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Previous Article | Next Article 
Infection and Immunity, December 2003, p. 7109-7118, Vol. 71, No. 12
0019-9567/03/$08.00+0 DOI: 10.1128/IAI.71.12.7109-7118.2003
Copyright © 2003, American
Society for
Microbiology. All Rights Reserved.
Phenotypic Switching and Mating Type Switching of Candida glabrata at Sites of Colonization
Paula J. Brockert,1 Salil A. Lachke,1 Thyagarajan Srikantha,1 Claude Pujol,1 Rudolph Galask,2 and David R. Soll1*
Department
of Biological Sciences, The University of
Iowa,1
Department of Obstetrics and
Gynecology, The University of Iowa Hospitals and
Clinics, Iowa City, Iowa 522422
Received 20 June 2003/
Returned for modification 22 July 2003/
Accepted 28 August 2003
 |
ABSTRACT
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|---|
Candida
glabrata switches spontaneously at high frequency among the
following four graded phenotypes discriminated on agar containing 1 mM
CuSO4: white, light brown, dark brown (DB), and very dark
brown. C. glabrata also contains three mating type loci with a
configuration similar to that of the Saccharomyces cerevisiae
mating type cassette system, suggesting it may also undergo cassette
switching at the expression locus MTL1. To analyze both
reversible, high-frequency phenotypic switching and mating type
switching at sites of colonization, primary samples from the oral
cavities and vaginal canals of three patients suffering from C.
glabrata vaginitis were clonally plated on agar containing
CuSO4. It was demonstrated that (i) in each vaginitis
patient, there was only one colonizing strain; (ii) an individual could
have vaginal colonization without oral colonization; (iii) phenotypic
switching occurred at sites of colonization; (iv) the DB phenotype
predominated at the site of infection in all three patients; (v)
genetically unrelated strains switched in similar, but not identical,
fashions and caused vaginal infection; (vi) different switch phenotypes
of the same strain could simultaneously dominate different body
locations in the same host; (vii) pathogenesis could be caused by cells
in different mating type classes; and (viii) mating type switching
demonstrated at both the genetic and transcription levels occurred in
one
host.
|
INTRODUCTION
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The extraordinary success of Candida albicans as both a
commensal and an opportunistic pathogen is due in part to the
phenotypic plasticity it derives from three developmental programs, the
bud-hypha transition (9,
15,
21), spontaneous
high-frequency phenotypic switching
(23,
24,
26), and a recently
discovered mating system
(4,
29). Until recently,
however, the second most successful Candida pathogen,
Candida glabrata, seemed devoid of these three developmental
programs. Like C. albicans, C. glabrata is a
relatively successful commensal
(15), particularly in the
elderly (13), and causes
recurrent vaginitis (2)
and bloodstream infections
(16). If the
developmental programs of C. albicans indeed play critical
roles in commensalism and pathogenesis, how could C. glabrata
mimic C. albicans without these programs? The answer that has
emerged in the past few years is that C. glabrata does in fact
possess similar developmental programs. Although C. glabrata
does not form compartmentalized hyphae, it does grow in the
pseudohyphal growth form
(1,
7) and extends tubes up to
four cell diameters in length
(7). It also undergoes two
forms of high-frequency phenotypic switching. First, it switches among
the following four colony phenotypes, colored in a graded fashion on
CuSO4-containing agar: white (Wh), light brown (LB), dark
brown (DB), and very dark brown (vDB)
(7,
8). These switch
phenotypes also show gradation in the frequency of switching,
coloration on phloxine B-containing agar, expression of the
metallothionein gene MT-II
(7,
8), and expression of the
mating type gene MTL
1
(31). This graded,
high-frequency switching system is referred to as the
"core" switching system. C. glabrata also
switches reversibly and at high frequency between the smooth colony
phenotypes of the core switching system and an irregular wrinkle
phenotype (7). Finally,
C. glabrata has recently been demonstrated to possess three
mating type-like loci (MTL1, MTL2, and MTL3)
with a configuration similar to that of Saccharomyces
cerevisiae (31,
32). Of the three mating
type-like loci, two (MTL2 and MTL3) appear to contain
silent a and cassettes, respectively, and the third
(MTL1), the putative expression locus, contains either an
a or an cassette that defines the mating type of the
cell (i.e., either a or ). In S. cerevisiae,
mating type switching involves a change in the expression cassette from
a to or to a by gene conversion with
a copy of the alternate silent donor cassette
(3).
