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Journal of Clinical Microbiology, March 1999, p. 715-720, Vol. 37, No. 3
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Genetic Multilocus Studies of Different Strains of
Cryptococcus neoformans: Taxonomy and Genetic
Structure
S.
Bertout,1
F.
Renaud,2
D.
Swinne,3
M.
Mallié,1 and
J.-M.
Bastide1,*
Laboratoire d'Immunologie Parasitologie
MENRT UPRES EA 2413, Université Montpellier I, Faculté de
Pharmacie, 34060 Montpellier Cédex 2,1 and
Centre d'Étude sur le Polymorphisme des
Micro-Organismes, CEPM/UMR CNRS-ORSTOM 9926, ORSTOM, 34032,
Montpellier Cédex 1,2 France, and
Institut de Médecine Tropicale Prince Léopold, 2000 Antwerp, Belgium3
Received 18 August 1998/Returned for modification 15 October
1998/Accepted 30 November 1998
 |
ABSTRACT |
The genotypes of 107 strains of Cryptococcus isolated
from the environment or from patients from various geographical areas were determined by multilocus enzyme electrophoresis (MLEE). We analyzed the relationships between genotype structure and serotype and
between genotype structure and strain origin. Twelve of the 14 enzyme-encoding loci studied were polymorphic, giving rise to 48 electrophoretic types. The genotypes of C. neoformans
and C. laurentii were very similar. MLEE could not
distinguish between these two pathogenic species. A correlation between
the genetic multilocus structure and the origin of the sample (from the
environment or patients) existed. A second analysis detected a
correlation between genotype distribution and serotype. The second
analysis considered three serotype groups (B, C, and A plus D plus
A/D), proving that serotypes A, D, and A/D are closely related. MLEE is
a useful epidemiological tool for improving our understanding of the
biology of this fungus.
| |
INTRODUCTION |
Cryptococcus neoformans
is a yeast-like fungus. This species has two varieties (14):
C. neoformans var. neoformans, which is
ubiquitous and which groups together serotypes A and D and the very
controversial serotype A/D (2, 4, 28), and C. neoformans var. gattii (serotypes B and C), which is
found mainly in tropical and subtropical regions.
This opportunistic pathogen causes cryptococcosis, a deep mycosis with
a specific tropism for the lungs and brain. It affects immunocompromised individuals and patients suffering from severe hematological malignancies (10). Its incidence has increased over the last 10 years because of the increase in the number of patients with AIDS and those undergoing transplantation
(10).
Epidemiological studies of cryptococcosis are essential if we are to
understand the biology of this fungus so as to obtain improved
treatments for this infection (9, 22). Serotyping was used
as an epidemiological tool for many years (1, 9). The
development of DNA typing methods increases the power of discrimination of strains. Random amplified polymorphic DNA analysis (5, 12, 22), karyotyping (11, 17), and fingerprinting
(12, 22) are routinely used to demonstrate intraspecific
diversity in the genus Cryptococcus. Multilocus enzyme
electrophoresis (MLEE), first used for the discrimination of
Candida strains (18, 19), has provided good
results for Aspergillus (20, 21) and
Cryptococcus (5, 6). It is a very effective
typing method (15, 21) and provides information about both
the phenotype and the genotype (5, 6, 18, 19, 20).
We typed 107 strains of Cryptococcus of very different
origins (patients and environment) and from different geographical areas by MLEE (5). Cryptococcus laurentii is
slightly pathogenic for immunocompromised individuals, and some strains
were included in this study to explore the genetic diversity of the
genus Cryptococcus.
This study was performed to obtain taxonomic information about the two
species C. neoformans and C. laurentii and to
identify any relationships between genotype information and the
serotype or origin of the strains. This should improve our
understanding of the biology of this fungus.
| |
MATERIALS AND METHODS |
The 107 strains tested in the present study were kindly donated
by the Institute of Tropical Medicine, Antwerp, Belgium. We used 48 strains that originated from the environment (e.g., soil, fruits, and
eucalyptus), 57 strains that originated from patients, and 2 reference
strains (Table 1). The isolates were
grown on Sabouraud agar (Biomérieux, Marcy l'Etoile, France) at
33°C.
Preparation of culture lysates.
