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JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1987, p. 1611-1622 0095-1137/87/091611-12$02.00/0
Vol. 25, No. 9
Copyright © 1987, American Society for Microbiology
High-Frequency Switching in Candida Strains Isolated from Vaginitis Patients DAVID R. SOLL,l* CAROL J. LANGTIMM,' JAN McDOWELL,' JAMES HICKS,2 AND RUDOLPH GALASK3 Department of Biology' and Department of Obstetrics and Gynecology,3 University of Iowa, Iowa City, Iowa 52242, and Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, California 920372 Received 4 February 1987/Accepted 5 June 1987
High-frequency switching and strain variability at the site of infection was assessed in Il patients with acute Candida albicans vaginitis. By cloning cells directly from the site of infection, it was demonstrated that 4 of the 11 isolates contained multiple-switch phenotypes at the site of infection and that 9 of the 11 isolates were in a high-frequency mode of switching (10-2 to 10-3). Isolates could be separated into four general categories of switching repertoires. To demonstrate that multiple phenotypes at the site of a single infection represented the same strain, EcoRI digests of total cell DNA were separated on agarose gels, and Southern hybridization patterns with two cloned midrepeat sequences were compared. Candida albicans remains the most persistent and pervasive yeast pathogen in humans, capable of invading virtually every tissue of the body (2, 11). Not only is it the major cause of vaginal and oral yeast infections (6, 7, 9, 16, 17, 20), but it has evolved into a major systemic pathogen of compromised hosts (2, 3, 5, 10, 12). Recently, it was demonstrated that a common laboratory strain of C. albicans possesses a high-frequency switching system which can be distinguished by colony morphology on the proper agar substratum (14). Cells of this strain switch spontaneously to six general colony phenotypes at a combined frequency of 10-4. This frequency is increased roughly 100-fold by a low dose of UV light which kills less than 10% of the population. Once a cell has switched, it continues to switch heritably and reversibly at a frequency of roughly 10-2 among the different phenotypes. Putative revertants with both the original phenotype and the original low switching frequency have been obtained (14; B. Slutsky, Ph.D. thesis, University of Iowa, Iowa City, 1986). In an analysis of isolates from the blood and lung of a patient with a systemic infection, a second high-frequency switching system has recently been identified (15; D. R. Soll, B. Slutsky, S. Mackenzie, C. Langtimm, and M. Staebell, in I. C. Mackenzie and C. A. Squire, ed., Oral Mucosal Diseases: Biology, Etiology and Therapy, in press). In this system, which is referred to as the white-opaque transition, cells switch heritably and reversibly between a white and opaque colony phenotype. At least three other heritable colony phenotypes are also generated in this system at a lower frequency. Perhaps the most unusual aspects of the white-opaque transition are the differences between white and opaque cells in cellular morphology, temperature sensitivity, developmental capabilities, and budding pattern (15). When the original switching systems of C. albicans were first described, it was suggested that they may play a role in pathogenesis (14, 15; Soll et al., in press) by providing cells with the plasticity to (i) develop resistance to antifungal agents, (ii) penetrate different tissues, (iii) sense changes in body physiology, and/or (iv) evade the immune system. It had been demonstrated that switching could result in changes in sensitivity to antifungal agents (Slutsky, Ph.D. thesis; Soll et al., in press; D. R. Soll, M. Staebell, S. Eisely,