Here, we have
examined whether core switching and mating type switching occurred at
sites of carriage in three patients presenting with vaginal C.
glabrata infections. In the reported studies, we tested (i)
whether these patients were colonized in both the oral cavity and
vagina, (ii) whether the same strain colonized both anatomical
cavities, (iii) whether high-frequency phenotypic switching occurred at
sites of colonization, (iv) whether a particular switch phenotype
dominated sites of infection, (v) whether a single mating type
dominated sites of infection, and (vi) whether mating type switching
occurred in the host. The results demonstrate that switching occurs at
sites of infection, that different switch phenotypes of the same strain
can prevail in different anatomical locations in the same host, and
that mating type switching can occur in the
host.
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MATERIALS AND
METHODS
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Isolation and maintenance of C.
glabrata strains.
C. glabrata isolates were
individually obtained from underneath the tongues, the inner cheeks,
the vaginal canals, and the vaginal pools of three vaginitis patients,
using sterile cotton swabs (Culturette; Becton Dickinson and Co.,
Sparks, Md.). The three patients exhibited symptoms in the vagina but
no signs of infection in the oral cavity. Each of the three patients
had previously been diagnosed with C. glabrata infections
based on the sugar assimilation patterns of the sampled yeast. For
assessing the intensity of colonization and for distinguishing switch
phenotypes, a strategy similar to one previously described for C.
albicans (27) was
used. Each cotton swab, containing a primary sample, was agitated in
sterile water to release yeast cells. These samples were then clonally
plated on YPD agar (1% yeast extract, 2% peptone,
2% dextrose, 2% agar) plates containing 1 mM
CuSO4 (8). The
plates were incubated at 25°C for 7 days, and the total number
of colonies, as well as the proportions exhibiting the different switch
phenotypes (Wh, LB, DB, and vDB), were scored. Isolates were verified
as C. glabrata first by testing cells from primary colonies
with the Vitek YBC system (bioMerieux Vitek Inc., Hazelwood, Mo.) and
then by DNA fingerprinting them using Southern blot hybridization with
the species-specific probe Cg6
(12).
"Colonization" refers to the presence of yeast
regardless of the disease state. "Carriage" refers to
commensal colonization. "Infection" refers to
colonization causing vaginitis
symptoms.
Measurements of phenotypic
switching.
Measurements of
the frequency of phenotypic switching of cells from primary colonies
were obtained as previously described
(8). Cells from a primary
colony visually homogeneous for a single switch phenotype were
inoculated into liquid YPD medium containing 1 mM CuSO4
(YPD-CuSO4) and grown at 25°C until they reached a
concentration of 
5 x 106 cells per ml. The
cells were then diluted and evenly distributed on agar plates
containing YPD-CuSO4 medium at a density of 50 to 70 CFU per
plate. The plates were incubated at 25°C for 5 to 7 days and
then scored for the proportions of the different switch
phenotypes.
DNA fingerprinting and
computer-assisted analysis.
DNA fingerprinting was performed
according to methods previously described
(12,
17,
25). Total genomic DNA
from each C. glabrata isolate was isolated using the methods
of Scherer and Stevens
(18). Three micrograms of
DNA was then digested overnight with EcoRI (4 U/µg of
DNA) for 16 h at 37°C, and the resulting DNA
fragments were separated in a 0.65% agarose gel at 45 mV.
Digested genomic DNA of the C. glabrata reference strain 7549
was run in the outer two lanes of each gel to facilitate
computer-assisted analysis of test strains run in the inner lanes
(25). The DNA was
transferred to a nylon membrane (Hybond N+; Amersham
Pharmacia Biotech, Little Chalfont, Buckinghamshire, England) by
capillary blotting, hybridized with randomly primed
[32P]dCTP-labeled Cg6 probe, and autoradiographed
(12).
Gel patterns
were analyzed according to methods previously described
(25,
28). Autoradiograms were
digitized into the data file of the DENDRON software program, version
2.0, using a Scanjet II cx flatbed scanner (Hewlett-Packard, Palo Alto,
Calif.). Distortions in the gels were removed with the unwarping option
of DENDRON, and the lanes and bands were identified automatically.
Southern blot hybridization patterns were compared using a similarity
coefficient (SAB) based on the band position for
every pair of patterns (samples) according to the following formula:
SAB = 2E/(2E +
a + b), where E is the number of
bands shared by strains A and B, a is the number of bands
unique to A, and b is the number of bands unique to B
(25). An
SAB of 1.0 represents identical patterns, and an
SAB of 0.00 represents patterns with no common
bands. Intermediate SABs in the range of 0.01 to
0.99 represent patterns with increasing proportions of common bands.
Dendrograms based on SAB values were automatically
generated by the DENDRON program based on the unweighted-pair-group
method (20). For
comparisons with collections of previously DNA-fingerprinted isolates,
SABs were computed from DENDRON band data files
(25).
