Jpn. J. Infect. Dis., 54, 229‑236, 2001

Original Article

Characterization of Phenotype‑Based Pathogenic Determinants of
Various Candida albicans Strains in Jordan
Khaled H. Abu‑Elteen*, Ali Z. Elkarmi and Mawieh Hamad
Department of Biological Sciences. Faculo, of Science and Arts. The Hashemite University,
PO. Box 330077, Zayqa 13133, Joydan(Received October 15, 2001. Accepted December 25, 2001)

SUMMARY: Sixty‑six chnical isolates of Candida albicans representing 14 different strain types were tested
for their phospholipase and proteinase activities in correlation with adherence to buccal epithelial cells (BECs)

and lethality to mice. Variationsin phospholipase and proteinase production as well as adherence to BECs wereobserved both among isolates of the same strain type and between isolates of different straintypes. All isolates
tested, irrespective of strain type, produced low levels ofphospholipase (0.5 mm for strain ‑BCD‑ and 2.7 mm forstrain ABC‑) and acid proteinase (0.6 mm for strain A ‑‑ E and 2.2 mm for strain ‑‑C‑). A correlation was noted
between adherence, phospholipase and proteinase production, and lethality to mice. C. albicans isolates, which

adhered most strongly to BECs, exhibited higher levels of phospholipase and proteinase activities as well as

higher pathogenicity･ This was most evident in strain type ‑‑C‑‑, which exhibited higher adherence ability(me.an
7 1 7 ± 2 1 yeasts/1 00 BEC), and proteinase activity(mean 2.2 mm), and relatively higher phospholipase activlty(mean 2.4 mm) compared with those or other strains. Additionally, this type was more prevalent and showedslgni点cantly higher levels of tissue colonizationinthe liver, kidneys, and spleen compared with most other strain

types in both subjects with healthy dentates and complete denture wearers. These results clearly demonstrate theslgnificant role of phospholipase and proteinase activities on the adherence of C. albicans and their overall

influence on the pathogenesis of Candida species.

immune system (8,12,17,18). The co汀elation between the

INTRODUCTION

degree of virulence and the levels of Saps production in both

Candida albicans, a human opportunisticfungus, is theC. albicans clinical isolates ( 1 1 , 1 9)and laboratory strains withcausative agent of several localized and systemic infections

altered proteinase levels (20,2 1 ) supports a potential role ofespecially in individuals with compromised immunity (1 ).

this family of enzymes in C. albicans virulence. lt has also

The increased incidence of candidal infections, mainly due tobeen suggested that highly proteolytic strains of C. albicans

C. albicans, in patients being treated for cancer, Organ trans‑

isolated from patientswith AIDS are more pathogenic thanplantation, and other forms of immunoincompetence (2,3 ),strains with less proteolytic activity (22,23), The importanceinaddition tothe emergence of azoleanti血ngal drug resistanceof Saps in establishing systemic and vaglnal C. albicans(4,5), make the effort to understand the mechanisms of C.
albicans pathogenesis all the more important. A number ofhas been shown that the proteinase inhibitor pepstatin A

infection is well demonstrated by several recent findings. It

putative virulence factors have been suggested in the enhance‑(a hexa peptide from Streptomyces) as well as espartic

ment of C. albicans pathogeniclty. These include yeast‑to‑proteinase antibodies play a protective role in experimental

hyphal transition, phenotype switching, thigmotroplSm,

animal models ( 1 8,24,25)･ Furthermore, proteinqse‑deficientmolecular mimicry, adhesion factors or surface hydropho‑

mutants were fわund to have attenuated vinllence ln an animal

bicity, phospholipase production, and the production or anmodel or disseminated candidiasis (20,26,28).

Adherence of Candida to the epithelial cells represents a

extracellular secreted aspanyl proteinase (Sap) ( 1 ,6‑ 12).
Phospholipases represent a class of lipolytic enzymes,

crucial step ln the pathogenesis of candidiasis in that it isacylhy血olases and phosphodiesterases, that have been exten‑

the initial step in colonization and subsequent infection ( 1 ).

sively characteri2:ed (9). C. albicans secretes lysophospholipase,

Furthermore, the phenotypic variability of Candida spp. andtransacylase,and exhibits phospholipase A, B,and C activities

strains may have some bearing On the differential pathogenic(1 3,14). These enzymes may promote penetration of host cellpotential of such species, especially with regard to adhesion

membranes and influence the adherence potential of the yeast

ability and toxin production (1 1 ,1 5,29).