The isolates were grown in
500-ml flasks containing 180 ml of Sabouraud agar medium (pH 8) at
33°C for 72 h. Yeasts were collected from the surface with glass
beads (diameter, 8 mm) in 12 ml of sterile deionized water.
The resulting suspension was centrifuged at 4,000 × g
for 30 min, and the yeasts were suspended in 8 ml of distilled water. The cells were mechanically disrupted in the cold by the glass bead
method (bead diameter, 0.25 mm) for 2 min in a Science tech MSK
homogenizer (B. Braun, San Francisco, Calif.). The cellular debris was
removed by centrifugation at 15,000 × g for 15 min at
0°C. The supernatant of each lysate was aliquoted and was stored at
20°C before use in MLEE.
Enzyme electrophoresis.
Starch gel electrophoresis and
specific enzyme staining were performed as described previously
(16, 18, 19, 20). In this study, the following 14 enzyme
systems were tested: peptidase A (PEP A; EC 3.4.11; substrate,
Val-Leu), peptidase B (PEP B; EC 3.4.11; substrate, Leu-Gly-Gly),
peptidase C (PEP C; EC 3.4.11; substrate, Leu-Ala), glucose phosphate
isomerase (GPI; EC 5.3.1.9), malate dehydrogenase (MDH; EC 1.1.1.37),
glucose-6-phosphate dehydrogenase (G6PD; EC 1.1.1.27),
phosphoglucomutase (PGM; EC 2.7.5.1), alcohol dehydrogenase (ADH; EC
1.1.1.1), fructokinase (FK; EC 2.7.1.4), fumarase (FUM, EC 4.2.1.2),
isocitrate dehydrogenase (IDH; EC 1.1.1.42), 6-phosphogluconate
dehydrogenase (PGD; EC 1.1.1.43), pyruvate kinase (PK; EC 2.7.1.40),
and sorbitol dehydrogenase (SDH; EC 1.1.1.14).
Twelve loci were polymorphic in the 107 strains examined, and each
polymorphic locus had three to six alleles (see Fig.
1).
We identified
58 alleles that gave 48 different electrophoretic
types (ETs) (see
Table
2). Alleles were numbered in decreasing
order of mobility. The
various polymorphic enzyme-encoding loci
were characterized for each
isolate. Distinct multilocus variants
were designated
ETs.
Statistical analysis. (i) Factorial correspondence analysis.
We analyzed the results by factorial correspondence analysis (FCA)
using the PRAXIS-p.c., version 2.0, software package (Praxème, R&D, Biométrie, Centre National de la Recherche Scientifique, Montpellier, France).
FCA involves the construction of a contingency table (samples versus
alleles), in which each isolate is represented in terms
of its allelic
composition. This method of analysis simultaneously
characterizes the
ET according to all the genetic variables (alleles)
and determines the
contribution of each allele to the overall
variability of the sample.
The calculation of variance is based
on the
2 test,
which measures the extent to which a sample distribution
deviates from
a theoretical distribution and is an integral part
of the
correspondence analysis (
3,
7). FCA was presented
as a plane
projection of the two most informative axes accounting
for the genetic
structure of the
ETs.
(ii) Canonical correspondence analysis.
We used canonical
correlation analysis (CCA) to compare and correlate genetic information
and the origin or serotype of strains of various ETs using the
PRAXIS-p.c., version 2.0, software package. This CCA was performed with
two contingency tables: one (X) in which each ET was
represented in terms of its allelic composition and a second one
(Y) in which the strains were organized into various groups
(sample origin and serotype) (23-25). The independence of
X and Y was testing by the
approximation-to-permutation test.
| |
RESULTS |
MLEE gave clearly reproducible results. Identical results were
obtained when isolates were subcultured many times on Sabouraud's medium and when electrophoresis was performed with samples from every
fourth isolate passage.
Genetic diversity and taxonomy.