Determination
of mating type class at the genetic and transcriptional
levels.
Southern analysis
employing specific probes was used to distinguish the three main mating
type classes of C. glabrata: class I (aa),
which contains MTLa genes at the MTL1 locus;
class II (a), which contains
MTL genes at the MTL1 locus; and class III
(a), which contains MTLa genes
at the MTL1 locus
(31). Genomic DNA of each
C. glabrata isolate was digested with XbaI at
37°C overnight. The resulting fragments were separated in a
0.8% agarose gel, transferred to a nitrocellulose membrane
(Hybond N+), and hybridized with one of the
following three radiolabeled probes: FuncP2, an antisense
oligonucleotide which binds to the MTL2 open reading
frame (ORF), which will be referred to as the MTL2
probe; a 395-bp PCR product spanning the 3' flanking region of
MTL1a2, which will be referred to as the
MTLa2 probe; and a 300-bp PCR product unique to the
MTLa1 ORF, which will be referred to as the
MTLa1 probe
(31). Northern blot
analyses were performed according to methods previously described
(31).
|
RESULTS
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Colonization
of the oral cavity and vaginal canal.
To assess yeast colonization, the cells
in primary samples collected on cotton swabs were suspended in buffer
and distributed evenly over the surface of an agar plate in order to
discriminate individual colonies. In patient P1, both the oral cavity
and the vaginal canal were colonized (Table
1). Colonization of the vagina was more intense than that of the oral
cavity, consistent with the vagina as the diagnosed site of infection.
In patient P2, only the vagina was colonized (Table
1). The samples from the
oral cavity contained no culturable Candida sp. cells (Table
1). In patient P3, both
the oral cavity and the vaginal canal were colonized, as in the case of
patient P1 (Table 1).
Colonization of the vaginal canal again was more intense than that of
the oral cavity, consistent with the vagina as the diagnosed site of
infection. These results demonstrate that women suffering from a
vaginal C. glabrata infection may or may not have concurrent
oral carriage.
Switch phenotypes in
primary cultures.
In vitro,
most strains of C. glabrata switch among Wh, LB, DB, and vDB
colony phenotypes, discriminated on agar containing 1 mM
CuSO4 (Fig.
1) (7,
8). As demonstrated in the
examples in Fig. 1,
plating cells from a clonal colony exhibiting a single switch phenotype
yields a majority of colonies with the original switch phenotype and a
minority with alternative (variant) phenotypes, as shown by the
frequencies of variant phenotypes presented along the arrows of
interconversion.
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FIG. 1. Core
switching system in C. glabrata. Cells switch spontaneously
among Wh, LB, DB, and vDB phenotypes discriminated on agar containing 1
mM CuSO4. In each box, low-magnification pictures of the
dominant phenotypes are presented with examples of switches (a and b in
parentheses). The frequencies of variant colony phenotypes are noted on
the arrows pointing to those variant
phenotypes.
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(i) Patient P1.
In the case of patient P1, 100%
of the colonies in the primary culture from the cheek exhibited the DB
phenotype (Table 1). In
marked contrast, 86% of the colonies in the primary cultures
from both the vaginal pool and the vaginal wall exhibited the DB
phenotype (Table 1). In
each of the last two cultures, 14% exhibited the vDB phenotype.
Based upon the frequency of variants in the vaginal samples, one would
have expected to find four vDB colonies in the oral sample if the
switching frequencies in the populations were equivalent. Using
Fisher's test, the difference between the proportions of
phenotypes in the oral cavity and vagina of patient P1 proved
significant (P = 0.012). The similarity in the
proportions of DB to vDB colonies in the vaginal-pool and vaginal-wall
samples (Table 1) attests
to the accuracy of the assay.
(ii) Patient
P2.
In the case of patient
P2, only the vagina was colonized. In both the vaginal-pool and
vaginal-wall samples, 100% of the primary colonies exhibited the
DB phenotype (Table
1).
(iii)
Patient P3.
In the case of
patient P3, a dramatic difference was observed between the proportions
of switch phenotypes in oral and vaginal isolates. Ninety-five and
93% of the primary colonies of the cheek and under-tongue
samples, respectively, exhibited the Wh phenotype, and 5 and 7%,
respectively, exhibited the DB phenotype (Table
1). Pictures of primary
cultures from the cheek sample (Fig.
2A and
B) and from the under-tongue sample (Fig.
2C and D) are presented.