and B. Slutsky, manuscript in preparation), alterations in the environmental constraints on the bud-to-hypha transition (15; Slutsky, Ph.D. thesis; Soll et al., in press), and changes in antigenicity (J. Anderson and D. R. Soll, manuscript in preparation). However, it was not known whether switching actually occurred at the site of infection, nor was it known whether different switching systems resulted in C. albicans infections at different body locations. In the present study, we clonally plated cells directly from the vaginal canals of 11 patients suffering from yeast vaginitis. We found that 4 of the 11 isolates contained multiple switch phenotypes at the site of infection, that 9 of the 11 isolates were in a high-frequency mode of switching, and that there were four general categories of switching systems into which the 1i strains could be grouped: (i) smooth white with no observable switching system (frequency less than 10-4; strains Pi and P2); (ii) a system analogous to the 3153A system first described (14; strain P3); (iii) systems similar to the white-opaque transition (15; Soll et al., in press; strains P5, P9, and Pll); and (iv) a new system which we designated the smooth white-heavy myceliated transition (strains P4, P6, P7, P8, and P10). In all of the systems but the first, the frequency of switching ranged between 10-2 and 10-3, and switching was completely reversible at high frequencies. To convincingly demonstrate that the multiple phenotypes of a single vaginal isolate represented switch phenotypes of the same strain, EcoRI digests of their total DNA were separated on agarose gels and hybridized with JH3, a mid-repeat sequence of C. albicans which appears to be relatively nonmobile, and JH7, a mid-repeat sequence which appears to be highly mobile (J. Hicks and D. R. Soll, manuscript in preparation). This study indicates for the first time that switching is occurring at the site of infection, provides a minimum estimate of the number and types of switching systems and strains which are involved in vaginal candidiasis, and suggests that transposition of the mid-repeat sequence JH7 may be occurring at extremely high frequency in C. albicans at the site of infection. MATERIALS AND METHODS or
*
Corresponding author. 1611
Isolation of strains. Samples were removed from the wall fluid (pool) of the vaginal canal with a sterile cotton swab
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TABLE 1. Proportion of different colony phenotypes after primary and secondary clonal platings of Candida specimens isolated from the vaginal canals of patients with yeast vaginitis Patient
Source of sample
No. of primary colonies and phenotype
No. of primary colonies
clonally
No. of
secondary colonies
Predominant phenotype of secondary colonies
phenotypesin secd colonies('
Switch
plated
Frequency of switch phenotypes in secondary colonies"
Pool Wall
65 smooth wtb 59 smooth wt
4 4
5,850 5,952
Smooth wt Smooth wt
O
P2
Pool Wall
6 smooth wt 3 smooth wt
4
5,250
Smooth wt
O
P3
Pool
15 stippled ring 1 ring 3 smooth wt
1
1,780
Stippled ring
42 smooth wt
2.3 x 10-2
1
2,548
Smooth wt
1.2 x 10-3
1
2,560
Stippled ring
2 stippled ring, wrinkled 7 smooth wt 12 star 14 petite smooth wt 2 ring
irregular stippled petite smooth wt flat top heavy myceliated heavy myceliated
2.1 x 10-3
Pi
1 petite smooth wt
P4
PS
Pool
220 smooth wt
4
7,075
Smooth wt
Wall
202 smooth wt
4
5,614
Smooth wt
Pool
943 143 3 1
1 1 1 1 (OS)'
2,520 1,365 1,350 487
Smooth Smooth Smooth Smooth
1 (WS)'
537
Smooth wt
4
4,930
Smooth wt
1 1
2,030 1,990
Heavy myceliated Heavy myceliated
998 smooth wt
1
1,860
Heavy myceliated
386 heavy myceliated
1
2,750
Heavy myceliated
3,980 smooth wt
4
3,626
Smooth wt
620 smooth wt
1
2,545
Smooth
629 heavy myceliated
1
2,165
Smooth
4
5,812
Smooth wt
Wall P6
Pool
Wall
P7
Vulva
P8
Wall
P9
Wall
smooth wt scalloped heavy myceliated opaque/wt sector
15 smooth wt
2,161 smooth wt 218 heavy myceliated
2,494 smooth wt
wt wt wt wt
0
8 3 1 2 5
1 opaque sector O O 149 opaque or opaque/ wt sector 14 opaque or opaque/ wt sector 5 opaque or opaque/ wt sector
276 smooth wt 56 smooth wt 1 stippled 1 stippled ring 30 smooth wt 1 fuzzy 44 smooth wt
1.4 x 10-2
8.9 x 10-4
3.9 x 10-4 3.1 x 10--4a 2.6 x 10-2 1.0
X
10-3
1.2 x 10-'" 3.5 x 10-2 1.4 x 10-2
2.2 x 10-1
27 heavy myceliated
7.4 x 10-3
837 heavy myceliated 3 flower 790 myceliated 2 ring 3 flower
3.3 x 10-1 3.7 x 10-1
4.0
x
10-'
16 heavy myceliated 12 petite smooth wt
6.9
x
10--
3 heavy myceliated
1.96
x
10-
23
opaque or
opaque/
wt sector
PlO
Wall
4,455 smooth wt
4
4,030
Smooth wt
Pli
Wall
1,805 smooth wt
4
3,490
Smooth wt
2
6 stippled 30 opaque or opaque/ wt sector "In some instances, the predominant phenotype of the secondary clonal plating switch phenotypes is that of the minority phenotypes. b wt, '
The
White. opaque sector
(OS) and white
sector
(WS)
were
individually cloned.
was
not
that of the first clonal plating.