(9,12,15)･ It has been shown that the level ofphospholipa苧e

1n this study, we characterized 66 clinical isolates of C.

activlty Secreted by each particular isolate of C. albicans IS

albicans, representing 14 different strain types in terms of

directly correlated with the pathogenicityofthe isolate ( 1 3, 1 6).
A correlation was also found between phospholipase activity,
cellular adherence, and pathogenicityof C. albicans ( 1 5).

their phospholipase and proteinase production in co汀elation

with the capacity to adhere to buccal epithelial cell (BBC)and their degree of lethality ln mice. The influence of these

Hydrolytic enzymes such as extracellularaspa〜l proteinase

putative virulence factors on the prevalence of the studied
strains in denture stomatitis patients was also evaluated.

enzyme (Saps) are believed to enhance the ability of C. albicans
to colonize and penetrate host tissues and to evade the host

MATERIALS AND METHODS*Corresponding author: Tel/Fax: +962‑6‑4 1 28772, E‑mail: Salma

Isolates: A total of 66 clinical isolates were obtained from@hu.edujo
229

the oral cavities of subjects with healthy dentates and complete

sterile PBS by repeated centr血gation.

denture wearers. Methods of isolation, identification, andstrain differentiation used in this study have been described

gentle rubbing of the mucosal surface of the cheekswith a

BECs were collected from healthy human volunteers by

elsewhere (30,31). A resistogram, a list of the chemicals tosterile tongue depressor. The epithelial cells were washedwhich the isolate is resistant, orthe strain types was based on

twice with sterile PBS and collected by centrifugation (500 g,

resistance of isolates to sodium selenite, boric acid, cetrimide,

10 min)･ Adherence assays were perfわrmed as previously

sodium periodate, and silver nitrate. Each of the five chemicals

described (34). Yeast cells were suspended in sterile PBS at

used is assigned a letter of the alphabet (From A to E), and1 X 107 cells ml‑); BEC were suspended in sterile PBS to a

the letters attached to the chemicals to which an isolate isconcentration or2 × 105 cells ml 1. Two ml of the yeast

resistant are then used in the descriptlOn Ofthe strain. Thus,suspension was mixed with 2 ml of the BBC suspension in a

isolates that are resistant to chemicals B, D, and E are describedsterile screw‑仁apped bottle. The mixture was shaken at 37oC

as strain ‑B‑DE (3 1). Candida albicans ATCC 36082, kindly

for 2 h then filtered through a 20 Elm filter (Retsch, ldar‑

provided by M. A. Ghamoum (Center fわr Medical Mycology,

Oberstein, Germany) to remove noヮIadherent yeast cellsI The

Mycology RefTerence Laboratory, University Hospital of Cleve‑land, Ohio, USA), was used as a control strain. All isolates
were maintained on Sabouraud Dextrose Agar (SDA) (Difco,

BEC on the filter were washed twice With 5 ml volumes ofsterile PBS and finally suspended in 5 ml of the same buffer.
A血op of this suspension was mounted on a glass slide, then

Detroit, Mich., USA) and stored at 4oC.

aiトdried, heat一缶xed and stained with cIYStal violet fわr 1 min.

Phospholipase assay: Phospholipase secretion from the

The adherence was detemined microscoplCally by countlng

various isolates of C. albicans was measured by the method

the mean number of yeast cells adhering to every 100 BEC.

of Price et all (32)･ The strain.s were cultured for 1 8 h at 300C

Each assay was carried out in duplicate, and student t‑testin Sabouraud broth containlng 2% (wt/vol) glucワse in an

was used to evaluate the differences in the adherence values;a P value of<0.05 was considered significant.

orbital shaker. Cells were harvested by centrifugatlOn (5000g, 30 min), washed twice with phosphate‑buffered saline

Mousevirulence test: Twelve‑week‑old Balb/C mice were(PBS), and resuspended at a denヲity of 1 X IOS cells ml‑)I Tenpurchased from the Jordan University Of Science and Tech‑microliters of the yeast suspenslOnS Were spotted onto SDA
plates (6.5% SDA, 5.84% 【wt/vol】 NaCl, 0.55% 【wt/vol】

nology, Irbid, Jordan. Mice wereused to establish a breedingcolony at the Hashemite Universityvivarium. Ten‑ to fourteen‑