In marked contrast, only 3 and 2% of the primary colonies of the
vaginal-pool and vaginal-wall samples, respectively, exhibited the Wh
phenotype, while 97 and 98%, respectively, exhibited the DB
phenotype (Table 1). Using
Fisher's test, the difference between the proportions of
phenotypes in the oral cavity and the vagina of patient P3 proved
highly significant (P = 2 x
10-100). Pictures of primary cultures from the
vaginal pool and vaginal wall are presented in Fig.
2E and F, respectively.
Hence, while the dominant phenotype in the oral cavity of patient P3
was Wh, the dominant phenotype in the vagina was DB.
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FIG. 2. For
patient P3, the Wh phenotype dominated in the oral cavity (cheek and
under tongue), while the DB phenotype dominated in the vaginal canal
(vaginal wall and vaginal pool). The low-magnification images are of
primary colonies plated directly from the original samples (swabs). (A
and B) Primary colonies of cheek sample (the arrows point to minority
DB colonies). (C and D) Primary colonies of under-tongue sample (the
arrow points to a DB colony). (E) Primary colonies of
vaginal-pool sample. (F) Primary colonies of vaginal wall
sample.
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The results
of the analysis of primary colony phenotypes revealed several
characteristics of C. glabrata colonization. First, colonizing
populations can be phenotypically homogeneous or heterogeneous. Second,
the DB phenotype dominated the site of infection (the vagina) in all
three patients, although DB could also be found in the oral cavity.
Third, the oral cavity may or may not be colonized in C.
glabrata vaginitis patients. Fourth, different switch phenotypes
can dominate the oral cavity and vagina of the same
patient.
Only one strain colonizes each
patient.
We assumed in the
plating experiments that the cells colonizing the oral cavity and the
vagina in the same patient represented a single strain and hence that
cells exhibiting different switch phenotypes in the same anatomical
location or the same host represented the same strain. To test this
assumption, cells from primary colonies sampled from different body
locations of the same patient and cells from different colony
phenotypes were DNA fingerprinted by Southern blot hybridization with
the complex species-specific probe Cg6
(12).
(i)
Patient P1.
Cells originating
from a primary DB colony isolated from under the tongue, from primary
DB and vDB colonies isolated from the vaginal pool, and from primary DB
and vDB colonies isolated from the vaginal wall of patient P1 were DNA
fingerprinted. All of the isolates exhibited similar patterns, but
there were minor differences (Fig.
3A). The hybridization pattern of the DB isolate from under the tongue
(DB-T) exhibited two major band differences from the patterns of the
vaginal-wall and vaginal-pool isolates (Fig.
3A). However, when the
isolates from patient P1 were compared with the patterns of the
isolates from patients P2 and P3 in a dendrogram based on similarity
coefficients, the oral and vaginal isolates of patient P1, including
that of DB-T, clustered (Fig.
4), and when compared with the patterns of an additional collection of
unrelated C. glabrata isolates, all of the patient P1 isolates
still clustered as a single genetically related group (Fig.
5). No isolates from the general collection penetrated this group (Fig.
5). These results
demonstrate that the isolates from the oral cavity and vagina of
patient P1 represent the same strain of C. glabrata. These
results also demonstrate that the major switch phenotypes DB and vDB in
the primary samples represented the same strain. Finally, these results
demonstrate that the strain colonizing patient P1 had undergone
microevolution.
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FIG. 3. Southern
blot hybridization with the DNA-fingerprinting probe Cg6 demonstrated
that only one strain of C. glabrata colonized each patient,
that the same strain colonized both the oral cavity and the vaginal
canal of each patient, and that the switch phenotypes represented the
same strain in each individual. 7549 is a reference strain for
normalization in computer-assisted analyses. T, under-tongue sample; C,
cheek sample; W, vaginal-wall sample; P, vaginal-pool sample. Wh1 and
Wh2 are two independent primary Wh colonies from the same patient. The
molecular masses on the left of the blots are in
kilodaltons. Note the examples of microevolution in DB-T of
patient P1, DB-P of patient P2, and Wh-P of patient
P3.
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FIG. 4. Dendrogram
generated for the DNA-fingerprinted isolates of patients P1, P2, and
P3. To generate the dendrogram, the similarity coefficients between all
pairs of the DNA-fingerprinted patterns of isolates from patients P1,
P2, and P3 were computed. Note that the isolates from each patient form
a single cluster, indicating that each patient's collection
represented an independent single strain of C. glabrata. See
the legend to Fig. 3 for
an explanation of the isolate names. The dashed line represents the
average SAB computed among all isolate
pairs.
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FIG. 5. Dendrogram
generated for the DNA-fingerprinted isolates from patients P1, P2, and
P3 and a large collection of unrelated C. glabrata isolates.
