Electrophoretic karyotyping of typical and atypical Candida albicans

(1)

Electrophoretic Karyotyping

of

Typical

and

Atypical

Candida

albicans

MONA

MAHROUS,'

T. J. LOTT,2 S. A. MEYER,'A. D. SAWANT,' AND D. G.

AHEARNl*

Laboratoryfor Microbial and Biochemical Sciences, Georgia State University,

Atlanta, Georgia

30303,

andDivision

of Mycotic

Diseases,

Centers

for

Disease

Control,

Atlanta, Georgia

303332

Received 28 September1989/Accepted 17 January 1990

Electrophoretic karyotypes of atypical isolates of Candida

albicans,

e.g., strains that were germ tube

negative, failed to express proteinase activity, demonstrated low virulence for mice, formed

hyperchlamydo-conidia, produced hyperhyphae, or weresucrosenegative (including the type

strain

of Candidastellatoidea),

were compared with those of typical C.

albicans.

Karyotypes of whole-cell DNA of classical C.

albicans

examined with transverse alternating-field electrophoresis under specific conditions werecomposed ofseven DNAbands with aspecific migration pattern. Certain atypical strains andrepresentativesof thethree serotypes of C.stellatoidea produced discrete karyotypes with5to 10 bands.Allisolates demonstrateda significantdegree of DNArelatedness,suggesting theirconspecificity. Densitometrictracings of DNA bands provided an objective andstandardized method for comparingbandswithin the gels.

Theincreased incidence of deepcandidiasis, particularly as anoftenfataliatrogenicdisease, has stimulated interest in

developingmore finite proceduresfor epidemiological

stud-iesofthe mostcommon etiological agent,Candidaalbicans.

Various methods, including differences in streak

morphol-ogy (2), resistance to chemicals (31), a combination of

physiological characteristics and resistances to chemicals

(20, 21), differencesincarbohydrate assimilation(33),

sero-typing (5), and sensitivity to yeast toxins (25), have been proposed for subdividing C. albicans into subgroups or

biotypes. Some success has been achieved, but ingeneral

the methods recognize either too many or an insufficient numberof subgroups forpractical use. Incertain instances the procedures havelacked reproducibility in different

lab-oratories (10, 22).

The development ofkaryotyping dataand endonuclease

digestion patterns of

genomic

DNA has recently offered

morefundamental andpossiblymorereproducible biotyping

capabilities

(27). Lott et al. (11) observed,

by

using field

inversion gel electrophoresis, five distinct chromosomal

mobilitygroups amongisolates ofC.albicans. Seventonine

bands with 14 distinct

electrophoretic

patterns were

ob-servedbyMerz etal. (15),who used

orthogonal-field

alter-nating-gel electrophoresisin an

epidemiologic

study of iso-latesofC. albicans from different

patients;

and

Magee

etal.

(12) resolved up to 11 DNA bands

by

applying

contour-clamped

homogeneous-field gel electrophoresis,

orthogonal-field alternating-gel

electrophoresis,

and field inversion

gel

electrophoresis.

These

procedures

in

conjunction

withgene

probes led Magee etal. to

postulate

thepresence ofseven

chromosomes. Laskeretal.

(9),

withasimilar

protocol

ina

study oftwo

isolates,

indicated thepresence ofatleast

eight

chromosomes.

Kwon-Chung

etal.

(7, 8)

withan

orthogonal-fieldalternating-gel

electrophoresis procedure

founddistinct

karyotype

patterns for two types of Candida stellatoidea

(considered

hereina

variety

ofC.

albicans).

One

form,

type

II, had sevenDNAbands in apattern similar to that of C.

albicans serotype

A;

the

other,

type

t,

serotypeB,had

eight

ornine bands.

In most

investigations,

conditions for

obtaining

karyo-*

Corresponding

author.

types have not been fullydescribed, andonly strains readily

identifiedas C. albicans or C. stellatoideahave been

inves-tigated. Atypical forms of C. albicans that lack classical

identification characteristics have been ofincreasing inci-dence among yeast cultures submitted to the Division of

Mycotic Diseases, Centers for Disease Control, for

identifi-cation. Karyotyping ofatypical C.albicans and the use of a transverse alternating-field electrophoresis (TAFE) system has not been reported. In this study we compare TAFE karyotypes of whole-cell DNA ofclassical and atypical C. albicans.

