Early and late heat-induced proteins during Leishmania mexicana transformation

Vol. 156, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages ]360-]357

November 15, 1988

EARLY AND LATE HEAT-INDUCED PROTEINS DURING Leishmania mexicana TRANSFORMATION

Antonio Alcina and Manuel Fresno~ Centro de Biologla Molecular Universidad Aut6noma,

Canto Blanco,

(CSIC-UAM) 28049 Madrid, Spain

Received September 30, 1988

During in vitro transformation of L. mexicana from promastigotes to amastigotes at 37-°C, a higher growing temperature, transforming parasites showed two different sets of polypeptides. One set of proteins was synthesized at the beginning of temperature-shift and corresponded to the so called "heat shock proteins" (hsp) being expressed by promastigotes mostly. The second set was expressed later on was specifically associated with the amastigotes stages and was strongly similar to the pattern of polypeptides synthesized by amastigotes from infected peritoneal macrophages. These results suggest hsp may play a function at a defined time in the transformation of the parasite. © 1988 A c a d e m i c Press, Inc.

L. mexicana amazonensis, American

cutaneous

distinct stages. infects

the etiological agent of

leishmaniasis, The

macrophages

promastigote, and

transforms

has a

two

morphologically

flagellated form, into

intracellular

Transformation

event is a crucial step for intracellular

of Leishmania

spp in

observations proteins

except for

(1-3).

(6-8)

the mammalian

the establishment (*) Correspondence

morphological

Although temperature

have

transformation,

host

been

"in vitro",

involved

called but it and

amastigote. survival

has not

been

ultrastructural

(4-6) and heat

indirectly

which

non-flagellated

round-shaped

studied in detail

parasite,

a

South

in

shock

parasite

the relevance of this phenomena

of a successful parasitism is still unclear. to M. Fresno.

Abbreviations used: Hsp, Heat shock protein; L.m., Leishmania mexicana; M~, macrophage; PBS, phosphate-buffered saline; SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis. 0006-291X/88 $1.50 Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

1360

to

Vol. 156, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

We report here the extra and intracellular we show that form of

mexicana

characterized

The

the

from promastigotes peritoneal

pattern of early

inducibility induced

by

conditions amastigote by

either

macrophages

proteins

groups:

to the hsp

(7,8), and late heat-induced

corresponded

MATERIALS

proteins

or by infecting

conditions. two

of the protein expressed

at high temperature

transformed

temperature

the same define

parasites

at least I0

L.

heat-shock

analysis

led

in

us

proteins

to

which

proteins.

AND METHODS

Leishmania mexicana amazonensis were and routinely grown in DEM (Dulbecco Minimal Eagle) medium supplemented with FCS (Fetal Calf serum) (Gibco) and antibiotics (i00 ug/ml streptomicin and I00 U/ml penicilin) at 259C (6). Originally they came from Dr K.P. Chang's Laboratory. Infection of macrophaqes. Resident peritoneal cells were collected from normal untreated Balb/c mice and macrophage population were enriched >95% by adherence to plastic dishes, as determined by staining with non-specific esterase methods, as previously described ( 9 ) . 24 hours later the cells were used in the infection experiments. Macrophage monolayers, on 1 x 1 cm coverslips in 24-well Linbro plates, were infected with stationary growth phase L. mexicana promastigotes. After 30 min of parasite-cell interaction at 35eC the cultures were washed three times with medium to eliminate all parasites not bound to or ingested by the macrophages. This was checked by phase contrast microscopy and, finally, plates were incubated at 37eC. After different times of incubation the level of infection was assessed by optical microscopy staining w i t h Giemsa or pulsed with (35S) methionine to analyze protein synthesis. Measurement of DNA or protein synthesis. Extracellular parasites in promastigote foms or cultures of infected macrophages were washed twice with methionine-free DME or normal DME medium supplemented with 50 uCi/ml of (35S) methionine (i000 Ci/mmol) or i0 uCi/ml ( 3H)thymidine (50 Ci/mmol), respectively, (Amersham). The pulse was carried out at 37~C for 1 h and the parasites and cells were washed twice w i t h PBS and resuspended in SDS-PAGE buffer (0.I M Tris-HCl, pH 6.8; 1% glycerol; 0.I M 2-mercaptoethanol). An aliquot was precipitated with cold 10% TCA to evaluate the level of DNA or protein synthesis. Samples were electrophoresed in 0.I mm thick slab gels as described (i0).

