Medical Mycology 2001, 39, 439±444
Accepted 31 October 2000
Analysis of restriction pro®les of mitochondrial DNA
from Sporothrix schenckii
M. MORA-CABRERA*, R. A. ALONSOy, R. ULLOA-ARVIZUy & H. TORRES-GUERRERO*
*Departamento de Microbiologia y Parasitologia, Facultad de Medicina, Universidad Nacional Autonoma de Mexico, Mexico City,
Mexico; yLaboratorio de Genetica Molecular, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autonoma de
Mexico, Mexico City, Mexico
Mitochondrial DNA (mtDNA) diversity was analyzed in 42 clinical isolates of
Sporothrix schenckii from México (n ˆ 29), Guatemala (n ˆ 4) and Colombia (n ˆ
9). Based on HaeIII restriction digestion proŽles, the isolates were classiŽed into
eight types. In addition to 24 mtDNA types previously reported in another study, 6
new types were found in this study. Most of the strains belong to type 14 and type 30,
the former restricted to Mexico, whereas the latter was distributed in Mexico,
Guatemala and Colombia. The new types (25–30) were identiŽed in Mexico,
Guatemala and Colombia. Restriction-fragment length polymorphism in mtDNA of
S. schenckii revealed high levels of genetic variation attributable to differences in
restriction sites as well as in mtDNA size. Based on genetic distances S. schenckii
types were clustered into two main groups.
Keywords
mitochondria, RFLP, Sporothrix schenckii, sporotrichosis
Introduction
Sporotrichosis is a subacute or chronic disease caused by
Sporothrix schenckii. The infection is usually limited to
the skin, subcutaneous tissue, and lymphatic glands.
Sporotrichosis has a worldwide distribution in temperate,
warm and tropical areas [1]. There is regional difference
in incidence of this fungal infection as it is the most
common mycosis in the central highlands of Mexico
[2,3].
IdentiŽcation of restriction-fragment-length polymorphisms (RFLP) in mitochondrial DNA (mtDNA)
has been used as a powerful tool for epidemiological
analysis of microbial pathogens [4–6] and as a sensitive
indicator of genetic relatedness or divergence within
taxonomic groups [7–10].
Isolates of S. schenckii have been classiŽed into 24
different RFLP types based on mtDNA restriction
patterns [8,11–14] and clustered into 2 groups based on
genetic distances [12]. The diversity of mtDNA from
different geographical origins described previously
ã
Correspondence: Haydee Torres-Guerrero, Departamento de
Microbiologia y Parasitologia, Facultad de Medicina, Universidad
Nacional Autonoma de Mexico, Mexico City, 04510, Mexico.
Tel.: (52) 5623-2305; fax: (52) 5623-2382; e-mail: [email protected]
2001 ISHAM
ISHAM, Medical Mycology, 39, 439±444
prompted us to work on the analysis of mtDNA in S.
schenckii isolates. Here we describe the mtDNA types
and the phylogenetic relationship between isolates from
Mexico, Guatemala, and Colombia, and compare them
with 20 types described previously [11,12]. Recently four
new types (21–24) were identiŽ ed by Kawasaki et al. [13]
and Ishizaki et al. [14]. These types, however, could not
be compared in this work.
Material and methods
The Mexican isolates used in this study were kindly
provided by M.C. Alejandra Espinosa, Universidad
Autonoma de Puebla, Mexico; Roberto Arenas, Hospital General Manuel Gea Gonzalez, Mexico City; and
Jorge Mayorga from Instituto Dermatologico, Jalisco,
Mexico. Dra. Concepcion Toriello, Facultad de Medicina-UNAM, Mexico City, provided the strains from
Guatemala and Dra. Angela Restrepo, Centro de
Investigaciones Biologicas, Medellin, Colombia, provided the strains from Colombia.