CaC12, SpPPlemented with 8% lvol/vol] egg yolk emulsion

week‑old Balb/C male mice with an average weight or 20 g

[Oxoid Ltd., Basingstoke, UK]) and the diameter of thewere used. C. albicans cells of each isolate were grown

preclpltation zone around each colony was measured a鮎r

separately at 37oC for 24 h on a rotary shaker in 50 ml quanti‑

incubation of the plates at 37oC fわr 72 h. Each assay wasties ofYNB supplemented with 2･5% (wt/γol) glucose andcarried out in triplicate, and the results were expressed as theadjusted to pH 6.0. Cells werethen harvested by centrifugation,

triplicate mean. Phospholipase activity (Pz) was detemined

washed twice with sterile PBS, and re‑suspended in 10 mlas the ratio ofcolony diameter to the diameter of the dense

PBS. This suspension was serially diluted, and the cells,

white zone ofprecipltation uslng the followlng formula:counted with a hemocytometer, were standardized to 1 X 107

cells m1‑I. Ten microliters of this suspension wereused forpz‑ 1‑i where

each intravenous叩eCtion through the tail vein; a group or

four mice were used for challenge by each respective isolate.

A ‑ diameter of colony + dense white zone ofprecIPltation
B ‑ diameter of colony.

Tissue colonization: Twenty‑four hours post inoculation
with C. albicans, the liver, kidneys, and spleen of injected

Proteinase assay: Proteinase production was evaluated byanimals were removed under sterile conditions. Superfluous

the method of Staib (33). Three flasks per isolate, each contain‑

tissue was trimmed away, and the remainlng tlSSue Was thenlng 50 ml orSabouraud broth, were incubated at 260C in an

homogenized in 5 ml PBS in a sterile glass homogenizer.

orbital shaker for 24 h. Cells were harvested by centrifugation

Serial 10‑fold dilutions in PBS were prepared from these(5000 g, 30 min), washed with P良S and resuspended at a

homogenates. One ml samples of approprlate dilutions wereplatedintriplicate onto SDA plates containlng Chloramphenicol

denslty Of 1 X lo島 cells m1‑.. Ten microliters of the cellsuspension were placed onto test medium (1% bactoagar(50 mg l 1), and the plates were le氏to dry then incubated at

37oC for 24‑48 h, after which the number of colony‑forming
units (CFU) (one colony ‑ 1 CFU) was determined. Values
were expressed as the mean CFU per organ based on datafrom four animals.
StatiStical analyses : Che‑way analysis of variance (ANOVA)lDifco], 0. 1% lwt/vol] KH2PO4, 0.5% lwt/vol] MgSO4, andl% 【W〟vol] glucose, pH 5.0) Containing 0. 16 (wt/vol) bovine

semm albumin (Sigma, Poole, UK) as the sole nitrogen source.
ARer 5 days of incubation at 37oC, the plates were fixed with

20% trichloroacetic acid and stained with 1.25% amido black(90% methanol, 1 0% acetic acid). Destaining was performed
with 1 5% acetic acid, and the clear zones around each colony

was camied out to test for slgnificant differences between thewere measured and used in the determination of the Pz value

the adherence of each C. albicans strain type. The examined

as described above. Each assay was carried out in triplicate

null (Ho) and altemative (Ha) hypotheses were:means or phospholipase and acid proteinase production, andon two separate occasions, and results were expressed as theHo:〟1‑〟2 ‑〝3‥.andtriplicate mean.

Ha: one of the means is different from the rest.

Adherence assay: Ovemight culture of C. albicans was
conducted at 37oC in a 0.67% (wt/γol) yeast nitrogen base

Additionally, multiple comparison uslng Fisher's least(YNB) (Difco) supplemented with 2.5% (wt/vol) glucose.

significant difference (LSD) with a ‑ 0.05 was camied out to

Flasks containlng 50 ml of the same medium were inoculated

determine which means were slgnificantly different from thewith I ml of the ovemight culture and grown fわr 24 h in arest.

shaking water bath (1 80 rpm) at 37oC. The cells were harvested
by centr血gation (1200 g, 10 min) and washed twice with230

range was 0.54 mm fわr strain ‑BCD‑ and 2.7 mm fわr strain

RES UIXS

ABC一一･ Overall, the level of phospholipase activities or all

Extracellularenzyme productiorL: A total of 66 clinicalC･ albicans isolates was consistently lower than that produced

isolates of C. albicans comprlSlng 1 4 resistogram strain types

by the ATCC 36082 C. albicans control strain (P2 ‑ 4. 1 mm),were screened for secretion of phospholipase and acidAs shown in ≠ble 2,仇e one‑way ANOVA test and LSD test ofproteinase. Fifty‑nine isolates were tested for production ofmultiple comparison clearly indicated a statistically significant

phospholipase secreted under standard conditions. Substantial

difference betwe?n strains ABC一一and A‑lD‑ (P ‑ 0･0300),

variations among various C. albicans isolates of similar anddifferent strain types were observed (Table 1 and Figure 1).