Twelve loci were polymorphic
in the 107 strains examined, and each polymorphic locus had three to
six alleles (Fig. 1). We identified 58 alleles giving 48 different ETs (Table
2). In the first FCA, strains formed
three clusters (Fig. 2) in the most informative plane (accounting for 26.78% of the total genetic variability). Cluster I consisted of one strain of C. laurentii, and the GPI 1 allele was responsible for this structure
(Table 2). Cluster II consisted of C. neoformans strains
only, and cluster III contained both C. neoformans and
C. laurentii strains. This projection shows the extensive
diversity of the C. neoformans species. The C. neoformans strains of cluster II are separated from those of
cluster III by axis 1, which is the most informative (15.78% of the
total variability). This shows that the strains from the various
clusters have very different genotypes. The inclusion of C. laurentii strains in a C. neoformans cluster suggests
that these species are genetically related and that there is greater genotype diversity in the C. neoformans sample than in the
C. laurentii sample. The alleles responsible for this
structure are indicated in Table 2 and are confirmed in Table
3 because the least frequent alleles
are the most discriminatory.
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FIG. 2.
First plane projection of FCA in two informative axes of
all ETs observed. Axes 1 and 2 represent 15.78 and 11.00% of the
overall variability, respectively. ETs represent the observed ET (Table
2). The ET group at the upper left (cluster III) includes C. laurentii strains.
|
|
We investigated genotype differences between the ETs of cluster III
using a second FCA. We obtained three clusters from the
original group
III (Fig.
3). Clusters III-1 and III-2
are separated
by axis 1 (accounting for 17.31% of the total
variability). Cluster
III-1 contains exclusively human serotype B
strains, and cluster
III-2 contains only human serotype C strains.
Cluster III-3 is
separated from the other two clusters by axis 2 (accounting for
15.36% of the total variability) and consists of both
C. neoformans (serotypes A, B, C, D, and A/D) and
C. laurentii strains. PEP
A5 (the numbers after the locus designation
indicate allele numbers)
was present only in isolates from cluster
III-1, and ADH 3 was
present only in those from cluster III-2 (Table
2).
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FIG. 3.
First plane projection of FCA in two informative axes of
ETs of cluster III (Fig. 2). Axes 1 and 2 represent 17.31 and 15.38%
of the overall variability, respectively. ETs represent the observed
ETs (Table 2). The ET group at the lower (cluster III-3) includes
C. laurentii strains.
|
|
Genetic structure and serotype.
A CCA was carried out to
identify a relationship between genetic structure and serotype. No link
between serotype and genotype was found, and the permutation test
revealed no significant relationship between genetic structure and
serotype (P > 0.1). Serotypes A, D, and A/D are
genetically similar, so we put them in the same cluster, in agreement
with previous studies (22). We considered three serotyping
groups (B, C, and A plus D plus A/D) (Fig.
4), and a correlation between the
genotype structures of the strains and their serotypes was identified
on the basis of a permutation test (P < 0.02).
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FIG. 4.
First plane projection of the CCA in two informative
axes of all observed ETs. Axes 1 and 2 represent 26.14 and 23.64% of
the overall variability, respectively. ETs are grouped as a function of
their serotypes. The ETs in boldface type are those of serotype A, D,
and A/D strains, the ETs at the upper left are those of serotype B
strains, and the ETs at the lower left are those of serotype C
strains.
|
|
Genetic structure and origin.
A second CCA showed a clustering
of the sample after the correlation between allelic composition and
sample origin (patients and the environment) was tested. This was
confirmed by a permutation test (P < 0.02). Isolates
were grouped into three clusters on the projection (Fig.
5). One cluster contained strains
isolated from both patients and the environment, the second contained
strains from patients only, and the third contained strains from the
environment only.
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FIG. 5.
First plane projection of CCA in two informative axes of
the observed ETs of C. neoformans. Axes 1 and 2 represent
52.43 and 47.51% of the overall variability, respectively. ETs are
grouped as a function of the sample origin. Boxed ETs represent those
of human strains, the shaded ETs at the lower right represent those of
environmental strains, and the ETs in boldface type represent those of
human or environmental strains.
|
|
| |
DISCUSSION |
We carried out a genetic analysis of 107 strains of
Cryptococcus. The results obtained showed that there is
extensive variation in this fungus, as reported in several other
studies (1, 2, 8, 9, 11, 12, 17). We found 48 allele
combinations among 107 isolates by studying 12 polymorphic loci. The
level of diversity observed was similar to that reported in previous studies (4, 5) and was due to the large number of enzymes tested and the extensive heterogeneity of the strains studied. MLEE is
among the numerous typing methods developed for the differentiation of
C. neoformans isolates. It has a relatively high degree of discriminatory power but also allows assessment of the structure of the
sample studied (4), including its genetic diversity. It was
therefore used as the main technique for the present study.