Isolation of total DNA
S. schenckii was cultured in liquid YEPD (1% yeast
extract, 2% peptone and 2% glucose) for 48 h at 28 oC in
440
Mora-Cabrera et al.
a water bath with constant shaking. Cells were collected
and suspended in 1 M sorbitol supplemented with lyticase
(Sigma Chemical Co., St. Louis, MO, USA) at a
concentration of 0¢5 mg ml¡1. After incubation at 37
o
C, spheroplasts were collected by centrifugation,
washed with 1 M sorbitol and suspended in 10 mM Tris,
1 mM ethylenediaminetetraacetic acid (EDTA) (pH 8¢0),
0¢1% sodium dodecyl sulfate (SDS) (Sigma). DNA was
obtained after phenol extraction and ethanol precipitation. The precipitate was suspended in TE (10 mM Tris, 1
mM EDTA, pH 8¢0). All DNA samples were stored at
¡20 oC.
Miller [15]. The average number of nucleotide substitutions per site (d) was estimated with the following
equation:
d ˆ ¡(2/r) ln (P)
where P is the probability of no mutation occurring at
restriction sites with r nucleotides, and this was obtained
by iteration of:
P ˆ [F(3 ¡ 2Pi)]1/4
where F is the band sharing index and P1 ˆ F1/4. Once dij
was estimated, the neighbor-joining methodology was
used for reconstruction of one unrooted tree [16].
Isolation of mitochondrial DNA
Mitochondrial DNA was obtained from S. schenckii
isolate EH217 as described previously [10]. Briey, cells
were disrupted by homogenization (B. Braun, Melsungen, Germany) for 30 s. Mitochondria were puriŽed from
the supernatant obtained after centrifugation at 1600g
for 10 min, followed by another centrifugation at 20 000g
for 30 min. The pellet was suspended in 100 mM Tris, pH
8¢0, 200 mM NaCl, extracted with phenol, phenol/
chloroform and chloroform. The DNA was precipitated
with two volumes of ethanol, washed with 70% ethanol,
dried, suspended in TE and quantiŽ ed.
Results
Previous work by Ishizaki et al. [11,12,14] and Kawasaki
et al. [13] demonstrated that clinical isolates could be
separated into 24 types on the basis of HaeIII restriction
digest patterns of puriŽed mtDNA. In this work, we have
used an alternative method, which involves analysis of
HaeIII digests of whole cell DNA and Southern blot
hybridizations, using puriŽed mtDNA as probe.
Labeling of mtDNA
PuriŽed mtDNA was used as probe for hybridization on
total genomic DNA. The molecule was labeled with
32
PdATP by random priming as described in the
Random primer kit (Roche Molecular Biochemicals,
Indianapolis, IN, USA) and Žltered through a Sephadex
G-50 column (Sigma).
Restricition endonuclease digestion, gel electrophoresis
and Southern hybridizations
Total S. schenckii genomic DNA was digested with the
restriction endonucleases HaeIII, MspI and HhaI
(Roche Molecular Biochemicals) according to the
manufacturer’s recommendations. The DNA fragments
were separated in 1% agarose gels in 1£ TAE buffer,
transferred to Hybond-N‡ Žlters (Amersham International, Buckinghamshire, UK) and hybridized to 32Plabeled mtDNA at 42 oC, overnight. Filters were washed
in 0¢1X SSC, 0¢1% SDS at 42 oC and exposed to Kodak
XAR-5 Žlm (Eastman Kodak Co., Rochester, NY, USA)
at ¡70 oC with intensifying screens.
Genetic distances analysis
Quantitative differences in restriction fragment patterns
of mtDNA were estimated by the method of Nei &
Fig. 1 Hybridization patterns obtained by using mtDNA labeled
with 32 P-dATP on total DNA restricted with HaeIII. Lane M
corresponds to DNA size markers (l EcoR1/HindIII). Lane 1, type
30; lane 2, type 8; lane 3, type 14; lane 4, type 25; lane 5, type 26;
lane 6, type 27; lane 7, type 28; lane 8, type 29.
