Vol. August

179,

No. 30,

BIOCHEMICAL

1, 1991

AND

BIOPHYSICAL

RESEARCH

Pages

1991

OVEREXPRESSION

OF THE ACTIN GENE IS ASSOCIATED HORPHOGENESIS OF GANDIDA ALBXGANS Vijay

Molecular Jawaharlal

Received

COMMUNICATIONS

July

Paranjape

Biology Nehrll

17,

and

Laboratory,School University, India

Asis

New

UITH

423-427

THE

Datta of Life Sciences, Delhi 110 067,

1991

A progressive increase in the synthesis of actin mRNA was observed by Northern blot analysis, when cells were induced form germ tubes at 370c by N-acetyl-D-glucosamine. to trifluoperazine, a calmodulin inhibitor, or Presence of at 25°C.or by N-acetyl-Dincubation of cells replacing glucosamine with glucose which inhibited germ tube formalowered this synthesis. Furthermore, in vitro tion translation of total RNA revealed an increase in the synthesis of actin (45 kDa) during germ tube formaThese results suggest for the first time the tion. that q orphogenesis expression of actin gene is regulated during of G. albicans. 0 1991 Academic Press, Inc.

Candida a I bicans, a pathogenic dimorphic yeast. can grow in yeast or hypha form depending on environmental conditions (Odds, 1988: Datta etal., 1989). Germ tube, an intermediate stage in yeast to hypha transition, can be induced by a number of fa.ctors including N-acetyl-Dglucosamine (GlcNAc) (Shepherd etal., 1980; Natarajan etal., There are several identifica1984). reports on the tion of morphology specific gene products in C. albicans by separation of yeast and hyphal cytoplasmic proteins using oneand twodimensional polyacrylamide gel electrophoresis (Odds, 1988). Unfortunately these proteins have not been characterised and their role in morphogenesis not known. is Hence a direct analysis of gene transcription investigation of RNA in yeast and germ tube by would help in the identification of morphology specific genes

in

C.

albicans.

We had

earlier reported that calcium and calmodulin regulate C. al bicans morphogenesis in (Paranjape etal., 1990) and other fungi (Paranjape and Datta, 1990). Calcium and calmodulin are aiso known to regulate cytoskeletal organization (Manalan and Klee. 1982) and actin polymerization (Greer and Schekman. 1982; Sobue etal., 1982). Actin granules are associated with the growth zones of yeast and of c. albicans (Anderson and Soil, 1986). It was hypha therefore of interest to investigate whether calmodu!in 0006-291X/91 423

$1.50

Copwight 0 I991 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol.

179, No. 1, 1991

is

BIOCHEMICAL

involved

in

thereby

controlling

Northern

blot

total

RNA

gene

C.

AND BIOPHYSICAL

regulation

of

we

gene

have

and

examined

expression

In

morphogenesis. hybridization

during

actin

RESEARCH COMMUNICATIONS

in

the

GlcNAc-induced

this

vitro

expression

germ

of

tube

and

paper, translation

by of

actin

formation

in

albicans. EXPERlflENTAL

PROCEDURES

C. albicans SC 5314 was maintained on a medium containing 1% yeast extract, 2% glucose and 2% peptone, 2% agar (all w/v). The cells were grown for 17 h in a 1 iquid 0.3% medium containing 1% glucose, 0.5% peptone and KH2P04 and then transferred to a new medium (with half the concentration of glucose) and grown for 11 h into stationary phase. Germ tubes were induced as described (Shepherd etal., 1980; Natarajan etal., 1984). Approximately 5x107 ccl Is/ml were incubated at370Cin a 20 an inidazole/HCl buffer (pH 6.6) containing 0.2 q fi tlnCl2 and 5 mfl GlcNAc as inducer. Total RNA was isolated at various times during germ tube formation by a published procedure (Chomczynski and Sacchi, 19871. RNA (B-10 fg) was denatured with glyoxal, and electrophoresed on 1.2% agarose and transferred to GeneScreen Plus (DuPont, USA) mergel brane. Prehybridization, hybridization and washing conditions were as described by the supplier of the membrane. The DNA used to prepare probe (Feinberg and Vogelstein, C. albicans 1984 1 was a 1.49 kb Cla I-EcoR 1 fragment of actin clone which was obtained from Dr. Joachia F. Ernst, FRG. It codes for a single aRNA species of about 1.5 kb (Losberger and Ernst, 1989). The blots were exposed to with a preflashed Kodak XAR film for l-5 days single intensifying For hybrid selected translation of screen. eta1 (19791 was actin mRNA. the procedure of Rlicciardi followed. Hybridized RNA was eluted and translated in vitro CB5Sl-labeled translated in rabbit reticulocyte lysate. product was separated on 12% SDS-PAGE. RESULTS Northern various there

is

mRN A

as

2-3

fold

addition ccl

cells

form

increase

in

of

1s

blot

blot stages of a progressive

form was

agarose lane

(data

tubes

quantitated

(Hirschmann).