and between stralPs ABC‑‑ and ‑BCD‑ (P ‑ 0･004)･ Strain‑‑C1‑ Showed a statlStically slgnificant difference in comparison

The mean phospholipase production (Pz) of 59 C. albicans

with strains A1‑D‑ (P ‑ 0.009) and ‑BCD‑ (P ‑ 0.0005).

isolates tested was I.91 mm irrespective of strain type; theAdditionally, statistically slgnificant dimerences were observed

Table L Correlation ofphospholipasc and prolcinasc production with adhcrcncc

in various strain types or C. albicanLV
Phosphol ipasc Protcinasc Yeast cell s

Strain lsolatc No.

(Pz)

in

mm

(Pz)

in

±SEa

mm

adhering

to±SEa lOOBEC±SE

C̲ albica17S

ATCC 36082
ABCD‑

12345′b70ノ 12345′▲U70ノ12tJ45AV7RU0 12つJ45′hV70n 1

4.3±0.2

3. 1±0. 1

420±1 61.9±0.01

1 21 01 0 2つJ2 12一I1 21 214‑ 0

54 tJつ4 5 tJ 4 丘U 075 ∠U85740n 70 2 32 ‑ 1‑2 1

慧洲批慧酬肌蜘捌慧批洲酬 洲 酬 軌釧 洲酬 NT 州側酬鮒NT批慧NT等

1.4±0.01
1.8±0.03

ABC一一

1.9±0.03
3.0±0.2

1 0 1 102I･ 1 I･ 1 0 1 0 1

3.2±0, 1

3.0±0. 1

1‑1‑l ‑

1.6±0.2

3.3±0.3<;

Lf C D ‑ E

1 1‑･ 1 12 2 2 2tJ tJつ 3 tJつ J4

2.5±0. 1

0.9±0. 1

1.4±0.2

3,1±0.7

I.1±0.04
3.1±0.01
1.5±0.02
3.1±0.01
3.4±0. 1

0.08±0.00 1
I.9±0. 1

0.07±0,01
2.6±0.2
4.2±0.3
4,1±0.1
4.8±0. 1

2.4±0.01

1.6±0.001

0,9±0.02NT
1.4±0.2
2.5±0.01
1.5±0.01

0.07±0,001
0,9±0.002
1.7±0.01NT
1.5±0.002

1.9±0.001
0.08±0.00 I

1.6±0.INT

231

つJ 2 0 0 2 2 2 0

三 ミニFf:.こi'IJF三諾憲誓.皿itj;'こ:.三二 至言諾≡憲ュニi'Y, 5. :読Table I.‑Continued
Pbospholipase Proteinase Yeast cells
StrainIsolate

No･

(Pz)

in

mm

(Pz)

± SEa

A一一一E

DU

C DE

adhering

to

100 BEC± SE

42

3,0±0.2

0.8±0.00 1NT

2.3±0.01

0.4±0.0 1

210±2144

1.4±0.01

0.6±0.00 1

200±2 14 4 4 5 ′nV 70.9±0.01

0, 6±0. 002

1 90±222.0±0. 1

1.9±0.01

310±21

3.0±0.06

I.0±0.02NT

0.07±0.00 1

1.7±0.02

0 0ノ 0 ‑ 2 tJ 4 5 ′0 7NTNT0.8±0.0 Ⅰ

2.0±0.0 1NTNT

0.6±0.00 1
1. 1±0.04

5 58 凸70 1‑ 2 つJ 4 5 ′0NT
180±19NT

I 90±212.3±0.5285±270. 6±0.002

0.3±0. 002

NT

1 ,4±0.00 I

140±16NT

120±150.8±0.03

0. 1±0.001

140±1 71.4±0.2

0.9±0.003

165±160.9±0.001
エU ′0 ′▲U ′0 ′h▼ ′LU ′▲U165±192 40±2 00.07±0.00 1

NT

D

mm

43

44555555 55

a

in

± SE8

NT

).6士0.02

NT

1 ,4±0.06

1.3±0.02

NTNT
1 95±20180±14NT

140±181.7±0.00 1

220±1 90.6±0.001

3.0±0.09160±171.6±0.01

1.2±0.001

1 80±20

I.5±0.02

0.4±0.00 1

1 90土222.0±0.03

a Values are the average of three separate experiments ± SE.

