INVOLVEMENT OF THE MITOGEN-ACTIVATED PROTEIN ( M A P ) KINASE SIGNALLING PATHWAY I N HOST CELL INVASION
BY TOXOPLASMA GONDII
ROBERT-GANGNEUX F.*, CREUZET C*, DUPOUY-CAMET J.* & ROISIN M.-P.*
Summary:
Little is known about signalling in Toxoplasma gondii, but it is likely that protein kinases might play a key role in the parasite proliferation, differentiation and probably invasion. We previously characterized Mitogen-Activated Protein (MAP) kinases in T. gondii lysates. In this study, cultured cells were tested for their
susceptibility to Toxoplasma gondii infection after tachyzoite pretreatment with drugs interfering with AMP kinase activation pathways. Protein kinases inhibitors, i.e. genistein, R031-8220 and PD098059, reduced tachyzoite infectivity by 38 ± 4.5 %, 85.5 ± 9 % and 56 ± 10 %, respectively. Conversely, protein kinases activators, i.e. bombesin and PMA, markedly increased infectivity (by 202 ± 37 X and 258 ± 14 %, respectively). These results suggest that signalling pathways involving PKC and AAAP kinases play a role in host cell invasion by Toxoplasma.
KEY WORDS :
Toxoplasma gondii, protein kinases, AAAP kinases, cell culture, intracellular signalling, cell invasion.Résumé :
RÔLE POSSIBLE D'UNE TRANSDUCTION DU SIGNAL VIA LES"MITOGEN-ACTIVATED PROTEIN" (MAP) KINASES DANS L'INVASION DE LA CELLULE PAR TOXOPLASMA GONDII
Il existe peu de données sur les voies de signalisation chez Toxoplasma gondii, mais il est probable que des protéines kinases jouent un rôle dans la croissance, la différenciation, mais aussi l'invasion du toxoplasme. Nous avons précédemment caractérisé des "mitogen-activated protein" (MAPI kinases dans le toxoplasme.
Dans cette étude, nous montrons que le pré-traitement des tachyzoïtes par des activateurs (bombésine, PMA) ou des inhibiteurs (génistéine, RO31-8220, PD098059) de protéine kinases intervenant dans la voie d'activation des MAP kinases, entraîne une augmentation ou une diminution de leur pouvoir infectieux pour des cellules 3T3 en culture, respectivement. Ces résultats sont en faveur d'un rôle des voies de signalisation impliquant des protéines kinases C et des MAP kinases dans l'invasion de la cellule par T. gondii.
MOTS CLES : Toxoplasma gondii, protéine kinases, MAP kinases, signalisation cellulaire, invasion, culture cellulaire.
INTRODUCTION
P
rotein phosphorylation o r dephosphorylation is involved in the enzymatic c a s c a d e s w h i c h trans- d u c e signals in eukaryotic cells. Signal trans- duction plays important roles in cell differentiation and proliferation, a n d in the regulation o f m e t a b o l i c path- ways. Phosphatases and kinases have b e e n widely stu- died in eukaryotic cells, but few data are available o n their p r e s e n c e a n d function in protozoan parasites. An important role o f protein kinases a n d signal transduc- tion has b e e n demonstrated in s o m e p r o t o z o a n para- sites, including Leishmania ( B e c k e r & Jaffe, 1 9 9 7 ) ,Trypanosoma cruzi ( O g u e t a et al., 1 9 9 6 ) , Entamoeba histolytica ( P e r e z et al, 1 9 9 6 ) , Giardia duodenalis ( C h e n et al, 1 9 9 6 ) , and Plasmodium falciparum (Dlu- zevski & Garcia, 1 9 9 6 ) . I n d e e d , it has b e e n d o c u - m e n t e d that the phosphorylation pattern o f Leishmania
* Laboratoire Signalisation et Parasites, Université Paris 5, Centre Hos- pitalier Universitaire Cochin-Port Royal, Pavillon Gustave Roussy, 27, rue du Faubourg Saint-Jacques, 75014 Paris, France.
Correspondence: Florence Robert-Gangneux.
Tel.: 0033 1.58.41.22.51 - Fax: 0033.1.58.41.22.45.
E-mail: florence.gangneux-robert@cch.ap-hop-paris.fr
and other kinetoplastids varies throughout their life cycle, and may play a k e y role in parasite survival, dif- ferentiation, and cell invasion (Valentine & Carter, 1993; H e r m o s o & Jaffe, 1 9 9 3 ; Dell & Engell, 1994; Par- s o n s et al, 1 9 9 5 ; W i e s e , 1 9 9 8 ) . Protein kinase inhibi- tors reduce the infectivity and growth o f Leishmania and Trypanosoma cruzi (Vieira et al, 1 9 9 4 ; B e c k e r &
Jaffe, 1 9 9 7 ) . Several protein kinases g e n e s h a v e b e e n identified in Toxoplasma, but their functions remain to b e determined (Ng et al, 1 9 9 5 ; Ng et al, 1997; Wast- ling & Kinnaird, 1 9 9 8 ) .