In
these instances, the frequency of
HIGH-FREQUENCY SWITCHING IN CANDIDA STRAINS
VOL. 25, 1987 SWAB FROM VAGINAL CANAL 'POOL' or 'WALL'
PRIMARY CLONAL COLONIES
1613
/ CELLS ARE SUSPENDED IN BUFFERED SALTS SOLUTION
,LBv~~~1
COLONY PHENOTYPES SCORED
CLONALLY PLATED ONNUTRIENT AGAR a a, r . J
SINGLE COLONIES ARE INDIVIDALLY SUSPENDED IN BUFFERED SALTS SOLUTION
SECONDARY CLONAL
rn . __w.AJ *l
-
1
1)Q)
---
AGAR CLONALLY PLATED ON NUTRIENT -
I
1
1
-
COLONY PHENOTYPES fl SCORED
1
FIG. 1. Method used to clonally plate cells directly from the vagina of patients with vaginal candidiasis.
and mixed into sterile water by agitation. The samples were then examined in a hemacytometer to obtain the concentration of spheres per milliliter and diluted or concentrated for plating. Cells were plated at a density of 100, 250, and 500 per plate because of low viability in some instances. The cells were plated on Candida agar, which was the amino acid-rich defined medium of Lee et al. (8) supplemented with arginine (70 p.g/ml) and zinc (9 ,uM) (19). The petri dishes were 10 cm in diameter. In most instances, bacterial contamination was low or absent and therefore not a problem. Vaginal epithelial cells were present in samples, especially in the wall samples. Cultures were incubated for 7 days at 25°C for initial assessment of colony phenotype. A representative sample of the original isolate was also streaked on sterile agar and stored at 25°C. A diagram of the method for generating primary clonal colonies is shown in Fig. 1. Serial cloning. To assess the switching frequencies of primary and subsequent colonies, cells were removed from individual clonal colonies of 7 to 9-day-old cultures, suspended in water, counted, and plated at 70 and 140 spheres per plate (Candida agar; 10-cm petri dish). These plates were also incubated for 7 days at 25°C. The scheme for secondary plating is shown in Fig. 1. Sugar assimilation patterns. Each strain was tested for carbohydrate assimilation patterns by using commercial kits purchased from Analytab Products, Plainview, N.Y. In all instances, the primary identification was C. albicans. Southern hybridization. DNA was isolated by a modification of the method described by Cryer et al. (4). DNA (1 ptg) from each of the strains was cut with 10 U of EcoRI enzyme (New England BioLabs, Inc., Beverly, Mass.) at 37°C for 1 h, and the fragments were separated on 0.7% agarose gels in TBE buffer (0.089 M Tris-borate, 0.089 M boric acid, 0.002 M EDTA). Electrophoresis was performed at 10 mA for 17 h. The gels were then soaked for 0.5 h in a solution containing 0.5 ,ug of ethidium bromide per ml in TBE. The gels were then destained in TBE and photographed. After electrophoresis, the gels were soaked in 0.25 N HCl for 15 min to depurinate the DNA, in 0.4 N NaOH for 30 min to denature the DNA, and in a solution containing 12 mM Tris-6 mM sodium acetate-0.3 mM EDTA (pH 7.5; transfer buffer) for 30 min to neutralize the gel. The DNA fragments were transferred to a GeneScreen Plus hybridization membrane (New England Nuclear Corp.). The transfer was performed in a Hoeffer electroblot transfer apparatus containing transfer buffer. Electrophoresis was performed for 1