MATERIALS ANDMETHODS

Cultures. Isolates were selected from cultures submitted for identification tothe Division ofMycotic Diseases,

Cen-ters for Disease Control (C isolates), from the American

Type Culture Collection (A isolates), and from the culture

collectionatGeorgiaStateUniversity (G isolates).Theyeast

isolates were screened with the API 20C

(Analytab

Prod-ucts, Plainville, N.Y.) yeast system andidentified by

stan-dardassimilation andfermentation testsand

morphological

studies of

pseudomycelium, chlamydospores,

and germ

tubes(1, 30).The sources andidentifications ofthecultures arepresentedinTable 1. Cultureswere

lyophilized

within 1 monthof theirsubmission to

Georgia

State

University,

and

working stocks were maintained on Sabouraud dextrose

agar.

Biotyping. Selected

characteristics,

namely,

resistanceto

flucytosine and

safranin,

citrate

utilization, pH

tolerance,

andproteinase

production

from the

procedures

of Odds and

Abbott (20), were

employed

for

biotyping.

Proteinase

pro-duction was detected

by

the bovine serum

albumin-agar

plate method of Ruchel et al.

(26).

Serotypes

were

deter-mined with the Candida Check RM 302 Iatron Kit

(Iatron

Lab, Inc.,

Tokyo,

Japan).

Strains

previously

serotyped by

standard

procedures

were

employed

ascontrols

(29).

Virulencetomice. C.albicans strainsweregrown in 50 ml

ofSabouraudbrothon a

reciprocal

shaker

(200

rpm)

at

37°C

for 24to48 h.Cellswereharvested

by

centrifugation

at

6,000

x gfor 10 min and washed three times withnormal saline

(0.9%

[wt/vol] NaCl).

Theinoculum

preparation

consisted of

cells

suspended

in salinetoan

optical

density

of 0.12 at 600 876

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TABLE 1. Physiological and morphological characteristics of selected isolates of C.albicans

Virulence' API Morphologyb

Strain Source Yrisolated Serotype to

mice

Proteinase

profile

G

C

H

G-30 Vagina 1977 A 0/10 + 2576170 + + +

G-M-44 Clinical 1988 A 0/10 + 6572170 + + +

G-9 Feces 1977 A 0/10 + 2560170C + + +

G-364 Mouse feces 1987 A 10/10 - 2576170

C-655 Skin lesion 1982 A 10/10 + 2572171 + + +

C-402 Clinical 1987 A 0/10 + 6776171C + + +

C-051 Lung 1987 A 10/10 - 2576171 + + +

C-448 Blood 1987 A 6/10 + 2566170 - - +

C-B-1071 Mouth Unknown B 0/6 + 2172170 + + +

G-1114 Clinical Unknown A ND + 2552150c + - +

A-11006d Vagina 1941 B + 2562150 + - +

C-B-1034e Skin C ND + 2546050C + - +

aNumber ofsurvivors/number inoculated. ND, Not determined.

bG, Germ tube; C,chlamydospores; H, hyphae.

CUnlisted oruncommon digit for C.albicans.

dType culture of Candida stellatoidea ATCC 11006. See

reference

8 forinformation on virulence in mice.

ePreviously described as strain J-1012 (23). Seereference23 for the year isolated.

nm(about 2x106 cellsperml).CFW WhiteSwiss mice from

Charles River Laboratories (Wilmington, Mass.) were

inoc-ulated with 0.1 ml of cell suspensions via the tail vein.

Autopsy was performed on mice that died and mice that

were sacrificed14 days after inoculation.

DNAcharacteristics. DNAwas isolated by the method of

Marmur (13) as modified by Meyer and Phaff(17) and the

base compositions (moles percent guanine plus cytosine)

weredeterminedat least twice for each strain by the thermal

denaturation method(14). The DNArelatednessof selected

strains was obtained by the

procedures

of Seidler and

Mandel (28)as modifiedby Kurtzman et al. (6).

Karyotyping by TAFE. Gel electrophoresis of whole-cell DNA was determined by a modification ofthe method of

Lott et al. (11). Cells grown for 14 to 18 h in 20 ml of1% (wt/vol)yeastextract-2% (wt/vol) peptone-1% (wt/vol) glu-cose at 30°C were harvested by centrifugation, washed twice, and

suspended

at

approximately

1:10

(vol/vol)

in0.05

MEDTA (pH 7.5, adjusted with NaOH). To 1.0 mlof this

cell

suspension,

100 ,ulofZymolyase60 T

(Miles

Laborato-ries,Inc.,

Naperville,

Ill.;2

mg/ml

in Tris-EDTAbuffer)and 1.0 ml of1.0% (wt/vol)LEagarose(Beckman Instruments,

Inc., Palo Alto,Calif.) in 0.125M EDTAwere added. This

suspensionwasvortexed and

poured

into molds(4

by

9

by

2

mm). After theagaroseinthemolds

solidified,

thesolidified

agarose plugs were incubated

overnight

at

37°C

in 0.5 M

EDTA-0.01 MTris(pH 7.5) with 7.5%

(wt/vol)