RESULTS Temperature-induced cellular

parasites.

temperature parasite

on

to

morphology

This

the

morphology

differentiate (II, 12).

promastigotes transformation

where from

in

vitro

transformation

study was based into

As shown incubated

of L.

mexicana,

a round-shaped, in the Table at

promastigotes 1361

of

extra-

on the effect of which

induced

amastigote-like

I, when cultures

379C, into

a

high

time

of

dependent

amastigote-like

Vol. 156, No. 3, 1988

Table I.

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Time-course of temperature induced in Leishmania mexicana

Time of incubation

transformation

(h)

1

37~C % of parasites 2 Promastigotes

25~C

0

1

2

5

24

48

24

98

94

90

75

20

5

30

1

3

8

21

68

82

51

Amastigotes

i) Promastigote forms of the parasite, 25x106/ml incubated at 259C in 96-well Linbro plates, were shifted at 379C and after 48 h of incubation the same cultures were back to 259C, for another 24 hrs. At the specified times, aliquots were taken, processed for counting under phase-contrast microscopy to differentiate between Found-shaped amastigotelike morphologies and long-flagellated promastigote forms. The percentage numbers were from triplicate cultures. 2) Parasites in transitional forms were not counted.

morphologies thought

was

to kill

viability However,

of

induced.

the

in our

hands

since

unable

synthesize

(Figure

as a s s e s s e d

parasites

treatment, to

Temperatures

L. m e x i c a n a

they

after

parasites

DNA

a 2-3

were

synthesized and

higher

than

by optical day

alive

proteins

replicate

treatment

after

a 2-day

although at

359C

were

microscopy

that

they

and

(13). heat were

temperature

i).

5

I,iJ

-z,.9 4 z I.-- ~ "'~

E

~ O

U

0

2

5

24

48 (hr)

3

\

K

Z

0

,

2

,

5

//----_u_~2

"

24

,

48 (hr)

Figure i. Effect of high temperature in the synthesis of DNA and proteins by L. mexicana. Aliquots of the cultures were taken at the indicated times after the temperature shipt and pulsed for 1 h with ( 35 S)-methionine (Top panel) or (~H)-thymidine (Bottom planel). The total radioactivity incorporated into proteins or DNA was estimated after TCA precipitation. 1362

V o l . 156, N o . 3, 1988

BIOCHEMICAL A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S

(hr) (hr)

]

2

2

5 2448

-

hsp

24 i

-

48 i --

i

L.m. AMA

ZNF MO proteins

:!!i!!

-- 110 8 3 -- ~ N

~-~--

70 . . . . . 65"-

~-110

...... .......

77 73

hip 8 3

65

hip 70--

._ 77 "- 73 65

~ ~

48

Actin

na

-- ~

-'- 48

39

b

39 35

x

35

:E

~

28

~

~

28

24 20

14.5 12.5--

14--

~

14

11 A

B

Figure 2. SDS-PAGE analysis of the £35S]-Met-labelled proteins synthesized by L. mexicana in axenical cultures (A) or inside of mouse peritoneal macrophages (B) during a cycle of complete transformation. The major L. mexicana proteins (L.m. AMA) synthesized by heat-induced amastigotes or by amastigotes inside macrophages (INF M~ proteins) are indicated by arrows. On the left are the major heat shock protein (hsp) expressed by yet untransformed promastigotes. Uninfected macrophages cultures (-) or infected macrophages (i) were pulsed for 1 h at the indicated times after infection with L. mexicana promastigotes.

Early promastigotes after most

started

shifting

in

hsp

cruzl

could

represssed.

A new

These

had

a maximum

heat

shock

shown

in Fig.

seen

seen on g e l s

(Figure

hsp

2). At the

related

parasites

first but,

polypeptldes

expression

time

T h e Mr

w i t h a Mr of

at 24 h of

was

after

48

of these 77,

65,

seen

time,

strongly found such

five h o u r s

afterwards,

incubation.

1363

same

were

has b e e n

the

cultured

immediately

synthesis

in

During

conditions.

2. P r o t e i n s

the m a j o r

on the gel

set of

"In vitro"

at l o w t e m p e r a t u r e

also

(10). be

proteins.

on p r o t e i n

but

under

clearly

temperature expressed

effect

Leishmania

induced

to s y n t h e s i z e

up the

This

Trypanosoma major

late h e a t

of the p r o t e i n s

inhibited. only

and

were 24

None

39,

h.

incubation

new proteins 48,

as

onlythe

they

after

h of

not

are

35 K d w e r e

of t h e m

have

Vol. 156, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table II.