W e previously demonstrated the p r e s e n c e o f mitogen- activated protein (MAP) kinase activity in T. gondii and putatively identified two MAP kinases h o m o l o g u e s o f the ERK (extracellular signal-regulated k i n a s e s ) family (Roisin et al, 2 0 0 0 ) . Gomez-Marin et al ( 1 9 9 8 ) have a l s o c h a r a c t e r i z e d mitogen-activated protein (MAP) kinase activity in T. gondii, that was decreased by INF-y.
As cell invasion and parasite replication within the host cell probably involve protein kinases, w e examined the impact o f protein kinases inhibitors and activators o n Toxoplasma infectivity, focusing on c o m p o u n d s acting at different steps o f MAP kinase activation pathways.
Activation o f MAP kinases (also k n o w n as extracellular signal-regulated kinases, or E R K ) is o n e o f the most
Parasite, 2 0 0 0 , 7, 9 5 - 1 0 1
Mémoire 9 5
Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/2000072095
ROBERT-GANGNEUX F., CREUZET C , DUPOUY-CAMET J. & ROISIN M.-P.
rapid cellular responses to various external stimuli, lea
ding to differentiation, growth and other cellular func
tions (Blenis, 1993). MAP kinase pathway activation o c c u r s through G protein-coupled receptors, calcium c h a n n e l - c o u p l e d receptors, and tyrosine kinase r e c e p tors (Treisman, 1996), and is highly d e p e n d e n t o n the intracellular calcium concentration and protein kinase C ( P K C ) activation ( C o b b & Goldsmith, 1995; R o b i n s o n
& C o b b , 1 9 9 7 ) . In previous experiments, w e o b s e r v e d that MAP kinase activity detected in toxoplasma lysates w a s stimulated by calcium or through PKC activation (Roisin et al, 1 9 9 9 ) . W e therefore tested Toxoplasma infectivity and growth in cultured cells after tachyzoite pretreatment with several c o m p o u n d s : 1) the tyrosine kinase inhibitor genistein, 2 ) the PKC inhibitor bisin- doylmaleimide or R O 3 1 - 8 2 2 0 (Chao et al., 1992), 3) the MAP kinase kinase (or MEK) inhibitor P D 0 9 8 0 5 9 (Alessi et al, 1 9 9 5 ) , 4 ) the PKC activator phorbol-myristyl a c e tate (PMA) ( 1 8 ) , and 5 ) the PKC and MAP kinase acti
vator b o m b e s i n (Pang et al, 1993). Our results point to a role o f MAP kinases and PKC in host cell inva
sion by T. gondii.
MATERIALS AND METHODS
CELL CULTURES
N
IH-3T3 cells w e r e u s e d for invasion experiments. Briefly, cells w e r e cultured o n glass coverslips in 12-well culture plates ( 7 0 , 0 0 0 cells per well) overnight at 3 7 ° C with 5 % C 02, in 1 ml Dul- b e c c o ' s Eagle modified m e d i u m ( D M E M ) containing 10 % fetal calf serum (FCS), 5 I U / m l penicillin and 5 Ug/ml streptomycin. O n e hour before invasion e x p e riments with Toxoplasma tachyzoites, the medium was removed and replaced with DMEM containing 5 % FCS.
PARASITES
T h e RH strain o f Toxoplasma gondii w a s maintained b y serial passages in I O P S - O F 1 Swiss m i c e (Iffa Credo, F r a n c e ) . T a c h y z o i t e s w e r e harvested from peritoneal fluid o f m i c e infected four days earlier. T h e fluid w a s centrifuged for 10 min at 1000 g. T h e pellet was washed and r e s u s p e n d e d in phosphate-buffered saline ( P B S ) containing 10 IU/ml penicillin and 10 U / m l strepto-
Fig. 1. - Biochemical targets of the different kinase activators (•-•) or inhibitors (•—I) used. Three main signalling pathways are concerned:
the tyrosine kinase receptors pathway, the G protein-coupled receptors pathway, and the voltage-dependent calcium channels pathway.
Abbreviations used: PLCy: phospholipase Cy; YP: tyrosine phosphorylated residue; PIP2: phosphatidylinositol-4,5-bisphosphate; DAG: dia- cylglycerol; IP3: inositol trisphosphate; PKC: protein kinase C; MEK: MAP kinase kinase.
96 Memoire Parasite, 2000, 7, 95-101
MAP KINASES AND TOXOPIASMA INFECrrVTTY
mycin. T a c h y z o i t e s w e r e c o u n t e d and their viability was a s s e s s e d b y trypan b l u e e x c l u s i o n method. Para
sites w e r e 9 5 % viable.