2-mercapto-ethanol. The

plugs

were rinsed with sterile distilled water

andincubated foran additional 12 to 18 hat

50°C

in0.5 M

EDTA-1%

lauryl sarcosine-0.01MTris

(pH

9.5)

containing

2 mgof

proteinase

K

(Sigma

Chemical

Co.,

St.

Louis,

Mo.)

perml.Theexcessbufferwas

removed,

and the

plugs

were

rinsed withsterile distilledwaterandwerestoredat

4°C

until

processed,

whichwas

always

within 2 weeks. The agarose plugswerecutintosmallerplugstofit the sizeof the wells in

gel plates

(1%

LE agarose in TAFE

buffer).

The smaller

plugswereheld insterile distilledwaterforabout 1 hatroom

temperature

before

they

wereloadedintothewells of the

gel

plates. The wells with

plugs

were sealed with melted

aga-rose, and the

gel plates

were

placed

in the

vertically

posi-tioned track in thechamber ofaTAFE system

(Beckman)

filled with 1x TAFEbuffer.The

temperature

of the chamber

was held at 10 to

13°C,

and the

voltage,

switch

time,

and

runningtimewerevariedfor the

optimal

resolution

of DNA

bands. Gelswere stained withethidium bromide, destained in distilledwater, and photographed under UV light.

The lanes on the

negatives

(a 1.6-mm width in the center of

thelane, every 20 ,um of migration distance) were scanned

with an Ultrascan XL enhanced laser densitometer (LKB

ProdukterAB, Bromma, Sweden). The data obtained were

analyzed with the computer software Gelscan XL (LKB)

under thefollowingconditions: peakwidth for peak search,

8; area rejected, 1.0;

integration

method, signal; and base

lineoption, common.

RESULTS

Phenotypes. The characterization of the isolates is

pre-sented in Table1. Sucrose-negativeC. albicans strainswere

considered C. albicans var. stellatoidea. This variety is

indicatedby theAPI20C

profile

thatterminatesin the

digits

50. Other variations in

profile

numbers thatindicated

differ-encesin assimilation of

glycine, xylose,

arabinose,

xylitol,

and

methyl-d-glucoside

fell within the

profile

codes for

possible C. albicans. The assimilation and fermentation

patterns(datanotshown) of all strains

by

standardtests

(30)

were in general agreement with those established for C.

albicans. Some reactions that were

negative

with the API

20C system (e.g.,

xylose

assimilation)

were

positive

in the

liquid assimilation media. Isolates that

readily

produced

germtubesand

chlamydospores

and

expressed

virulencefor

mice, in

conjunction

with

compatible

physiological

charac-teristics (e.g.,

G-30,

G-M-44),

wereconsidered classical or

typical

isolates ofthe

species.

These

typical

isolates were

proteinase

positive,

showed toleranceto

pH

1.4,

werecitrate

positive,

and were resistant to safranin and

flucytosine.

These

properties

grouped

the

typical

isolateswithtwoofthe more common

biotypes

in the scheme of Odds and Abbott

(20). These characteristics for

distinguishing

the common

biotypeswere not

always

reproducible;

e.g.,distinction ofa

positive

or

negative

reaction was

arbitrary

for the

atypical

strains ofC. albicans.

Allstrains

readily

expressing

proteolysis,

except

forstrain

C-655, a

hyphal form,

were virulent for mice. A

repre-sentative ofclassical C.

albicans

(G-30)

produced

in mice

intensivetissueinvasionandnecrosis.The

yeast

was

recov-ered from the

kidney,

heart,

and

spleen

and occurred in

tissues in

hyphal

andyeastforms.Detailsof the virulence of

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TABLE 2. G+Ccontentand DNArelatedness ofselected

isolates of C. albicans

Paired DNA(molto G+C) _relatedness%DNA

G-364 (36) +G-30 (35)... 100

G-655(36) +G-364 (36)... 100

C-448(36) + G-30(35)... 95

A-11006(34) +G-1114 (36)... 98

G-1114(36) + G-30(35)... 91

this strain (G-30, also known as CA 30) for mice were

presented by Cole et al.

(3).