germ

of tube increase

in

revealed

3

h

RNA

total formation

isolated indicates

the

in

germ tubes the level of

by Ethidium

gel not

analysis

(GlcNAc)

inducer germ

AND DISCUSSION

level

(Fig. actin

1). mRNA

(Fig.

I).

About

(data

not

shown).

densitometry bromide comparable

shown).

424

amount

of There in 3 QO-95% The

with staining

at that actin

a

was

h

a after

of

the

Nothern

densitometer

of

a

of

RNA

parallel in each

Vol.

179, No. 1, 1991

BIOCHEMICAL

123

Fig.l.Time

4

course formation Total

of in RNA

electrophoresed transferred was hybridized total to 4, spectively, Densitometric

Germ

tubes cells

at

replacing

as

by

yeast

yeast

and

germ

using

rRNA

was of

a

by

ethidium

actin

germ

tube morphogenetic

and

Datta

In

order

germ

tube

using

rabbit

SDS-PAGE

to

in

kDa

and of

28 these

compared

a

that change

calmodulin and is

proposed

in

inhibitor

1990)

calmodulin

the

gene

actin involved

earlier

in (Paran-

in

kDa)

vitro

germ

the encoded mRNA

translated tubes

species

and

synthesis

protein synthesis

of by

of

actin

during

translated

vitro in Mannheiml.

(Boehringer,

1 ysate of

the

synthesis

RNA was

total

from in

tion

study

reticulocyte

isolated

increase

as

level

19901. formation,

differences

the

suggests with

of

we

of (Fig.ZB)

1s

This

etal, that as

intensity in

ccl

associated

pathway,

analysis

RNA

The

these Northern

staining

(Fig.2A,C).

indicates

all by

increase

effect

formremain

from

forming

(Paranjape

(Fig.21

this

fold

is

from

analyzed

(Fig.2).

the

in

cells

isolated

were

by incu-

by

Furthermore, cells

bromide

the

formation

expression

RNA

tube

gene

either

formation)

conditions

probe

observed

ocked or

prevented

2-3

Furthermore,

morphology.

jape

as

germ

tube

three cells

a

in

cells

expression

on

gene whereas

actin

also Total

observed

b 1990).

germ

these

forming

mRNA

yeast

mtl)

viable.

actin

unchanged

of

etal, of

all

tube

bands,

was to

form and

blot

In

be inhibitor,

(Paranjape

(5

tubes.

can

calmodulin

(inducer

glucose

germ

GlcNAc

by a

25OC

GlcNAc

medium ing

actin mRNA synthesis during germ tubs Candida albicans induced by GlcNAc. isolated at indicated times was on 1.2 % agarose gel. Resolved RNA Plus and GeneScreen membrane to c34;labeled actin probe. A. Lane 1 RNA isolated at 0, 60, 120, 100 min. reafter addition of GlcNAc. 9. blot. scan of the Northern

(TFP),

bating

A

induced

trifluoperazine

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

three RNA,

the during

425

germ

products yeast pattern proteins indicated tube

ccl

of 1s (Fig. (50 the

formation.

total revealed 3).

An

kDa, accumula-

45 The

Vol.

179, No. 1, 1991

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

C

r--l

!Llnl0

1

2

3

3

4

Northern blot analysis of actin mRNA of yeast and germ tube forms of C. albicans. Germ tube formation at 37OC and inhibited induced was by GlcNAc trifluoperazine (20 JIM) or by either by adding incubating cells at 25OC or by replacing GlcNAc in the medium with Glucose. Total RNA was isolated from both morphological forms and sub$ected to as described in the legend blot analysis Northern to Fig.1. incubated at 37'C (Germ A. Lane 1, cells tube); Lane 2, cells incubated at 37OC in presence (yeast); Lane 3, cells of 20 JIM trifluoperazine at 25OC (yeast); Lane 4, cells incubated incubated in presence of glucose instead of GlcNAc 37OC at (Yeast). total B. Ethidium bromide staining of the RNA samples run on a parallel agarose gel. C.Densitothe Northern blot. metric scan of

Fig-P.

