., 1988).

Population structure

Studies based on electrophoretic karyotypes, multi­locus enzyme electrophoresis (MEE), URA5 sequences, RFLP and RAPD analyses suggest a clonal population structure of e. neoformans var. neoformans (CHEN et aI., 1995; BRANDT et aI., 1996; BOEKHOUT et aI., 1997; FRAN­ZOT et aI., 1997). In this study, the genetic relatedness and genetic homogeneity among many of the studied iso­lates belonging to genotypes 1 and 2 appear to support this clonal concept. Using multiple molecular techniques such as URA 5 sequencing, RFLP and karyotyping, FRANZOT et al. (1997) reported the presence of a global population structure because of concordant patterns among some e. neoformans var. neoformans isolates from New York and Brazil. They also observed however, local genetic diversity among Brazilian and New York City isolates. Evidence for a local clonal structure of e. neoformans var. neoformans has also been suggested by BOEKHOUT et al. (1997), who found relatively homoge-

Molecular Sequence Analyses of the Intergenic Spacer (IGS) 543

neous karyotypes and RAPD patterns in isolates from AIDS patient in Rotterdam, The Netherlands. However, we encountered genetic differentiation in these clinical isolates as they were grouped into three different geno­typic groups: genotype 1a (AVB2, AVB3, AVB4, AVB5, AVB7, AVB10, and AVB12); genotype 1b (AVB1, AVBO, AVBll, and AVB13) and genotype 2a (AVB6).

Recent evidence appears to contradict the clonal ex­pansion model, as variable banding patterns were identi­fied in serotypes A and D of C. neoformans var. neofor­mans isolates (BOEKHOUT et a!., 1997; COGLIATTI et a!. (1999a,b); BOEKHOUT et a!., 2000). Based on sequence analysis of RAPD markers and flow cytometric analysis of nuclear DNA content, COGLIATTI et a!. (1999a,b) showed the presence of diploidy and heterozygosity among some C. neoformans var. neoformans isolates. In our analysis the clustering of strains in genotypic groups, which do not necessarily correspond to their serotype classification may suggest that C. neoformans can under­go hybridization. Also, AFLP analysis confirmed the pres­ence of hybrid genotypes probably due to recombination (BOEKHOUT et a!', 2000). Among the strains that followed a hybrid pattern were RV 53794 (serotype D, genotype 1a), RV 52733 (serotype D, genotype 1a), RV 61756 (serotype AD, genotype 1a), BA3 (serotype AD, genotype 2c), BA4 (serotype AD, genotype 2c) CBS 464 (serotype A, genotype 2c) and CBS 1584 (serotype A, genotype 2c). Genotype 3 (c. neoformans var. gattii) appears to have a common ancestor to a sister clade of genotypes 4 and 5 (Fig. 1). However, BOEKHOUT et a!. (2000) suggested that this genotype 3 strains may represent hybrids generated through sexual recombination between isolates of geno­types 4 and 5. Their interpretation was based on AFLP analysis showing the occurrence of 14 bands, which agreed in size with bands of genotypes 4 and 5.

Geographic distribution

In this study, no significant differences were seen in the genetic structure of C. neoformans var. neoformans strains isolated from different geographic locations. Simi­lar observations using RAPD analyses have been report­ed for C. neoformans var. neoformans isolates that origi­nated from different continents (BOEKHOUT et a!., 1997). However, the presence of a geographic population sub­structure among clinical isolates of C. neoformans var. neoformans has been reported in Central Europe where serotype D is more prevalent than in America (BENNETT et a!., 1984; DROMER et a!., 1996). Genetic concordance among some geographically distant strains of C. neofor­mans var. neoformans has led investigators to suggest the presence of a widespread global distribution of some C. neoformans var. neoformans strains (FRANZOT et a!., 1997). The homology in sequences of strains isolated from geographic locations as diverse as Europe, Africa and America, (ie. genotypes 1a and 1b) might be associ­ated with a global dispersal of certain clones. This global distribution may have been facilitated by the introduc­tion of the European and Northern African pigeon, Columba livia, throughout the world (JOHNSTON, 1992).

Eolian transport and bird migrations are other factors being linked to the widespread of strains (FRANZOT et a!., 1997). Although, pigeon excreta has been associated as the main ecological niche for C. neoformans var. neofor­mans, arboreal sources, such as wood of trees in Brazil, has been identified as another ecological niche for C. ne­oformans var. neoformans (FORTES et aI1999).

Our data also show that there is a geographical differ­entiation among C. neoformans var. gattii isolates. The genetic divergence between the Spanish and American goat isolates is an example of this genetic variability that might result from strains originating from different geo­graphic locations (BOEKHOUT et a!., 2000). Additional suggestions of the geographic distribution of strains are genotype 3 and genotype 5, which are strains isolated from America (Possible exemptions of unknown origin are CBS 5758 and CBS 6996). The genetic divergence be­tween American isolates (genotype 3) and those originat­ed from Australia/Asia/ Africa/Spain (genotype 4) can be associated with the introduction of Australian/Asian/ African Eucalyptus camaldulensis along with C. neofor­mans var. gattii into the Americas. The presence of a geo­graphic substructure among populations of C. neofor­mans var. gattii has also been reported by BOEKHOUT et a!. (1997). Different geographic ecological niches can promote the necessary separation for divergence to occur. More samples representing different geographic locations need to be analyzed to have a better under­standing of genetic divergences.

Conclusions

In summary, we suggest the presence of two taxa: C. neoformans and C. bacillisporus. Our results are corrob­orated by physiological studies that also separate these two varieties, based on differences in the assimilation patterns of canavanine, d-proline, fumaric, I-malic, and succinic acids (KWON-CHUNG et a!., 1987; NISHIKAWA et a!., 1996; KWON-CHUNG, 1998). Other documented dif­ferences are the susceptibility to antifungal agents (SHADOMY et a!., 1987), EDTA inhibition of urease (KWON-CHUNG et a!., 1987) and polysaccharide compo­sition and structure of the capsules (CHERNIAK and SUND­STROM, 1994). The genetic diversity in C. neoformans strains may result from the diverse ecological niches they occupy and microevolution events as the strains pass through a variety of environmental and host stresses. Also, sexual recombination events can lead to genetic di­versity. The mechanisms of action that influence this inter-strain diversity have yet to be elucidated. Even though the primary mode of C. neoformans reproduc­tion in the environment is asexual (budding) (TIBAYRENC et a!., 1991; BRANDT et a!., 1996; FRANZOT et a!., 1997), the occurrence of hybrids, as reported by BOEKHOUT et a!. (2000), and COGLIATTI et a!. (1999) indicates that sex­ual recombination can be a viable mode of reproduction in natural populations. The reproduction strategy, whether sexual and/or asexual will have implications on the spread of virulent genes.

544 M. R. DIAZ et al.

Acknowledgements The authors want to acknowledge the assistance of Dr.

FRANCOISE DROMER, who kindly performed serotyping analysis for some of the studied strains and to Dr. J. M. RODRIGUEZ for providing the spanish goat isolates. We are also grateful to Dr. K. J. KWON-CHUNG and Dr. MEAD Mc CABE for critical reviews of the manuscript. We thanks. This work was funded by a grant from the Ocean Sciences Division of the National Sciences Foundation and Genetic Vectors Inc., Miami.

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Corresponding author: JACK W. FELL, Rosenstiel School of Marine and Atmospheric Science, University of Miami, 4600 Rickenbacker Causeway, Key Biscayne, FL 33149 USA Tel: (305)361-4608; Fax: (305)361-4600; e-mail: jfell@rsmas.miami.edu