h at 10 V and 5°C. The voltage was then increased to 40 V for 2 h at 5°C. JH3, a hybrid lambda bacteriophage containing three EcoRI restriction fragments of C. albicans 3153A (2.5, 2.8, and 4.0 kilobases) hybridized to an array of at least 15 restriction fragments; JH7, a hybrid lambda phage containing a single EcoRI restriction fragment of C. albicans 3153A (9.5 kilobases) hybridized to a different array of restriction fragments (Hicks et al., in preparation). JH3 and JH7 were nick translated by the method described by Rigby et al. (13). The membrane was sealed in a plastic bag along with a prehybridization solution containing 1% sodium dodecyl sulfate, 1 M sodium chloride, and 10% dextran sulfate for 1 h at 65°C. The radioactive probe (final concentration, 105 cpm/ml) and 1 mg of calf thymus DNA were denatured by immersion in a boiling water bath for 10 min. The denatured DNA preparation was then added to the hybridization bag and incubated for 16 h at 65°C. At the end of the incubation period, the membrane was washed twice with 2x SSC (lx SSC is 0.15 M NaCI plus 0.015 M sodium citrate) at room temperature for 5 min, twice with 2x SSC containing 1% sodium dodecyl sulfate at 65°C for 30 min, and twice with 0.1x SSC at room temperature for 30 min. The membrane was then pressed against XAR-5 film (Eastman Kodak Co., Rochester, N.Y.) with a Cronex Lightning-Plus (Du Pont
FIG. 2. Smooth white phenotype exhibited by colonies of strain Pi. (A) Single colony; (B) lower magnification of a number of colonies. Note the homogeneity of colony size in panel B.
1614
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FIG. 3. Examples of the switching repertoire of strain P3 (a 3153A-type system). (A) Stippled-ring (SR) and smooth white (SW) colonies; (B) closer view of stippled-ring morphology; (C) irregular morphology; (D) star colony; (E) extremely myceliated colony; (F) switch from stippled-ring to smooth white colony; (G) switch from stippled-ring to star (ST) colony; (H) switch from star to stippled-ring and petite (P) colonies; (t) switch from petite to stippled-ring colonies.
Co., Wilmington, Del.) intensifying screen in a lightproof exposure chamber. RESULTS Phenotypes of original vaginal isolates. To obtain an estimate of the number of switch phenotypes at the site of infection, it was necessary to clone cells from the original isolate (Fig. 1). Of the 11 original sets of isolates, 4 exhibited
multiple colony phenotypes (Table 1). The four included P3, P5, P6, and P8. The P3 isolate included 15 stippled-ring colonies (see Fig. 3A), 1 smooth-ring colony, 3 smooth white colonies (see Fig. 3B), and 1 petite smooth white colony (see Fig. 3D and F). The P5 isolate included 943 smooth white colonies, 143 scalloped colonies (see Fig. 5A), 3 heavy myceliated colonies (see Fig. 5B), and 1 smooth white colony with an opaque sector (see Fig. 5C). The P6 isolate included 2,161 smooth white colonies and 218 heavy myceliated colonies. The P8 isolate included 620 smooth white
VOL. 25, 1987
HIGH-FREQUENCY SWITCHING IN CANDIDA STRAINS
PRIMARY f COLONIES L
I
|11
STIPPLE-RING
1 CLONE
4
SMOOTH-WT|
1
1615
El RING
PETITE S-W
-2
(1780 COLONIES) f2.3x10
_ 1738 STIPPLE-RING
f
4 CLONES
42 SMOOTH-WT
(2548 COLONIES)
fl.1x103 SESECONDARY CON DARY< q
1 CLONE (2560 COLONIES) -2
fl~7w7 '.4xl0 1.x0 STIPPLE-RING| | IRR. WRINKLED|| 1~~~2545 SMOOTH-| W TI [2 ~ I G~3IK E
COLONIES
2525
STIPPLE-RING|
F14
PETITE S-W
12 STAR7 SMOOTH-WT|
2
1 CLONE (500 COLONIES) f5.8x 1o2
J
472
1 CLONE
PETITE SW
8STIPPLE-RING|
(412 COLONIES)
f3.4x102
TERTIARY
COLONIES
_ STA | STIPPLE-RING E3i98 |602 STIPPLE-R
1 CLONE (607 COLONIES)
PETITE S-W| G
f8.2x 103
L4 STAR| 1MOTTLED-MvC]
FIG. 4. Flow sheet ofthe switching repertoire of strain P3. The method for initial isolation and clonal plating is shown in Fig. 1. For each plating in the scheme, the number of clones, the number of colonies originating from those clones, and the frequency (f) of variant colonies (differing from the phenotype of the original clone) are shown. Smooth-wt, smooth white; petite s-w, petite smooth white; irr. wrinkled, irregular wrinkled; mottled-myc, mottled-myceliated. Examples of several of these phenotypes are shown in Fig. 3.