Isolate G-364

(germ

tube

negative), even in conjunction with an adjuvant (gastric

mucin),wasrecoverablefrom the

kidney

of

only

1 sacrificed mouse outofthe 10examined.

Microscopic

examination of tissuesfromthis mouse revealed

negligible

tissue invasion. Therecoveredyeastisolateshowedthesamecharacteristics (i.e.,nogermtubes,proteinase

negative, etc.)

asthe inocu-lum.

The moles percent G+C ofthe DNA and DNA

reassoci-ations ofselected isolatesaregiven inTable2. ClassicalC.

albicans G-30 showed

significant (>90%)

DNA relatedness with therepresentative

morphologically

andphysiologically atypical strains, including C. albicans var. stellatoidea

(G-1114).

Karyotypes. Inpreliminary studieswith C. albicans

G-30,

the karyotype patterns obtained with pulsed-field electro-phoresis showed differences in the numberofDNA bands

(and theirresolution), varying mainly with alterationsin the

running time, voltage, and switching time. Maximal and

usuallycomparable resolution ofbandsoccurredat72to96 h with test conditions of50 mA and a 10-min switch time. Under theconditions of50 mA anda10-minswitchtimefor

96

h,

isolates G-30, G-9, G-M-44, and C-B-1071

demon-stratednearlyidentical

karyotype

patternsconsistingoffour

obvious electrophoretic mobility groups or clusters,

indi-cated as A1,2, B 3, C4,5, and D 6,7. Twoofthese strains are shown in Fig. 1, lanes 1 and 3. With conditions recom-mended by the manufacturer (Beckman) of 140 mA and a

60-s switch time for 18 h for DNA from Saccharomyces

cerevisiae (334pulsed-fieldelectrophoresiscertified) and 38 mAanda 60-min switch time for 140 h for

Schizosaccharo-mycespombe(Y-12796), 13and 3 bands,respectively,were

produced. Only 11 and 2 bands,respectively, of DNA from

these specieswere obtained with the standardconditionsfor C. albicans (datanotshown).

The

resolution

of DNA bands andcomparisonsof karyo-types were facilitated by densitometric tracings. Computer

analysis of scan data, under conditions that recognized

sevenbandsforG-30, indicatedthat the broad and diffuse A

cluster of C-402 and the D cluster ofC-B-1071 were

com-posed of two bands(Fig. 1, cluster D of lane 3 and cluster A of lane 4; Fig. 2, C-B-1071 and C-402). Karyotypes and

densitometer scans of the same strain which were prepared

separatelyandfrom

different-age

cultureswerenearly iden-tical.

Some atypical strains haddistinct

karyotypes

from those of theclassical group. This distinct

karyotype

was

reproduc-ible. Only sixdefinite bands wereresolvedforisolate

G-364

lacking

one band in cluster A; probably this is a

result

of

comigrationofbands A 1,2 (Fig 1, lane 2). Isolate C-448 had

additionalbands ingroupsBandD(Fig. 3).Asmall shoulder correspondingin location to band 10 of C-448 was observed in G-30. This slight shoulder was observed for a few other

strainsatthe same location, but our standard

densitometric

1

2

3

4 ___-_!l

A

t.

B

c

FIG. 1. Ethidium bromide-stained gel showing chromosome sizes of C. albicans DNA separated by TAFE at 50 mA with a 10-min switch time for 96 h. Lanes: 1, strain G-30; 2, G-364; 3, C-B-1071; 4, C-402.

conditions did notrecognize it as a peak except forC-448,

which was theonly strain whereband 10 was detectableon

photographs ofgels. The karyotype of C. albicans C-655

(hyphal form) was uniqueunder avariety of testconditions

because it gave a maximum of only five bands (Fig. 4).

Three serotypes of C. albicans var. stellatoidea

(G-1114,

serotypeA;A-11006, serotype B; andC-B-1034, serotypeC) producedthreedifferent electrophoretic karyotype patterns (Fig. 5).Photographsof the karyotypinggel of strainG-1114

appeared to haveonly a single band at cluster A. However,

with densitometric analysis the karyotype of G-1114 was similar to that of classical C. albicans (G-30) (Fig. 5).

0 10 20 30 40 50 60 70

MIGRATIONDISTANCE(mm)

FIG. 2. Densitometric tracings ofchromosome-size DNA bands separatedby TAFEat50mAwith a 10-min switch time for 96 h.

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0 10 20 30 40 50 MIGRATIONDISTANCE (mm)

60 70

FIG. 3. Densitometric tracings of chromosome-size DNA bands separatedby TAFEat50mAwith 10-min switch time for 96 h.