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2001 ISHAM, Medical Mycology, 39, 439±444
Sporothrix schenkii mtDNA restriction pro®les
In order to analyze the variability of the mitochondrial
genome, a total of 42 different S. schenckii isolates were
evaluated by RFLP. These isolates were obtained from
patients in Mexico (n ˆ 29), Guatemala (n ˆ 4) and
Colombia (n ˆ 9). The strains were classiŽed according
to 20 previously described types [11,12]. After HaeIII
digest, the 42 strains revealed eight different restriction
proŽles (Fig. 1), including types 8 and 14 which had been
previously reported, and the new types 25 to 30. No
further subtypes were identiŽ ed after digestion with
HhaI and Msp1 endonucleases. The length of the S.
schenckii mitochondrial genome was estimated to range
14–27 kb (Fig. 1), based on the mtDNA fragments
generated by three endonucleases. The mtDNA restriction with HaeIII generated 7–8 bands, some of which
were common among the eight mtDNA types (1¢25, 0¢75
and 0¢7 kb). Types 14 and 26 shared four additional
bands of 7, 4¢8, 4¢5 and 2¢9 kb; however, a 3¢6 kb band
present in type 14 was absent in type 26. Types 25, 28, 29
and 30 shared the 2¢9 and 3¢6 kb bands. Type 25 has a 5¢1
kb band that was not present in any of the other types
described in this work. Types 28, 29 and 30 shared three
Table 1
Type of
mtDNA
441
bands of 4¢5, 3¢6 and 2¢9 kb. The difference among them
is that type 29 has a 9 kb band, type 30 has a 7 kb band,
and type 28 lacks a high molecular weight band. Type 8
and type 27 share four bands, and both lack the 2¢9 kb
band, but type 27 has an extra band of 1¢6 kb.
New types of mtDNA were identiŽed in isolates from
Mexico (type 25 [n ˆ 1], type 26 [n ˆ 1] and 30 [n ˆ 7]),
Guatemala (type 30 [n ˆ 3]) and Colombia (types 27 [n
ˆ 2], 28 [n ˆ 1], 29 [n ˆ 1] and 30 [n ˆ 5]). This analysis
revealed that type 30 has a wide distribution, it was
identiŽed in isolates from the three countries, while type
14 was found with high frequency only in Mexican
isolates (Table 1).
An unrooted tree has been constructed on the basis of
the genetic distances among the different types (Fig. 2)
[16]. The dendrogram was obtained with the types
described in this work (25–30) and data on types 1–20
taken from previous reports [11,12]. In the tree the
mtDNA types were clustered into two main groups:
Group B contained most of the Japanese isolates and
Group A most of the American types. It is interesting
that Group A is more branched compared with Group B,
Distribution and frequency of mtDNA types of S. schenckii
Mexico*
1
–
2
–
3
–
4
–
5
–
6
–
7
–
8
3
9
–
10
–
11
–
12
–
13
–
14
17
15
–
16
–
17
–
18
–
19
–
20
–
25
1
26
1
27
–
28
–
29
–
30
7
Total strains 29
Guatemala* Colombia* USAy
–
–
–
–
–
–
–
1
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
3
4
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
2
1
1
5
9
Brazily
1
–
12
–
–
1 (A)
2
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
1 (B), 2 (C) –
1
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
19
1
Venezuelay Argentinay Costa Ricaz France§
Japan
–
–
13(B)
6
–
–
–
–
–
–
–
–
–
–
–
–
–
1
3
6
–
–
–
–
–
–
29
3§
10§
1§
54§
151§
20§
12§
1§
4§
1§
–
1z
1z
–
–
–
–
–
–
–
–
–
–
–
–
–
259z,§
–
–
2 (A)
4
–
–
–
–
–
–
–
–
–
–
–
4
3
–
–
–
–
–
–
–
–
–
13
–
–
1
–
–
1
–
–
–
–
–
–
–
19
–
–
–
–
–
–
–
–
–
–
–
–
21
–
–
–
–
–
–
–
–
–
–
1
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
1
*, This study; y, Ishizaki et al. [12]; z, Ishizaki et al. [11]; §, Takeda et al. [9]. Letters in parentheses (A, B, C) are the subtypes described by Ishizaki et al. [12].
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2001 ISHAM, Medical Mycology, 39, 439±444
442
Mora-Cabrera et al.
Fig. 2 Genetic distance tree obtained for restriction fragment
length polymorphisms of mtDNA. Genetic distances were calculated as in Nei & Miller [15] and the dendrogram was generated
with the neighbor-joining method [16].
where most of the types seems to be more closely
genetically related. The new types reported in this work
were clustered in Group A (type 26 28, 29 and 30) and
Group B (type 27).