NT‑Not tested,

between strain A‑D‑ and the respective strains ABC一一and

1 ). The result of the one‑way ANOVA (Table 2) and LSD test

‑C‑. This strain type also showed a significant differenceof multiple comparison carried out to determine possible dif‑from strain AB‑D‑ (P ‑ 0.037). Overall, the correlation

ferences between variousC. albicans resistogram strain types

between phospholipase activities and adherence was goodshowed a statistically slgnificant difference in adhesion(r‑ 0.49,P<0.001).

between strain ‑‑C‑‑ and all other strain types (P ‑ 0.002).

strain types was 1.72 mm, which ranged from 0.6 for strain

Strain ABCD‑ showed a statistically significant difference
from respective strains A‑‑DE (P ‑ 0.01 5), A‑‑D‑ (P ‑ 0.0004),
A‑E (P ‑ 0.008), ‑BCDE (P‑ 0.025), ‑BCD‑ (P ‑ 0.0007),

A一一一E to 2･2 mm fわr strain一一C一一. Acid proteinase production

and strain ‑‑‑ (P ‑ 0･0005)･ Strain ‑‑て‑ Showed a statisticallyby the ATCC 36082 C. albicans control strain was 3.1 mm

significant difference from respectlVe Strains ABC‑‑ (P ‑0.03), ABID‑ (P ‑ 0.0001), and strain ‑CM (P ‑ 0.000). The
mean adherence value of C. albicans isolates per 100 BECs
varied from 130 adherent yeast cells in strain ‑B‑D‑ to 717

The mean of acid proteinase production established uslng

a group of53 C･ albicans isolates representlng all resistogram(Table 1 and Figure 1). No statistically significant differenceswere fわund among the 14 strain types in terms of mean

proteinase production (P ‑ 0. 196). The largest zone (4.4 mm)
was fわund in strain HCH, isolate # 25; this isolate exhibited

adherent yeast cells in strain一一C一一(Figure 1 ). Strains AB‑D‑,

the highest levels ofphospholipase production (4.8 mm) and

ABCD‑, and ‑‑C‑‑ showed high levels of adherence to BECs
as compared with that of the ATCC C. albicans control strain

the highest degree of adherence to BBC (982 ± 23) whencompared with other isolates of the same strain type and with

(420 ± 16). Overall, the correlation between the phospholi‑

different strain types, respectively (Table 1 )･ The c?rrelationpase and proteinase activities and adherence to BECs was

between proteinase production activlty and capaclty tO ad‑

good. However, the co汀elation between the phospholipase

here to BECs among all 53 C. albicans isolates tested wasand proteinase activities and a曲erence to BECs in strain一一C一一found to be good (r ‑ 0.37,P< 0.014).
Adherence of various strain types to BECs in vitro: The
adherence ability of a representative group of 58 selected C.

higher prevalence rate than those of most other strain types.

albicans isolates representing all 14 resistogram straintypes

in the murine system: The pathogenlClty Of23 isolates fromand having variable ranges of phospholipase and proteinase

activities was dete‑ined･ As was the case in both phosph0‑

12 C･ albicans strains was evaluated 24 h post intravenous
inoculation into adult Balb/c mice. As shown in Table 3, the1ipase and proteinase activities, Variation in adherence wasmean CFU recovered from tissues of mice inoculated withobserved both among lSOlates of the same strain type anddifferent isolates was calculated. C. albicans was recovered

between isolates of different strain types (Table 1 and Figure

from the liver of all animals infected irrespective of the isolate

was not significant, although this strain (type ‑C‑) had a

Pathogenicity and Lethality of var!0.us C･ aEbicans strains

232

A)

7, q> 5 4 3 2
0 0 0 0 0 00 0 0 0 0 0

3山gO LO10U aJOuPeueaM

ABCD‑

uo!pnpoJd￠st2u!Ot0Jd

00∧UOO

1111■

ABC一一

A8‑D一

･一C‑

A一一DE

A一一D･

A‑∈

･BCDE

･

BC

D･

8642 0

ABCD.

ABC‑

A8‑D･

ABCD.

ABC一一

‑(>‑

A･‑DE

A‑‑D･

A一一･E

‑BCDE

･BCD･

uo!73nPOJdOst2d!10LJdsoLJd
0. 0. 0. 2 一｢ 一つ 0

5 0

AB‑

D･

‑C･‑

A･‑DE

A‑･B

A一一一∈

･BCDE

‑BCX>

Various C. albicans strains

Fig. 1. Plot ofadhcrence to buccal epithelial celts (A), proteinasc production (B), and phospholipasc production (C) by
variousC. albicans strains.