DISCUSSION

The karyotypes of strains of C. albicans determined by TAFE varied with changes in electrophoretic conditions and, in some instances, between runswith the same

condi-tions(gel-to-gel variations). A control DNA ofaclassical C.

albicans (G-30) therefore was included in each gel.

Densi-tometric tracings of gels faciitated the detection of bandsnot readilyobservedonphotographs of gels, the discernment of

broad ordiffuse bands in the gels, and the comparison of

karyotypes obtainedindifferentruns.Classical isolates of C.

albicans produceda distinctive karyotype of 7 DNA bands

in four mobility groups, whereas some atypical strains

produced differentpatternsof 5 bands (C-655, hyphalform), 9 or10 bands (C-448), or 6 bands (G-364, avirulent form).

Though the karyotypes of each of the three representatives ofC. albicans var. stellatoidea were distinct, C. albicans

A

B

G-30 1

12 34

C-655 2

0 10 20 30 40 0 60 70

20 30 40 50

MIGRATION DISTANCE (mm)

FIG. 5. Densitometric tracings of chromosome-size DNA bands separatedby TAFEat50 mA witha10-min switch time for 96h. and C. albicans var. stellatoidea demonstrated significant

DNA relatedness, supporting their conspecificity as noted

by Meyer (16). Kwon-Chungetal. (7) suggested thattypeII C.stellatoideawas asucrose-negativemutantofC. albicans

serotype A. Probably, type I C. stellatoidea strains of

Kwon-Chung etal. (7) (represented hereinby A-11006)are

derivedfromC. albicansserotype B.

In comparison of DNA of S. cerevisiae with molecules

thatmaybe withinarangeof 260to2,200 kilobases (18, 24)

c

0 10 20 30 40 50 60 70

D

G-30 2345

10 20 30 40 50 60 70

MIGRATION DISTANCE (mm)

FIG. 4. Densitometrictracingsofchromosome-sizeDNA bandsseparatedbyTAFE as follows:(A)60mA,5-min switchtime,72 h;(B)

50mA,5-min switchtime, 96h; (C)50mA, 10-minswitchtime,96h;(D)38mA,60-min switchtime, 140h.

G-30 3

C-655 2

. ..34

C-655 4

i 3

2

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andSchizosaccharomyces pombe with molecules of 3,000to 9,000 kilobases (4), we estimated that chromosome-size DNA molecules of classical C. albicans range from about

1,000 to 5,000 kilobases. Perfectet al. (24) with a

contour-clamped homogeneous-field gel electrophoresis procedure

estimatedthatchromosome-size DNA moleculesof C.

albi-cans range from 900 togreater than 2,200 kilobases. How-ever, forreasons cited in the paragraph below, particularly conformational influences, direct size comparisons of DNA bands fromdifferent species may be somewhat inaccurate.

C. albicans, which has not demonstrated a sexual cycle,

hasbeenreportedtobediploidwith strainsshowing possible

aneuploidy (2n + n) (32). Magee et al. (12) reported that

homologous chromosomesmaymigrateseparately, possibly because of deletions, translocations, or differences in the amountordistributionofrepetitive DNA. Chromosomesof nearly the same molecular weight may comigrate as one band inagel (15). Conversely, similarly sizedchromosomes

could differ inconfiguration andthe stereochemical state of

their DNA and therefore migrate differently (19). Some of

our data suggest that large DNA molecules or complexed DNA molecules in the wells may failto enter the gel. This

was possibly the case with C. albicans C-655. Also, the

DNA bands inour system did not migrate at the samerate throughout the entire gel distance. For example, the first groupofbandsentering thegel (the D cluster) in classical C. albicans contained three closely packed bands at 150 mA witha90-sswitch timefor24h;thesebandsconvergedin the

gel to two bands by 96 h. Therefore the number of DNA bands inourTAFEsystem(as noted byotherswithdifferent

systems) does not necessarily represent the number of

chromosomes.

Thecurrent statusofgel electrophoretic karyotypingof C.

albicansinvolvesvariedproceduresthatprovideDNAband

patternsof apparent value inbiotyping, but thepatternsare notcomparablebetween laboratories. Toourknowledgethis is the firstpresentation of karyotypes for C.albicans witha

TAFE procedure. It permitted distinction of strain types amongphenotypicallydiverseisolates of C. albicanswhose identification had been verified by molecular procedures.

Furtherstudies withestablishment ofkaryotypedata banks

and refinement of procedures for additional species are

required before karyotyping can be employed in practical

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