Note that even in this larger dendrogram, the clusters of P1, P2, and
P3 isolates remain intact, supporting the conclusion that each patient
is colonized by a single strain of C. glabrata.The dashed line represents the average SAB computed
among all isolate pairs for patients 1, 2, and
3.
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(ii) Patient P2.
Cells originating from a primary DB
colony from the vaginal pool and a secondary vDB colony emanating from
a primary DB colony from the vaginal pool, as well as from a primary DB
colony from the vaginal wall and a secondary vDB colony emanating from
a primary DB colony from the vaginal wall, were DNA fingerprinted. The
hybridization patterns of three of the four isolates (DB-W, vDB-W, and
vDB-P) were identical, while that of the fourth (DB-P) differed by a
single band change (Fig.
3B). When compared to the
patterns of isolates from the other two patients (Fig.
4) and when further
compared to the patterns of the additional collection of unrelated
isolates (Fig. 5), the P2
isolates clustered into a group not penetrated by any other isolate in
the collections, demonstrating that the DB isolates from the vagina
represented a single strain. The results also revealed microevolution
in the infecting population.
(iii) Patient
P3.
Seven of eight primary Wh
and DB isolates from the cheek (Wh1-C, Wh2-C, and DB-C), under the
tongue (Wh-T and DB-T), the vaginal pool (DB-P), and the vaginal wall
(Wh-W and DB-W) exhibited identical DNA fingerprinting patterns (Fig.
3C). One primary isolate
(Wh-P) exhibited a pattern with two band differences (Fig.
3C). A secondary vDB
colony emanating from a primary DB colony of the vaginal pool (vDB-P)
had the same DNA fingerprint as the majority of isolates (Fig.
3C). When compared in
mixed dendrograms to the patterns of isolates from the other two
patients (Fig. 4) and when
further compared to the patterns of the additional collection of
unrelated isolates (Fig.
5), the P3 isolates
coclustered, again demonstrating that they represented the same strain.
These results also revealed microevolution.
Together, these
results demonstrate that one strain colonized both the oral cavity and
the vagina of each of the three patients, that the different colony
phenotypes in the same host and in the same anatomical cavity of that
host represent switch phenotypes of the same strain, and that there was
microevolution in all three colonizing strains. The mixed dendrogram in
Fig. 5 further revealed
that relatively unrelated isolates of C. glabrata from
different deep-rooted groups caused vaginal candidiasis and underwent
switching between core
phenotypes.
Switching characteristics of
primary isolates.
To confirm
that multiple colony phenotypes in the same anatomical location or the
same host were in fact the result of switching, we analyzed the
switching characteristics of primary isolates. A mixture of cells from
two individual primary colonies with the same switch phenotype and from
the same anatomical location were plated onto agar containing 1 mM
CuSO4, and the frequencies and types of the variant colonies
formed were measured. In the case of patient P1, primary DB cells
switched primarily to the vDB phenotype at frequencies ranging between
6.7 x 10-3 and 2.6 x
10-2 (Table
1). Primary vDB isolates
switched to LB at lower frequencies of 6.2 x
10-4 to 8.7 x 10-4 (Table
1). An example of a switch
from vDB to LB is presented in Fig.
6A. In the case of patient P2, primary DB cells switched primarily to vDB
at frequencies ranging from 3.2 x 10-2 to
4.3 x 10-2 (Table
1). An example of a vDB
sector in a DB colony is presented in Fig.
6B. In the case of patient
P3, primary Wh cells switched to DB at frequencies ranging between 3.4
x 10-3 and 4.4 x
10-3, and primary DB cells switched to Wh at
frequencies ranging between 1.2 x 10-3 and
5.2 x 10-3. Examples of switches from Wh to
DB and from DB to Wh for cells from patient P3 are presented in Fig.
6C and D, respectively. To
demonstrate that all cells are capable of switching, secondary colonies
generated from cells of primary Wh colonies obtained from
the under-tongue samples from patient P3 were plated and examined at
different times. Early in development (6 days), the colonies were
uniformly Wh (Fig. 6E),
while later in development (12 days), each colony exhibited multiple
sectors of the other variant phenotypes (Fig.
6F). Together, these
results demonstrate that cells of all primary phenotypes switched
spontaneously to other phenotypes in the core switching system and
suggest that the multiple phenotypes in a patient arose through
high-frequency, reversible phenotypic
switching.