TREATMENT OF TACHYZOITES AND INVASION EXPERIMENTS
T h e site o f action in the MAP kinase activation pathway, o f the different compounds used, is reported in Figure 1.
For drug treatment, tachyzoites ( 5 x 1 06) w e r e resus- p e n d e d in a final v o l u m e o f 5 0 ml containing various concentrations o f drugs, diluted either in DMEM (geni- stein (Alexis), R O 3 1 - 8 2 2 0 (Alexis, b o m b e s i n (Sigma), and PMA [Sigma]), o r in 7 % b o v i n e serum albumin ( P D 0 9 8 0 5 9 (Alexis]), and incubated for 15 min (all drugs e x c e p t P D 0 9 8 0 5 9 ) o r 3 0 min (PMA and P D 0 9 8 0 5 9 ) at 3 7 ° C with 5 % C 02. Untreated control tachyzoites w e r e i n c u b a t e d for the s a m e times at 3 7 ° C. For inva
sion e x p e r i m e n t s , 1 06 treated or untreated tachyzoites w e r e inoculated per well ( p a r a s i t e x e l l ratio 10:1). T h e volume o f inoculum was adjusted s o that the final dilu
tion o f the drug in the well w a s at least 1:100 o f the initial concentration, thus rending unlikely a direct effect o n cell cultures. T h e a b s e n c e o f any effect o n cells o f all the drugs applied at such concentrations had b e e n c h e c k e d previously. After t w o hours o f incuba
tion at 3 7 ° C, Toxoplasma tachyzoites w e r e r e m o v e d and cells w e r e w a s h e d extensively with DMEM then reincubated overnight. T h e cells w e r e fixed for 3 0 min in 4 % paraformaldehyde then w a s h e d and kept in PBS until i m m u n o f l u o r e s c e n c e staining. Each drug e x p e r i ment w a s r e p e a t e d at least three times.
QUANTIFICATION OF HOST CELL INVASION
• I m m u n o c y t o f l u o r e s c e n c e staining and m i c r o s c o p i c quantification
Host cell invasion w a s quantified b y counting after i m m u n o c y t o f l u o r e s c e n c e ( I F ) staining o f cell c o v e r s - lips, using specific anti-Toxoplasma antibodies, as pre
viously d e s c r i b e d (Creuzet et al., 1 9 9 8 ) . Coverslips w e r e also incubated for 3 0 min with phalloidin (diluted
1:200 in P B S containing 2 % FCS and 0.25 % Triton X - 1 0 0 ) in o r d e r to visualize p r e c i s e l y cell structure.
C o v e r s l i p s w e r e m o u n t e d in V e c t a s h i e l d ( V e c t o r , Biosys) and e x a m i n e d under an epifluorescence micro
s c o p e ( N i k o n ) . At least 1 0 0 0 cells w e r e c o u n t e d per coverslip. T h e p e r c e n t a g e o f infected cells and the m e a n n u m b e r o f tachyzoites per infected cell w e r e recorded. T h e data allow a precise quantification o f the efficacy o f invasion. Results are e x p r e s s e d as the rela
tive reduction or relative i n c r e a s e in cell infection induced by tachyzoite treatment, relative to untreated tachyzoites. Results are presented as m e a n (± SE) infec
tion rates determined in three i n d e p e n d e n t experi
ments.
• 3H-uracil incorporation assay
In s o m e experiments, cell infection was also deter
mined by 3H-uracil incorporation assay, as previously described (Pfefferkorn & Pfefferkorn, 1977). In this latter case, identical numbers o f cultured cells ( 1 04 c e l l s / c m2) w e r e s e e d e d on coverslips. Cells w e r e infected with 2 x 1 05 treated or untreated parasites per well for 2 h, then w a s h e d and incubated for the next 24 h in labe
ling medium containing dialyzed FCS ( 3 % ) and (5-6)- [3H]-uracil (2 uCi/ml, A m e r s h a m ) . T h e medium was then discarded, cells w e r e lysed in PBS containing 1 % sodium dodecyl sulfate and 1 mM uracil, and protein was precipitated with trichloracetic acid ( 1 0 % final c o n c e n t r a t i o n ) . Precipitates w e r e w a s h e d o n G F / C fil
ters ( W h a t m a n n ) , and the radioactivity o n filters w a s counted in a scintillation counter. This technique allows to quantify the parasite mutiplication, and thus reflects roughly the efficacy o f invasion. Results are e x p r e s s e d as the ratio incorporation rate o f control tachyzoites : incorporation rate o f treated tachyzoites. T h e results are presented as the m e a n s ( ± S E ) o f at least three inde
p e n d e n t experiments.