Summary of the Leishmania mexicana response high temperature conditions

to

Incubation at 37eC Promastigotes (Axenical culture) 2h 48h ~ DNA Synthesis Protein Synthesis Hsp Synthesis Growth Reversibility 2

+ +

Amastigotes (Inside macrophages) 2h 48h

-

+

-

+

+ + +

+ +

+

I) The form of the parasites at that time was amastigote. 2) Parasite growth after returning to 25eC (Table I).

the same apparent were

expressed

molecular weight as by

cells

These parasites did not when

further

with

at

were

had

detectable

almost

not

of infection a yet

Therefore

they

proteins

morphologies.

became

specific

immediately promastigotes

although

flagellated

(Table II).

transformation. after

infection

(Figure 2B).

remaining parasite

in

form

of

However

proportion of intracellular

transformed

the

25eC,

in intracellular

peritoneal macrophages with after 2 h

amastigote-like

forms and started to replicate

Heat shock proteins Hsp

These

replicate at h i g h temperature,

incubated

promastigote-like

the hsp.

parasites

promastigote responsible

stage. for

the

production of the hsp is not clear but assuming that

macrophages

(Fig. 2B, u n i n f e c t e d MZ after 2 h of incubation)

amastigotes

(Fig.

2A,

axenically

incubation)

induced

amastigotes

contribute minimally to

and

after

48

h

of

hsp synthesis, hsp would

be

most likely attributable to the promastigotes. After 48 promastigotes morphologies

h

were

at

37eC all

already

(Table I).

transformed

in about 24

stage because

of

into

extracellular amastigote-like

On the other hand, promastigotes,

being internalized by macrophages, amastigotes

populations

h and kept intracellularly

they were

not able

cultures at 379C were returned

after

were completely transformed to

to replicate.

in a

resting

However

to 359C C they started to

when divide

(Table II). Interestingly, intracellular comparing

the

induced

the set of

proteins expressed by heat

amastigotes

proteins

synthesized 1364

were by

very

similar.

axenically

or By

induced

Vol. 156, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

INF M~ L.m. AMA

L1 ~

~i

I-Z I- 3 L4

;

1-5

t-6 IM1

-

L7

-

IMz~ IM 3

L8

IM 6 -

j

L9 LIO

Figure 3. Comparison of the proteins synthesized by heat induced amastigotes (L.m.AMA) and intracellular amastigotes (INF MZ). It is indicated as L1 to LI0, the major proteins synthesized by heat-induced amastigotes which were also found to be synthesized by intracellular amastigotes. IM indicates the proteins specifically induced in infected macrophages (M~), and M the ones produced by MZ. All of these proteins have been differentially deduced from the gels of figure i.

amastigotes

and

amastigotes,

from figure

the

ones

produced

2A and 2B

expressed by u n i n f e c t e d macrophages the

infection

process

(IM),

amastigote-like morphologies

(L)

by

intracellular

there were clearly

induced proteins

(M),proteins associated and

(Fig.

those

with

synthesized

by

3).

DISCUSSION This

work

was

undertaken

changes during transformation of on the

proteins

temperature (Table I).

in In

synthesized

biochemical

L. m e x i c a n a and to

concentrate

the parasites

b o t h promastigotes addition, as and

and in

heat shock

affected not only the expression the physiology

investigate

by

to

the

heat

shock

macrophages

temperature

of proteins

morphology of

at

infected

strongly

(7, 8) but also

parasites

(13,

14),

to we

wanted to define the capability of the parasites to divide and to grow at this condition. 1365

Vol. 156, No. 3, 1988

Several

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

groups

temperature from promastigote

have

26eC to

to

reported

that

33-359C caused

the amastigote

synthesis of the hsp (6-8).

forms

in

the

a transformation

from

(5,

a

Ii)

rise

and induced

the

Due to the shift in temperature

and

metabolism during the life cycle of this parasite, hsp have

been

suggested to play a role in its adaptation to the new environment (6).

Promastigotes

expressed

a

placed at 379C, which induced transformation,

similar

protein

profile

to

that

of

amastigotes and suggest that heat or intracellular follow similar pathways. with previous

results

synthesize a

internal

transformation

First, they synthesize hsp in agreement

(6-8).

Later,

different set of

after a

proteins.