Table 2. One‑wayanalysis of variance (ANOVA) results for phospholipase and proteinase production
and adherence

Effect df Efrcct MS Effect df Error MS Error F P‑Icvcl
Phospholipasc

43*01010314*

21558869*

Proteinase

99*0.0 1 0396

0.026391*

40

0.007日71.460775

Adberencc

9*2969 1 8.2*42*

1 2406.85*

df ‑ dcgrecs offrecdom

MS‑ Means sum orsquares
F‑F‑test*‑ Significant difference

233

23.93 1 80*

0･018790*
0. I 96002
0.000000*

Table 3. Number ofcolony‑forming units (CFU) ofycast in mousethat variations in these virulence factors exist both among

tissues 24 h after separate intravenous challcngc using 23 different
isolates of C albicans.

isolates of the same strain type and between isolates ordifferent resistogram strain types. All C albicans isolates

Straintype Isolate No,a Kidneyb Liverb Splccnb
ABCD‑

2

0.4±0.07

1±0. 19tested in this study, irrespective of strain type or source, were0.3±0.03

capable of secretlng phospholipase. lt has been shown thatthe quantlty Of this enzyme varied with the specific isolate

30.8±0. 1 6

3.6±0.26

0.58±0. 14

5 ′LU

0.3 1±0.04

1±0.2

0. 1±0,04

0.6±0. 14

5.9±0.76

0.4±0. I 8

and is correlatedwith the site of infection (9). Barrett‑Bee etal･ (15) were the first to evaluate the role ofextracellular
candidal phospholipases in vimlence by uslng a munne model

13

4±1.8

9.8±0.94

3. 1±0.34

I 8

4.2±2. 1日±1.5

3.4±0.5625

5.8±2.5

20±2.926

3.7±1.9

5.6±0.93.9±1.8270.95±0.06

1.9±0.26

0.6±0.04

albicans was explored by lbrahim et al. (10). These authors

showed that blood isolates of C. albicans produced higherof candidiasis. The C. albicans isolates (four strains of C.
albicans) that adhered most strongly to BEC were most path0‑

4.2±3.5

genic to mice and had the highest phospholipase activities(15). The role orphospholipases in the pathogenesis of C.
A A D DE Il l l I

3 1

1.4±0,24

2,2±0.37

1.0±0.8533

1.6±0.3

5, 1±0.22

1.9±0.28

extracellular phospholipase levels than did commensal isolates.

37

1.5±0. 1

4.3±0.09

1.2±0.3

Furthemore, it was shown that isolates with high extracellular40…C C E D D
A CP 良EIl4,6±0.4

0.6±0.07430.35±0.09

0.9±0.0 1

3.0±0.48

0.28±0.04

46

0.39±0. 1 1

4.3±0.55

phospholipase activity were not (1 0). ln this context, it was
48

ND

53

0.8±0,06

55
nD B 叩
lII

EP…a

一

phospholipase activity Were invasive in the infant mousecandidiasis model, whereas isolates with low extracellular

57

ND

ND

concluded that of the common virulence factors (phospholipase

ND

and proteinase production, adherence, germination, growth rate,

0.6±0.07

and ability to damage endothelial cells), Only extracellular

ND

phospholipase activitywas found to be predictive of mortality

1.0±0.29
1.6±0.29
0.36±0.09

0.64±0. I 2

1.2±0.24(10). Results presented here collaborate previous findings;

ND

namely, that strain type一一C一一seems to have a substantial
58

ND

ND

ND

number of isolates with relatively high phospholipase activity61

ND

0.3±0.

163

0.4±0. 14

3, 1±0.4566

0.65±0,23

1 ,6±0.42

ND

and greater adherence properties as compared with most strain

0,01±0.025

types; thistype also seems to have greater capacity to colonize

ND

in tissue. Interestlngly, Some isolates of this strain type hada Isolates, refer to those in Table 1, column 2.

low phospholipase activlty; this paradoxical finding cannot

bRcsu]ts arcthe mean CFU /organ i: SD X 103.

be readily explained. Nonetheless, the overall correlation

ND‑ Not detectable.

between the phospholipase activityof the clinical C. albicans
isolates representlng the 14 resistogram strain types and theadherence to BEC was good (r ‑ 0.49, P ‑ <0.001). Althoughthese findings suggest a correlation between phospholipase

Or strain type used fわr inoculation. The only exceptlOn Was

isolate number 58, ‑B‑, where clearance in all tissues tested

activlty and candidal virulence, the strains used were not

was evident. The kidneys ranked second in tems ofcoloniza‑

genetically identical. lt is worth mentionlng that future
research should include an evaluation of the role orphosph0‑

tion, followed by the spleen.