Genetic diversity and taxonomy.
The first FCA (Fig. 2)
produced three clusters. ET 7 consisted of one strain of C. laurentii. Cluster II contained only C. neoformans
strains, whereas cluster III contained both C. neoformans and C. laurentii strains. There was more genetic divergence
between some strains of C. neoformans than between C. laurentii and some strains of C. neoformans. The
C. neoformans strains from clusters II and III differ more
from each other than the C. neoformans and C. laurentii strains of cluster III do. Therefore, these fungi from
different species are presumably closely related genetically.
A second FCA (Fig.
3) was used to identify differences between the ETs
of cluster III. Most human strains of serotypes B and
C were well
separated on the projection. Serotypes A, D, and A/D
were grouped
together, and
C. neoformans and
C. laurentii were
not always separated on this projection. This raises important
taxonomic questions about the notion of species because
C. neoformans and
C. laurentii have very similar
genotypes. The most informative
enzyme systems (FUM, SDH, ADH, PGM, and
PEP A) should be tested
first to type an isolate and to determine to
which cluster it
belongs, but these enzymes cannot discriminate between
some of
the
C. neoformans and
C. laurentii
strains used in this
study.
Correlation between genotype and serotype.
Several studies
have shown that multilocus genotypes correlate with variety (C. neoformans var. neoformans and C. neoformans var. gattii) and serotype (5, 6). However, the
CCA, which tested for a possible link between serotype and genotype,
detected no correlation when strains of the five serotypes (serotypes
A, B, C, D, and A/D) were considered separately. This absence of a
correlation was also confirmed by a nonsignificant value obtained in
the approximation-to-permutation test (P > 0.1). If
serotypes A, D, and A/D, which are clustered together in the FCA (Fig.
2), were placed in the same group, there was a correlation between the
serotype and the genotype for the three resulting serotype groups (Fig.
4). Thus, MLEE cannot differentiate between serotypes A, D, and A/D,
indicating that these serotypes are closely related genetically. Some
studies (4, 6) have shown that MLEE differentiates serotypes
A, D, and A/D and consider them to be genetically different. The
results of our work are consistent with the preliminary results of
other studies (27) indicating that strains readily switch from serotype A to serotype D via the intermediate serotype A/D.
Correlation between genotype and strain origin.
CCA for the
genotypes and origins of the strains provided completely new
information. The sample gave three clusters on the projection. There
was a strong relationship between the genotypic information and the
origin of the strain, in contrast to the results of other studies
(13). This was confirmed by the significant value obtained
in the permutation test (P < 0.02). The first cluster, which was genetically diverse, exclusively contained strains isolated from patients. The second cluster contained strains only from the
environment (Fig. 5). The third cluster contained strains isolated
either from patients or from the environment. These results are of
epidemiological importance, because most of the environmental strains
had particular isoenzyme profiles which were different from those of
strains from patients. So, the genotypes of the strains were correlated
with their origins. Some studies (11, 12) have demonstrated
karyotype changes in Cryptococcus after multiple infections
in the mouse. This suggests that the fungus acquires a characteristic
genotype after infection. Our data suggest that environmental strains
are only slightly pathogenic, if at all, for humans. The genotypes of
the strains responsible for infection and those isolated from the
environment were very different.
Thus, MLEE is a powerful discriminatory method. Analysis of the
relationship between ETs should improve our understanding
of the
genetic polymorphism and the genotypic similarity of clinical
and
environmental
C. neoformans isolates. This should help us
to
understand the transmission of this parasite and identify the
risk
factors for contamination so that more effective prevention
measures
can be
implemented.
| |
FOOTNOTES |
*
Corresponding author. Present address:
Laboratoire d'Immunologie et Parasitologie, Faculté de
Pharmacie, 15, Av. Charles Flahault, 34060 Montpellier Cédex
2, France. Phone: 33.4.67.63.52.02. Fax: 33.4.67.41.16.17. E-mail:
Jean-Marie.Bastide{at}pharma.univ-montp1.fr.
| |
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Journal of Clinical Microbiology, March 1999, p. 715-720, Vol. 37, No. 3
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