3.~~~5F$GErne~;a;;;;~

Fig.

o:a~~l~d~rooftf~~~~at~~~

E;zduci;

mRNA species isolated by hybrid selection with immobilized DNA of actin clone. Germ tube formation was induced by GlcNAc and inhibited as described in the legend to Fig.2. Total RNA was isolated and translated in vitro in rabbit reticulocyte ly12% sate. Translated products were analysed on gels and fluorographed. polyacrylamide Dried gels exposed to preflashed Kodak X-ray film at -2OOC were days. Lane 1, RNA hybrid selected with DNA for 3-4 RNA from ccl Is of actin clone; Lane 2, total RNA from trifluforming germ tubes; Lane 3, total total RNA from operazine treated cells; Lane 4, incubated cells at 25OC. Position of molecular weight standards are indicated on the right. Arrow indicates the position of actin.

identity

of

50

present.

A

protein

actin

since

total

RNA

that ccl

the Is

it level

as

supported

by confirm

during

and

28 of

of

comigrated actin

the

is kDa

morphogenesis

more ccl

of the

actin

is the

of

Is. Northern

426

These

albicans.

at as

product This

germ

of

C.

known

identified

clone.

in

expression

not is

translated

the

the

yeast

results that

protein

with by mRNA

to

kDa Mol.wt.45

selected

compared

further

regulated

is

hybrid

kDa

of

suggests

tube

forming

observations, anlaysis, blot actin

gene As

germ

is tube

Vol.

179, No. 1, 1991

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

formation is accompanied by an increase in the cell volume, the increase in the level of actin in germ tubes is most probably required to accomodate the increasing size of the cells (MC Cairns et al., 19841. Increased level of mRNA could be the result of either increased rate of transcription or enhanced stability of actin mRNA accompanying change in morphology. It will be of interest to study the stability of actin mRNA and also determine whether the same or different upstream sequences control the transcription of actin gene during morphogenesis. To our knowledge this is the first report of a differentially expressed gene during mophogenesis of C. albicans.

ACKNOULEDGllENTS

We acknowledge Dr. Joachim F. Ernst, FRG for the gift of actin clone from C. albicans and Dr. K. Natarajan for doing the hybrid selection translation experiment and providing translated product. This work has been supported by a the from Council of Scientific and Industrial Research, .

REFERENCES 1.

Anderson, 01.

132,

2. Chomczynski, 182,

J.H.,

2035-2047.

158-159.

P..

and Soil,

D.R.

and Sacchi,

(19861 N.

(1997)

J.

Gen.

Anal.

HicrobiBiochem.

3. Datta, A., Ganesan, K and Natarajan. K. (19891 Adv. Microbial Physiol. 30, pp.53-98. Academic Press, London. 4. Feinberg, A.P., and Vogelstein, B. (19841 Anal. Biothem. 137, 266-287. 5. Greer, G., and Schekman, R. 119B21 Mol. Cell. Biol. 2, 1279- 1286 6.

Losberger, C.. and Ernst, J.F. (19891 Nucleic Acid Res. 17, 22. 7. HJ.nJ.lan, A. S. , and Klee, C.B. (19821 Adv. Cyclic Nucleotide Protein Phosphorylation Res. 18, 227-278. 8. Hccairns, E., Fahey, D., Muscat, G.E.U., Murray, H., and Rowe, P.E. (19841 Mol. Cell. Biol. 4, 1754-1760. A. (19843 Rai, Y.P., and Datta. 9. Natarajan, K., Biochem. Int. 9, 735-744. 10. b&p, F.G. (lSB81 In Gandida and Candidiasis, pp.42-59. liere Tindal, London. V.D., Gupta Roy, B., and Datta, A. (19901 J. 11. Paranjape, Gen. Microbial. 136, 2149-2154. V.D., and Datta, A. (19901 In Calcium as 12. Paranjape, Eucaryotic Microbes, an Intracellular Messenger in Day Ed.), pp. 362-374. ASH Publication. USA. (D.H.0' R.P., Millers, J.S., and Roberts, B.E. t19791 13. Ricciardi, Proc. Natl. Acad. Sci.(USAl 76, 4927-4931. H.G., C,hi;w. Y.Y., Ram, S.P., and Sullivan, 14. Ehzpherd, 26, 21-20. . . (1980) Can. . Microbial. 15. Sobue, K., Horimoto, K., Kanda. K. , Maruyama, K., and Kakiuchi, S. (19821 FEBS Lett. 138, 289-292. 427