Mice Injected with C. albicans strain ‑CM gave the maximal

1ipases and candidal virulence and should be confirmed byyeast colon)‑ counts, followed by those recovered from mice

infected with strain A‑‑D‑. Isolate number 25, strain HC‑‑,

the use of an isogenic strain pair that differs only in phosph0‑
1ipase production. Molecular cloning of the candidal gene(S)which exhibited high levels ofphospholipase and proteinase

encoding the extracellular phospholipases is the essential firstactivities and had the highest adherence capability whenstep ln the molecular genetic dissection of the role of thesecompared with most other isolates, gave the maximal yeast

enzymes in pathogenesis.

colonization in the organs tested ( Table 3). Only isolate number

Proteinase production is believed to enhance the ability of

53from strain ‑BCD‑ showed colonization in all organs tested;

C. albicans to colonize and penetrate host tissues and to evade

the rest (isolates 48 and 55) showed colonization only in thethe host immune systPm (8,12)･ Previously it was indicatedliver. The remainlng Strain types showed variable degrees orthat the aPpartyl protelnaSFS (Saps) are rare or absent in n?n‑tissue colonization in the liver, kidneys, and spleen.

pathogenlC Strains or specleS Of Candida (1 2). Mutant stralnSof C. albicans that do not secrete the proteinase enzyme show
significant reduction in lethality to mice (20,35,36). TheDISCUSSION

presence of co汀elation between the level or proteinase activ‑

Understanding the mechanisms of C. albicans pathogenesis

ity in clinical C. albicans isolates (1 1,19,29) or C･ albicans

has become more important in recent years as the incidence oflaboratory strains with altered proteinase levels (20,2 1 ) andserious fungal infections, particularly in immunocompromised

C. albicans virulence supports the contention that thispatients, is on the rise (I)･ Here, We sought to determipe any

enzyme may have some role in influenclng the degree ofrelationships that might exist between the productlOn Of

virulence of C･ albicaTs･ Ghannoum and Abu‑Elteen (1 1)

phospholipase and proteinase and the adherence potential ordemonstrated that partlCularly strong proteolytlC Strains orvarious C. albicans isolates with the degree ofprevalence of

C. albicans adhered more readily to human BEC in vitro. Also,

specific resistogram strain types in both healthy dentates andVariation in proteinase production and adherence has been

complete denture wearers, and, simultaneously, to attempt toshown to exist both among Isolates of the same strain typecharacterize some of the phenotype‑based pathogenic deter‑

and between different straintypes. Similar findings have beenminants of C. albicans prevalent in Jordan. The results showed

reported by other researchers (29)･

234

4. Berrouane, Y. F., Herwaldt, L A. and Pfaller, M. A. (1999):
Trends in antifungal use and epidemiology ofnosocomial

All C. albicans strains evaluated in this study were shown

to be capable of secretlng acid proteinase. A reasonably goodyeast infections in a universityhospitalJ. Clin. Microbio1.,

co汀elation between proteinase levels and adherence to BBC

was obtained for the 53 selected C. albicans isolates represent‑

37, 53ト537.

ing all the resistogram strain types (r ‑ 0.37, P <0.014). Strain

5. Rex, J. H., Rinaldi, M. G. and Pfaller, M.A. (1995) Resis‑

type一一C‑, which showed relatively high enzyme activity

along with greater adherence properties as compared withtance of Candida species tofluconazole. Antimicrob.
Agents Chemother., 39, 1‑8.

other strain types, showed higher levels of tissue colonization.

6. Cutler, J. E. (1991): Putative virulence factors ofCandida

albicans. Annu. Rev. Microbio1., 45, 1 87‑218.

This was clearly evident in isolates exhibiting high phosph0‑
1ipase and proteinase levels combined with higher adherence

7. Wu, T., Wright, K., ‖urst, S. 氏 and Mo汀ison, C. ∫. (2000):

capabilities. In this regard, adherence of C. albicans to human

Enhanced extracellular production of aspartyl proteinase,muc?sa (28,37), epidermal comeocytes (38), and epidemala virulence factor, by Candida albicans isolates fbllowlng

keratlnOCyteS (39) has been shown to be i血ibited by pepstatin

growth in subinhibitory concentrations of nuconazole.