Hsp

lag period were not

they

detected

between the ones induced by heat at later times of incubation and therefore, as part of the proteins synthesized for the amastigote forms;

if

they had

would

be

in

any role

the

early

in the

steps.

We

similarity between the different between extracellularly peritoneal macrophages, expressed

at

the

transformation process cannot

demonstrate

types of amastigotes,

heat-induced

or

induced

conditions,

and

when

is,

infecting

but by comparing the profiles of

same

the

that

by

it

protein

reverting

such

stressing situation, checking up for viability; thus, we can assume that both forms are morphological and biochemical alike. The biological

significance of these results could be

as

did

parasites

addition,

not

replicate

temperatures higher

spp belonging

at

However, not assay but also promastigotes

and mucocutaneous

the parasites

they could be (Table II),

temperature-shift.

than 35eC caused death

the cortical

only did

after

questioned

survive

In

Leishmania

groups

(13).

within the

2-day

transformed back into

replicating

indicating that other unknown

factors

may affect the transformation process. Our cutaneous

results

suggest

leishmaniasis,

that

could

L.

mexicana,

which

be affected in their

causes

replication

capability but not in its survival at 37~C.

This observation

of interest because several

in which

occurred, have been The metastasis

caused by

of parasites

affected by several

clinical cases,

some cutaneous in the

host

factors including,

metastasis

leishmanias

body is

is

(15).

known to

at least, the

be

different

sensitivity of Leishmania spp to the serum complement system (12, 16),

the

thermo-sensitivity

interaction

and

functional

glycoprotein

of

Hypothetically

infected

parasites

of

parasites

activity with

macrophages 1366

of

(17) the

macrophages could

move

and major (12, from

by

the

surface 18). cortical

Vol. 156, No. 3, 1988

areas in which the body and

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the parasite may contribute to the

replicate to internal parts spread of the disease,

of

although

replication would take place at locations where temperature

were

permisive. ACKNOWLEDGEMENTS The project was supported by grants from CAICYT and FISS. A.A. was supported by an FIS fellowship. REFERENCES i. 2. 3. 4. 5. 6. 7. 8. 9. i0. ii. 12. 13. 14. 15.

16. 17. 18.

Akiyama, H.J. and Taylor, J.C. (1970). Am. J. Trop. Med. Hyg. 19: 747-754. Akiyama, H.J. and Mc Quillen, N.K. (1972). Am. J. Trop. Med. Hyg. 21: 873-879. Chang, K.P. (1978). Am. J. Trop. Med. Hyg., 27: 1084-1096. Pan, A.A. and Pan, S.C. (1986). Exp. Parasitol., 62: 254-265. Hunter, K.W.Jr., Cook, C.L. and Hensen, S.A. (1982). Acta Trop., 39: 143-150. Van der Ploeg, L.H.T., Giannini, S.H., and Cantor, C.R. (1985). Science, 228: 1443-1445. Hunter, K.W., Cook, C.L. and Hayunga, E.G. (1984). Biochem. Biophys. Res. Commun. 125: 755-760. Lawrence, F.L. and Robert-Gero, M. (1985). Proc. Natl. Acad. Sci. USA. 82: 4414-4417. Alcina, A. and Fresno, M. (1987). J. Immunol. Methods. 105: 1-8. Alcina, A., Urzainqui, A., and Carrasco, L. (1988) Eur. J. Biochem., 172: 121-127. Darling, T.N. and Blum, J.J. (1987). J. Protozool. 34(2): 166-168. Mosser, D.M. & Edelson, P.J. (1987) Nature. 327: 329-331. Sacks, D.L., Barral, A. and Neva, F.A. (1983). Am. J. Trop. Med. Hyg., 32: 300-304. Chang, K.P. (1980). Science 209: 1240-1242. Mauel, J. and Behin, R. (1982). Leishmanlasis: immunity, immunopathology and immunodiagnosis. In "Modern Parasitology". Cox, F.E.G., Ed. pp 311-319. Backwel 1 Scientific Publication. Mosser, D.M., Burke, S.K., Coutavas, E.E., Wedgwood, J.F., and Edelson, P.J. (1986). Exp. Parasltol., 62: 394-404. Smejkal, R.M. & Edelson, P.J. (1987) Nature. 327:329-331. Nogueira, N., Medina, E., Espinosa, M., Quintao, L. and Block, P.K. (1987). In "Moleclar Strategies of Parasitic Invasion", pp 301-310. Alan R. Liss, Inc.

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