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FIG. 6. Examples
of in vitro switching. Cells from primary colonies were replated
(secondary colonies). (A) Colonies from primary DB-VP
colonies of patient P1 (the arrowhead points to switch to vDB sector
and the arrow points to switch to LB colony). (B) Colonies
from primary DB-VP colonies of patient P2 (the arrow points to switch
to vDB sector). (C) Colonies from primary Wh-C colonies of
patient P3 (the arrow points to switch to DB colony). (D)
Colonies from primary DB-VP colonies of patient P3 (the arrow points to
switch to Wh). (E) Colonies from primary Wh-C colonies of
patient P3 secondary plating incubated for 6 days (note no visible
sectoring). (F) Colonies from primary Wh-C colonies of
patient P3 secondary plating incubated for 12 days (note sectoring in
every
colony).
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Mating type classes and
switching.
Recently,
Srikantha et al. (31)
identified and characterized the mating type loci (MTL1,
MTL2, and MTL3) of C. glabrata. Based on the
MTL gene distribution among the three loci in a collection of
independent C. glabrata isolates, four classes were
distinguished (15). The
three major classes, I, II, and III, accounted for 97% of all
tested isolates. The three classes were readily distinguishable by
Southern blot hybridization with an antisense oligonucleotide that
hybridizes specifically to the 5' end of the
MTL2 ORF, referred to as the MTL2
probe (Fig.
7A) (31). Each class
exhibited a distinct hybridization pattern with the
MTL2 probe. For class I cells (reference strain
7549), which contained MTLa1 and MTLa2
at the putative expression locus MTL1, the
MTL2 probe hybridized only to the MTL3 band;
for class II cells (reference strain PB921), which contained
MTL1 and MTL2 in the MTL1
locus, the MTL2 probe hybridized to the MTL1
and MTL3 bands; and for class III cells (reference strain
J432285), which contained MTLa1 and
MTLa2 at the MTL1 locus, FuncP2 hybridized to
the MTL3 and MTL2 bands
(31) (Fig.
7A). To assess the mating
type class of the strains colonizing patients P1, P2, and P3, Southern
blots were probed with the MTL2 probe for the same
sets of isolates that had been DNA fingerprinted with the complex probe
Cg6 (Fig. 3). Four of the
5 P1 isolates exhibited the class I pattern (Fig.
7B), all 4 of the P2
isolates exhibited the class II pattern (Fig.
7C), and all 10 of the P3
isolates exhibited the class I pattern. Hence, class I cells (patient
P1 and P3 strains) contained MTLa1 and
MTLa2 in their putative expression locus MTL1
and were therefore tentatively classified as mating type a
cells, while class II cells (patient P2 strain) contained
MTL1 and MTL2 in their expression
locus MTL1 and were therefore tentatively classified as mating
type cells. These results, therefore, indicate that
pathogenesis (vaginal infection) is not limited to a single mating
type. The results also demonstrate that in each host, the majority of
cells exhibited the same mating type. The single exception was a vDB-P
isolate from patient P1 (Fig.
7B). This single isolate
exhibited a class II pattern, while the other four isolates from
patient P1 exhibited a class I pattern, suggesting a mating type switch
through gene conversion at the MTL1 locus. DNA fingerprinting
of vDB-P demonstrated that it was closely related to the other P1
isolates (Fig. 3C,
4, and
5), supporting the
conclusion that a mating type switch had occurred in the host at the
MTL1 locus. To verify both the mating type and mating type
switching, the Southern blots of the P1 isolates were hybridized first
with a probe unique to the MTL1a2 5' flanking
sequence and then with the MTLa1 ORF
(31). The probe for the
MTL1 locus hybridized to a single MTL1 band that was
of the same molecular weight in class I and class III isolates but of
slightly higher molecular weight in class II isolates due to the fact
that the MTL1 sequence is 185 bp longer
than the MTL1a sequence (Fig.
8A). The four class I isolates from patient P1 exhibited the class I
MTL1a band position, while the one class II isolate
from patient P1, vDB-P, exhibited the class II MTL1
band position (Fig. 8B).
The probe for MTLa1 hybridized with the MTL1
and MTL2 bands for all class I isolates with similar molecular
weights but with only the lower-molecular-weight MTL2 band for
the class II isolate (Fig.
8C),
indicating that a mating type switch had occurred at the
MTL1 locus. Because the majority of isolates from patient P1
were class I (a) while a minority were class II (), one
might assume that the switch event was from a to , but
the reverse could just as well have been the case if a had a
selective growth advantage.
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|
FIG. 7. Southern
analysis of mating type classes reveals that infecting strains can be
a or and that mating type switching occurred in
patient P1. Southern blots of test isolates from each patient were
probed with FuncP2, an antisense oligonucleotide that binds to a unique
5' end sequence of the MTL2 ORF. This
MTL2-specific probe discriminates among the three
major classes of C. glabrata, class I (a), class II
(), and class III (a)
(31). (A)
reference strains for the three classes representing the three
discriminating patterns. (B) isolates from patient P1.