Discussion
The diversity of mtDNA in S. schenckii is outstanding, as
is the fact that in a relatively small sample new groups
may still be identiŽ ed. HaeIII restriction of total DNA,
followed by hybridization with mtDNA, revealed eight
different mtDNA RFLP types, six of them not previously reported. The number of HaeIII fragments did
not vary substantially among most of the studied isolates;
however, the mitochondrial genome size ranged 14–27
kb. It has been reported that generation of mtDNA
diversity can involve point mutations, length mutations
and rearrangements [17,18]. It seems that the variability
in S. schenckii mtDNA is due in part to restriction site
changes; however, the variation in size could also
indicate length mutations. mtDNA of Saccharomyces
cerevisiae is characterized by clustering of G‡C-rich
regions [19] that contain recognition sites for restriction
enzymes such as HaeIII and MspI; these enzymes split
sequences which contain only G‡C residues. These
G‡C clusters have been identiŽ ed as potentially
involved in intramolecular recombination [17]. The
mitochondrial genome of S. schenckii may have some
G‡C-rich regions dispersed randomly in the genome,
and these could be involved in the mechanism producing
the size diversity observed. Surprisingly, when isolates of
each type were plated onto medium with glycerol
(YEPG), all of them grew as well as in the medium
with glucose (data not shown). This suggests that some
polymorphic long stretches of S. schenckii mtDNA are
not required for aerobic respiration.
The Žnding of two main different mtDNA genetic
groups revealed by our genetic distance analysis between
S. schenckii types, was consistent with previous reports
[11,12], where isolates from Japan were clustered in
Group B and types from America in Group A.
Comparison of the dendrogram published by Ishizaki
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2001 ISHAM, Medical Mycology, 39, 439±444
Sporothrix schenkii mtDNA restriction pro®les
ã
et al. [12] with the one obtained in this work showed
small differences in branching; however the distribution
of the mtDNA types of the two main groups is the same,
giving strength to our results. The difference found
among the dendrograms could be due to the different
methods of analysis used and to the sample size. The
neighbor-joining method was applied in this work
because it has been shown to reconstruct the original
tree [20] more accurately than the method of Fitch &
Margoliash [21].
In this and previous works, the amount of information
is rather small, and this could generate differences in
branching among dendrograms when new data are
incorporated. It should be noted that Group A is more
branched compared with Group B. This is not surprising
since most of the isolates clustered in Group B were
from Japan, suggesting a closer genetic relationship
among them. More variability is found in Group A,
probably because the isolates have different geographical origins, although most of them come from the
American continent.
The distribution of mtDNA types of S. schenckii and
its geographical origin is controversial, since only some
mtDNA types showed a consistent correlation between
their geographical origin and their group in the
dendrogram. Examples include types 14 and 18 which
appeared to be restricted to the American continent
(Group A) [11, this study]. In contrast, types 3 (Group
A) and 4 (Group B) are common in different areas,
suggesting a worldwide distribution [8,12]. Such Žndings
hamper the use of the mtDNA–RFLP type as an
unambiguous molecular marker of geographic origin;
however, mtDNA–RFLP has shown to be useful marker
in molecular epidemiology [10,22].
As was mentioned above, the samples used in this and
in previous studies have been limited in number, and
mtDNA types present at low frequency may be underestimated or not detected. The analysis of a larger
sample will improve our understanding of the relationship between the mtDNA types and the geographic
origins of the major and minor groups.
The molecular evolutionary explanations for the high
variability of S. schenckii mtDNA are not clear. It has
been suggested that this variability is ancient and
originated before and during the separation of the
present continents in continental drift (Panagea hypothesis), resulting in a worldwide distribution of at least
some of the different mtDNA types [11]. Experimental
data, however, are not currently available to support this
hypothesis. The evolutionary history of mtDNA of
S. schenckii will start to emerge with the molecular
study of other genetic characters such as DNA sequences
of mitochondrial and nuclear genes.
2001 ISHAM, Medical Mycology, 39, 439±444
443
Acknowledgements
This work was supported by grant IN207296 PAPIIT,
UNAM.
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