A, a hexa peptide from Streptomyces with strong anti‑Saps

Antimicrob. Agents ChelllOther., 44, 1 200‑ 1208.
8･ Hube, B. (2000): Extracellular proteinases of humanpotential. This suggests that surface proteins may be targets of

the aspanyl proteinases. The role of Saps as virulence factors

pathogenicfungi･ p. 126‑137. In Emst, J. F. and Schmidt,
A･ (eds･), Dimorphism in Human Pathogenic andwas confirmed recently by the finding that the virulence ofnull mutants, Sups 1‑3 and a triple mutant of SAPs 4‑6 Were

Apathogenic Yeasts. γol.5･ Karger, Base1.

9. Ghamoum, M. A. (2000): Potential role ofphospholipases
in virulence and fungal pathogenesis. Clin. Microbiol.
Rev., 13, 122‑143.
10. Ibrahim, A. S., Mirbod, F., Filler, S. G., Bamo, Y, Cole,G. T., Kitajima, Y., Edwards Jr., J. E., Nozawa, Y. and
Ghamoum, M･ A･ ( 1 995): Evidence implicating phosph0‑
1ipase as avirulence factor of Candida albicans. Infect.

all attenuated in a systemic mouse infection model, and agulnea Plg infection model,and in terms of adherence to human

BECs (26‑28).

Strain type ‑‑C‑‑ ranked first among the 14 straintypes intems of proteinase and adherence values and was third interms of phospholipase production; desplte this, few of theisolates belonglng tO this strain showed low levels with regard

to all three parameters. At face value, this suggests that, forlmmun., 63, 1993‑1998.

the most part, this strain type has a much higher tendency

1 1. Ghannoum, M. A.andAbu‑Elteen, K. H. (1986): Correla‑

towards pathogeniclty. This may also partly explain thetive relationship between proteinase production, adherence

obseⅣation that strain type一一C‑ is more prevalent than otherand pathogenicity of various strains of Candida albicans.

strain types in bothsubjectswith healthy dentates and complete

J. Med. Vet. Myco1., 24, 407‑413.
12. Chaffin, W., Lopez‑Ribot, ). L., Casanova, M., Gozalbo,
D. and Martinez, ). P. (1998): Cell wall and secreted

denture wea.rers (3 1 )･ lt is worth mentioming that strain ‑C‑‑
was predomlnant in cancer patients undergolng therapy, while

the least predominant was strain A‑C‑ (40).

proteins of Candida albicans: identification, function,and expression. Microbiol. Molecular Biol. Rev., 62, 130‑

In conclusion, the results presented here indicate that a180.

strain‑dependent correlation between phospholipase and13. Mirbod, F., Banno, Y., Ghannoum, M. A., lbrahim,

proteinase activlty and adherence to BECs on one hand, andcontribute to the pathogeniclty Of C. albicans. That said,

A. S., Nakashima, S., Kitajima, Y., Cole, G. T. andNozawa, Y. (1 995): Purification and characterization oflysophospholipase‑transacylase (h‑LPTA)from a highly

several other factors not addresseed here, such as host immune

virulent strain of Candida albicans. Biochem. Biophys.

tissue colonization on the other, lS Present. The effect of theseparameters, separate or di飽rentially‑combined, may variably

status, therapy ( ) ), hydrophobicity, and environmental condi‑

Acta, 1257, 18ト188.

14. Hube, B., Stehr, F., Bossenz, M., Mazur, A., Kretschmar,
M. and Schafer, W. (2000): Secreted lipases of Candida

tions that promote adhesion and production or phospholipase

and proteinase (41), are not to be discounted.

albicans : clonlng, Characterization and expression analysis

ofa new gene family with at least ten members. Arch.

ACKNOWLEDGMENTS

Microbio1., 174, 3621374.

This work was supported by a.research grant (KH Abu‑
Elteen 98/99) from the Hashemlte Universlty College ofGraduate Studies and Scientific Research.
The authors wish to thank Drs. N. lsmail and M. Mubarak

15. Ba汀ett‑Bee, K., Hayes, Y, Wilson, 良. G. andRyley, J. F.

(1985): A comparison ofphospholipase activity, cellularadherence and pathogenicity ofyeasts. J. Gen. Microbio1.,
131, 1217‑1221.

16. Leidich, S. D., lbrahim,A. S., Fu, Y., Koul,AJessup, C.,

for their critical reading of the manuscnpt･Vitullo, J., Fonzi, W., Mirbod, F., Nakashima, S., Nozawa,
Y･ and Ghannoum, M･ A･ (1998): Clonipg and disrup‑

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