(C) isolates from patient P2. (D) isolates from
patient P3. The mating type classes and MTL1 genotypes are
provided below the blots. Note that the prevailing patterns of the
isolates from patient P1, P2, and P3 are I (a), II (),
and I (a), respectively. Note also the single mating type switch
from class I to class II in isolate vDB-P of patient P1. In Fig.
3A, it was demonstrated by
Cg6 DNA fingerprinting that the five P1 isolates, including vDB-P,
represent the same strain. See the legend to Fig.
3 for an explanation of
the isolate names. The positions of MTL1, MTL2, and
MTL3 bands are noted to the right of the blots
(31).
|
|
To test whether the apparent genetic
switch from a to at the MTL1 locus resulted
in a change in mating type, the set of P1 isolates was analyzed for
MTLa and MTL expression by Northern
analysis with an MTLa1-specific probe and an
MTL1-specific probe. While the isolates that were
a at the MTL1 locus (vDB-T, DB-W, vDB-W, and DB-P)
expressed MTLa1 and not MTL1, the one
isolate that was at the MTL1 locus (vDB-P) expressed
MTL1 and not MTLa1 (Fig.
9). These results demonstrate that the genetic switch at the MTL1
locus in the P1 strain resulted in a change in mating type
expression.
To test whether mating type switching also occurred
in vitro, cells from a secondary DB colony of the P1 isolate DB-P
(class I; aa) were clonally plated. Fifty of these
colonies were then analyzed by Southern blot hybridization with FuncP2.
All exhibited the class I pattern of the original DB-P isolate (data
not shown). Cells of the DB-P isolate were then mass cultured through
200 generations in four separate flasks; the cells were pooled every 12
generations and analyzed by Southern blot hybridization with the
MTL1 probe. The gels were exposed for various lengths
of time to monitor the appearance of the MTL1
hybridization band of class II (Fig.
7A), which would indicate
a mating type switch from MTL1a to
MTL1 at the MTL1 locus. The sensitivity of
the overexposed gels would have identified one class II cell per 100 to
500 class I cells. No MTL1 band was observed (data
not shown), suggesting an extremely low in vitro frequency of switching
from class I to class II in DB-P
cells.
|
DISCUSSION
|
|---|
The results presented
here first suggest that only one strain of C. glabrata
colonized each of the three vaginitis patients, as has been
demonstrated to be the case for C. albicans in vaginitis
patients colonized by that species
(10,
11). In two of the
patients (P1 and P3), the same strain colonized both the oral cavity
and the vaginal canal. The higher level of C. glabrata in the
vaginal canal in both patients was consistent with the clinical
diagnosis of the vagina as the site of infection. One patient (P2) had
no oral carriage, even though she had a high level of vaginal
colonization, suggesting that in some vaginitis patients, the oral
cavity may not support, or may suppress, carriage. Similar differences
between oral and vaginal colonization of C. albicans in the
same host have been demonstrated both in healthy individuals
(30) and in vaginitis
patients
(27).
Here, we
provide evidence that the oral cavity and the vaginal canal can be
colonized by different proportions of switch phenotypes of the same
strain. In the case of patient P1, the primary sample from the oral
cavity was composed entirely of cells exhibiting the DB phenotype,
while the primary samples from the vaginal wall and vaginal pool were
composed of 86% DB and 14% vDB. This difference was
significant. If one applied to an initial homogeneous population of 100
DB cells of patient P1 a simple model based on the relatively high in
vitro frequency of DB switching to vDB and the relatively low in vitro
frequency of switching from vDB to DB, the population would reach the
proportions observed in the primary vaginal isolates after only eight
cell doublings and would continue to decrease in favor of vDB cells.
The fact that we did not observe a single vDB colony in the primary
plating of the oral-cavity sample from patient P1, and only 14%
in the primary plating of the vaginal sample, indicates a strong
selection bias for DB in the vaginal canal and an even stronger bias in
the oral cavity or inhibition of switching from DB to vDB in both
locations.
Because DB cells from patient P2 switched to vDB at
relatively high frequencies in vitro, one would have expected the
vaginal population of this patient to contain vDB cells. The primary
vaginal samples from patient P2, however, were devoid of vDB cells.
Similarly, the in vitro frequency of switching of DB cells from patient
P3 to Wh was similar to the in vitro frequency of switching of Wh cells
to DB, yet the oral cavity was dominated by the Wh phenotype while the
vaginal canal was dominated by the DB phenotype. These results clearly
demonstrate that the in vivo situation does not reflect in vitro
frequencies of switching.