colonies and 629 heavy myceliated colonies. If we consider smooth white as the basic colony phenotype, then the proportion of the cell population exhibiting switch phenotypes at the sites of infection in patients P3, P5, P6, and P8 were 85, 13, 9, and 16%, respectively (Table 1; in the calculations for P5 and P6, pool and wall figures were combined). Of the 11 sets of isolates, 7 exhibited only one colony phenotype at the site of infection (Table 1). In all instances, the phenotype was smooth white (e.g., see Fig. 2A and B). Switching capabilities of original isolates. To assess the switching capabilities of the original isolates, individual primary colonies were resuspended and clonally plated according to the scheme shown in Fig. 1. Of the 11 sets of isolates, 2 (Pi and P2) exhibited no variant phenotypes. Of the 5,850 secondary colonies plated from four primary smooth white colonies from the pool sample of Pi, all
exhibited the original smooth white phenotype (Table 1 and Fig. 2A and B). The same was true for the 5,952 secondary colonies from the wall sample of Pi and for the 5,250 secondary colonies from the pool sample of P2 (Table 1). Therefore, the switching frequency of the Pi isolate was below i0-', and that of the P2 isolate was below 2 x 10-'. The remaining nine sets of isolates exhibited high switching frequencies, ranging from 10-2 to 5 x 10-4 (Table 1). There appeared to be three general switching categories into which these nine sets could be separated. (i) Original 3153A-type switching system. Strain P3 exhibited a switching system with phenotypes (Fig. 3) roughly analogous to those observed in the originally characterized switching system in laboratory strain 3153A (14). As noted above, the primary colonies of P3 included 15 stippled-ring,
_ ` a _ _ _
_ _ _
FIG. 5. Examples of the switching
repertoire of strain PS (a
WO-1-type system). (A) Scalloped colonies; (B) myceliated ring colony; (C) smooth white (W) colony with an opaque (Op) sector; (D) cells cloned from an opaque sector (note the difference between the smaller white and larger, grayer opaque colonies).
1616
SOLL ET AL.
PRIMARY COLONIES
J. CLIN. MICROBIOL.
{
SECONDARY COLONIES
TERTIARY COLONIES
QUATERNARY COLONIES
FIG. 6. Flow sheet of the switching repertoire of strain Pli (a WO-1-type system). The method for initial isolation and clonal plating is shown in Fig. 1. For each plating, the number of clones, the number of colonies originating from those clones, and the frequency (f) of variant colonies are shown. Smooth-wt, smooth white; op, opaque; op/wt, opaque with white sector or white with opaque sector; heavy myc and h.myc., heavy myceliated; op/st.r., opaque with stippled-ring sector or stippled ring with opaque sector; dark op, dark opaque; op-wrinkled, opaque wrinkled.