The results obtained with patient P3
samples suggest for the first time that both the oral cavity and the
vaginal canal can select for different switch phenotypes of the same
strain. There are several possible ways this could be achieved. First,
selection could be through the differential inhibition of cell
multiplication. The oral cavity would selectively inhibit DB cell
multiplication, while the vaginal canal would selectively inhibit Wh
cell multiplication. Second, selection could be the result of the
nutrient compositions of the fluids in the two locations. While the
oral cavity would provide nutrients that selectively support Wh cell
multiplication, the vagina would selectively provide nutrients that
support DB cell multiplication. In vitro analyses have revealed no
differences in the growth rates of the core switch phenotypes in a
synthetic medium (8), but
observations of the nutrient requirements of the white and opaque
phenotypes of C. albicans suggest that growth in rich
synthetic medium may be irrelevant to the in vivo situation. Although
white- and opaque-phase C. albicans cells exhibit similar
growth rates in synthetic medium, they exhibit profound differences in
their sugar assimilation patterns
(22). Similar differences
may prove to exist for the core switch phenotypes of C.
glabrata. Third, selection could be due to differences in adhesion
or the capacity to form biofilms on the surfaces of the different
cavities. In C. albicans, it has been demonstrated that while
white-phase cells do not colonize skin, opaque-phase cells are highly
proficient (6). Hence,
there is precedent for selective colonization of switch phenotypes.
Finally, selection could be due to the in vivo inhibition of switching
in one direction (e.g., inhibition of switching from DB to vDB). In
C. albicans, a variety of environmental conditions have been
identified that affect the frequency of switching, including
temperature (19),
oxidants (5), culture age
(26), and low doses of UV
(14). Particular
anatomical locations may selectively or generally inhibit switching or
induce switching in one direction. Experiments to identify the
mechanisms for the phenotypic selection of switch phenotypes of C.
glabrata in the oral cavity and vaginal canal are now in progress.
In addition, we are pursuing the possibility that the DB phenotype,
which dominated the site of infection in each of the three vaginitis
patients, may be a more pathogenic phenotype, although it should be
noted that DB did not cause infections in the oral
cavity.
Finally, our results demonstrate that both class I and
class II cells, representing mating type a and cells,
respectively, can cause similar vaginal infections and that one mating
type dominates in each colonizing strain. More interestingly, the
results from patient P1 revealed a mixture of mating type a
(class I; aa) and mating type (class II;
a) cells in the vagina. Southern analysis
revealed that the differences in the distribution of MTL genes
at the MTL1 locus of the mating type a and
isolates was consistent with conserved gene replacement at the
MTL1 locus in a single strain. Northern analysis demonstrated
that this genetic change resulted in a change from MTLa
to MTL expression. DNA fingerprinting of the strains
verified that the MTLa and MTL
isolates from patient P1 represented the same strain. These results
demonstrate for the first time a mating type switch at the
MTL1 locus. Furthermore, these results demonstrate that mating
type switching occurs at the site of colonization. Remarkably, mass
culturing of the mating type a isolate (class I;
aa) through 200 generations in vitro did not result in
a switch to mating type (class II;
a), suggesting either that the strain
colonizing patient P1 had undergone far more than 200 generations in
vivo for a switch to have occurred or that mating type switching occurs
more frequently in vivo.
In conclusion, our analysis of the
colonizing populations of C. glabrata in three vaginitis
patients has revealed both high-frequency phenotypic switching and
mating type switching in the host. Our results also suggest either that
these processes do not occur at the same frequencies in vivo as they do
in vitro or that host environments select for particular switch
phenotypes or mating types. Finally, our results demonstrate for the
first time that different phenotypes of the core switching system of a
single strain can dominate different anatomical locations of the same
host.
|
ACKNOWLEDGMENTS
|
|---|
This research was supported
by National Institutes of Health grant DE014219.
We are indebted
to Karla Daniels for help with the figures.
Paula J. Brockert and
Salil A. Lachke contributed equally to this
work.
|
FOOTNOTES
|
|---|
*
Corresponding author. Mailing address: Department of Biological
Sciences, 302 BBE, The University of Iowa, Iowa City, IA 52242, Phone:
(319) 335-1117. Fax: (319) 335-2772. E-mail:
david-soll{at}uiowa.edu. 
Editor:
T. R. Kozel
|
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Infection and Immunity, December 2003, p. 7109-7118, Vol. 71, No. 12
0019-9567/03/$08.00+0 DOI: 10.1128/IAI.71.12.7109-7118.2003
Copyright © 2003, American
Society for
Microbiology. All Rights Reserved.
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Abstract
Introduction