4 smooth white, 1 petite, and 1 ring colony (Table 1 and Fig. 4). Of 1,780 secondary colonies plated from a single primary stippled-ring colony, 1,738 were stippled ring (Fig. 3A and B) and 42 were smooth white (Fig. 3A). Therefore, the frequency of switch phenotypes (in this instance, smooth white) in this primary colony was 2.3 x 10-2. Of 2,548 secondary colonies plated from a single primary smooth white colony, 2,545 were smooth white, 2 were stippled ring, and 1 was of irregular morphology (Fig. 3C). This represents a frequency of 1.2 x 10-'. When cells from the single petite smooth white primary colony were clonally plated, the predominant phenotype of the secondary colonies was stippled ring (Table 1 and Fig. 4). This suggests either that the petite colony was in fact a stippled-ring colony to begin with or that there was unusually high enrichment or differential viability by the time the petite colony had matured. The latter possibility is suggested by the unusually high proportion of petite smooth white colonies (Table 1 and Fig. 4). The switch phenotypes in this instance included 7 smooth white, 12 star (Fig. 3D), 14 petite smooth white (Fig. 3H and 31), and 2 ring (Fig. 4). This represents a combined frequency of
1.4 x 10-2. To test the heritability of the switch phenotypes, secondary colonies were disaggregated, and cells from the individual colonies were plated (Fig. 4). One secondary petite smooth white colony gave rise to primarily petite smooth white colonies, demonstrating that petite smooth white is a distinct and heritable phenotype in the switching repertoire
of this strain. In addition, one secondary star colony gave rise to predominantly star colonies, demonstrating the heritability of this phenotype (Fig. 4). The secondary petite smooth white and secondary star colonies gave rise to other switch phenotypes at frequencies of 5.8 x 10-2 and 3.4 x 10-2, respectively, suggesting a high frequency of reversibility (Fig. 4). One secondary ring colony, with a smooth interior (as opposed to the mottled interior of stippled-ring colonies [Fig. 3A]), gave rise to predominantly stippled-ring colonies, suggesting that rings are in fact stippled rings and therefore do not constitute a separate phenotype. This ring clone gave rise to four star colonies and a previously unobserved phenotype, mottled myceliated (Fig. 4). Exampies of switching are shown in Fig. 3F to I. (ài) White-opaque transition. Three sets of isolates (PS, P9, and P11) exhibited the white-opaque transition first observed in strain WO-1, isolated from the blood and lung of a bone marrow transplant patient (15). The primary colonies from the pool sample of PS included 943 smooth white colonies, 143 scalloped colonies (Fig. SA), 3 heavy myceliated ring colonies (Fig. SB), and 1 smooth white colony with an opaque sector (Fig. SC and Table 1). Of 2,520 secondary clonal colonies emanating from a smooth white primary colony, 2,519 exhibited the original smooth white phenotype and 1 was a smooth white colony with an opaque sector (Table 1). All 1,365 secondary colonies emanating from a single scalloped colony exhibited the smooth white phenotype, and all 1,350 secondary colonies emanating from a
VOL. 25, 1987
HIGH-FREQUENCY SWITCHING IN CANDIDA STRAINS
1617
FIG. 7. Examples of the switching repertoire of strains in the smooth white-heavy myceliated transition group. (A) Heavy myceliated colony; (B) smooth white (S) and heavy myceliated (HM) colonies; (C) flower (F) colony with smooth white sector; (D) smooth white and flower colonies; (E) wrinkled morphology; (F) stippled-ring colony with heavy mycelia; (G) very heavy myceliated colony; (H) switch from smooth white to stippled-ring (R) colony; (t) switch from stippled-ring to smooth white colony.
single heavy-mycelicated colony also exhibited the smooth white phenotype (Table 1). For the scalloped colony, if the scalloping was the result of peripheral sectoring from white to opaque, it is not surprising that the predominant phenotype was smooth white since opaque cells have been demonstrated to exhibit high death rates in the stationary phase under select conditions (15). Scrapings of the opaque and white sections of the primary sectored colony were also clonally plated. Of 487 secondary colonies emanating from the opaque sector, 338 were smooth white and 149 were completely opaque or sectored (Fig. 5D). Of 537 secondary colonies emanating from the white portion of the colony, 523
exhibited the smooth white phenotype and 14 exhibited the opaque phenotype or were smooth white with opaque sectors. The 15 primary colonies from the wall sample of P5 were
smooth white. Of 4,930 secondary colonies emanating from four primary clones, 4,925 exhibited the original smooth white phenotypes and 5 were either opaque or smooth white with opaque sectors. Therefore, the pool and wall isolates exhibited the same switching system. All 2,494 primary colonies from the wall sample of P9 and the 1,805 primary colonies from the wall sample of Pli were smooth white. Of the 5,812 secondary colonies emanating
1618
J. CLIN. MICROBIOL.
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PRIMARY COLONIES
-
SECONDARY COLONIES
{
TERTIARY COLONIES
[
FIG. 8. Flow sheet of the switching repertoire of strain P8 (a smooth white-heavy myceliated transition system). The method for initial isolation and clonal plating is shown in Fig. 1. For each plating, the number of clones, the number of colonies originating from those clones, and the frequency (f) of variant colonies are shown. Smooth-wt, smooth white. heavy-myc, heavy myceliated; petite s-w; petite smooth white.
from four primary clones of P9, 5,789 were smooth white and 23 were opaque or white with opaque sectors. Of the 3,490 secondary colonies emanating from four primary clones of Pli, 36 were opaque or white with opaque sectors, 3 were heavy myceliated, and 6 were stippled (Table 1). To investigate in depth the switching repertoire of a strain exhibiting the white-opaque transition, secondary colonies of strain Pli were serially plated (Fig. 6). Of 1,464 tertiary colonies emanating from secondary smooth white colonies, 908 were smooth white and 556 were opaque or sectored. Of 1,624 colonies emanating from secondary opaque colonies, 1,589 were opaque and 35 were smooth white or white with an opaque sector. Tertiary colonies were also generated from the smooth white and opaque sectors of a secondary colony. Again, smooth white colonies, opaque colonies, and smooth white colonies with opaque sectors were obtained in both instances. To demonstrate the continued reversibility between the white and opaque phenotypes, quaternary colonies were plated from a tertiary smooth white colony. Quaternary colony phenotypes included smooth white, opaque, white with opaque sectors, and stippled ring (Fig. 6). If the lineage of switch phenotypes is traced to a final quaternary opaque colony, the following lineage is obtained: smooth white (10)
opaque
(2°)
->
smooth white
(3°)
>
(4°). The frequencies of switch phenotypes emanating from the primary, secondary, and tertiary colonies in the smooth white lineage (Fig. 6) were 1.2 x 102, 3.8 x 10-1, and 3.4 x 10-2, respectively. These frequencies are similar to the frequency of switch phenotypes for the original systemic isolate WO-1 (15; Soll et al., in press). Strains which undergo the white-opaque transition also produce variant phenotypes other than opaque at a relatively high frequency. With Pli (Fig. 6), six primary colonies exhibited a stippled-ring phenotype and three quaternary colonies emanating from the opaque sector of a primary colony also exhibited this phenotype. To assess the switching repertoire of this phenotype, one primary stippled-ring colony was clonally plated. Of 655 secondary colonies, 601 were stippled ring, 1 was smooth white, 15 exhibited opaque sectors in stippled-ring colonies, and 38 were stippled ring opaque
and heavy myceliated. One secondary colony which was stippled ring with an opaque sector was clonally plated. Of the 605 tertiary colonies emanating from the stippled-ring portion of the colony, 350 were stippled ring, 221 were dark opaque, 32 were stippled ring with an opaque sector, 1 was opaque wrinkled, and 1 was smooth white. Of the 507 tertiary colonies emanating from the opaque sector, 358 were dark opaque, 139 were dark opaque with lighter opaque sectors, and 10 were opaque wrinkled. These results suggest that when Pli switches to the stippled-ring phenotype, the subsequent switching pattern changes to include several new phenotypes. Variant phenotypes other than white and opaque were also observed in strain WO-1 (15). (iii) Smooth white-heavy myceliated transition. Five sets of isolates (P4, P6, P7, P8, P10) exhibited a general pattern of switching which included a heavy myceliated colony morphology, in which the colony proper is ringed by mycelia penetrating the agar periphery and in turn forming secondary colonies along the hyphal tracks (Fig. 7A and B). None of the strains loosely placed in this category formed opaque sectors or colonies. All, however, exhibited the two major colony morphologies, smooth white (Fig. 7B, D, H, and I) and heavy myceliated (Fig. 7B). In addition, two of these strains generated a "flower" morphology (Fig. 7C and D), three generated a ring or stippled-ring morphology with heavy mycelia (Fig. 7F, H, and 1), two generated a petite smooth white morphology, one generated a wrinkled morphology (Fig. 7E), and four generated a very heavy myceliated morphology (Fig. 7G). Although all five strains formed smooth white and myceliated phenotypes, there were pronounced differences in the propensities for the two phenotypes. For instance, when a smooth white colony and heavy myceliated colony of P4 were clonally plated, most clonal colonies exhibited the parent colony phenotype, smooth white for the former and heavy myceliated for the latter. However, when a smooth white colony and a heavy myceliated colony of P8 were clonally plated, both produced predominantly smooth white phenotypes. In contrast, when a smooth-white colony and a heavy myceliated colony of P6 were clonally plated, both produced predominantly heavy
HIGH-FREQUENCY SWITCHING IN CANDIDA STRAINS
VOL. 25, 1987
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