RESULTS
EFFECT OF PROTEIN KINASE INHIBITORS ON CELL INVASION AND PARASITE GROWTH
T
oxoplasma tachyzoites incubated with the PKC- inhibitor RO31-8220 showed reduced infectivity for 3T3 cells, in a dose-dependent manner (Fig. 2A).T h e p e r c e n t a g e o f infected cells c o u n t e d after IF stai
ning d e c r e a s e d b y 55 ± 8 % (t Student test, p < 0 . 0 1 ) w h e n parasites w e r e submitted to 1 0 0 pM R O 3 1 - 8 2 2 0 for 15 min before cell contact. In addition, the n u m b e r o f parasites per infected cell, c o u n t e d after overnight i n c u b a t i o n o f i n f e c t e d c e l l s , s h o w e d a m a r k e d d e c r e a s e , from 6 ( ± 0 . 8 ) tachyzoites/cell ( c o n t r o l ) to 3.5 (± 0.6) ( 1 0 0 pM R O 3 1 - 8 2 2 0 ) , suggesting reduced parasite replication (not s h o w n ) . T h e assessment o f parasite growth by [3H]-uracil incorporation supported these data by demonstrating a marked dose-dependent fall in radioactivity incorporation, to 6.4 ± 2 . 1 % o f control at 1 0 0 uM (t Student test, p < 0 . 0 1 ) , and reflec
ting b o t h r e d u c e d infectivity and r e d u c e d parasite replication ( T a b l e I ) .
Parasite incubation with genistein, a tyrosine kinase inhibitor, r e d u c e d infectivity by 3 8 ± 4.5 % c o m p a r e d to the control (t Student test, p < 0 . 0 1 ) , but only at a high concentration (Fig. 2 B ) . Similarly, [3H]-uracil incor
poration s h o w e d a slight d e c r e a s e ( T a b l e I ) . Parasite replication was not significantly altered, as the m e a n n u m b e r o f parasites per infected cell was not different from the control value (not s h o w n ) .
Parasite, 2000, 7, 95-101 Mémoire 97
R O B E R T - G A N G N E U X F . , C R E U Z E T C , D U P O U Y - G A M E T J . & R O I S I N M , P .
Data are means ± SE of three independent experiments.
Table I. - Effects of protein kinase inhibitors and activators on the intracellular development of T. gondii in vitro. Quantification by [3H]- uracil incorporation after 24 h culture.
B y contrast, parasite preincubation for 3 0 min with P D 0 9 8 0 5 9 , a MEK inhibitor, led to a marked d e c r e a s e in Toxoplasma infectivity ( - 50 % at 10 uM) (Fig. 2 C ) {t Student test, p < 0 . 0 0 1 ) .
EFFECT OF KINASES ACTIVATORS ON PARASITE INFECTIVITY AND GROWTH
T a c h y z o i t e preincubation for 15 min with b o m b e s i n resulted in a marked increase in Toxoplasma infecti
vity, w h i c h r e a c h e d 2 0 2 . 6 ± 37 % o f the control value at 5 0 nM {t Student test, p < 0 . 0 0 1 ) (Fig. 3A). Quanti
fication o f parasite replication b y [3H]-uracil i n c o r p o ration yielded similar but l o w e r results ( T a b l e I ) . Similarly, the PKC activator PMA led to an increase in infectivity, w h i c h r e a c h e d 2 5 8 ± 14 % o f control at 500 nM after 3 0 min o f stimulation (t Student test, p <
0 . 0 0 1 ) (Fig. 3 B ) . PMA also i n c r e a s e d infectivity after 15 min o f stimulation, but to a lesser d e g r e e (+ 150 % ) . W h a t e v e r the incubation time, higher PMA c o n c e n t r a tions had n o effect on infectivity. This type o f d o s e res
p o n s e is c o m m o n l y o b s e r v e d with PMA, the effect o f w h i c h is b o t h d o s e - and time-dependent. [3H]-uracil g a v e similar results ( T a b l e I ) .
DISCUSSION
I
t is likely that protein kinases play a k e y role in m a n y cellular p r o c e s s in Toxoplasma, as well as in other eukaryotic cells. In this study w e e x a m i n e dseveral signalling pathways leading to MAP kinase activation, b y pre-treating parasites with several pro
tein kinases inhibitors and protein kinases activators.
T h e quantification o f host cell invasion w a s a s s e s s e d
Fig. 2. - Effects of protein kinase inhibitors on Toxoplasma infecti- vity.
Toxoplasma tachyzoites were preincubated with increasing concen
trations of RO31-8220 for 15 min (A), genistein for 15 min (B), or PD098059 for 30 min (C).
The efficacy (if invasion was evaluated by microscopic counting of the percentage of infected cells. Results are expressed as relative percentages, corresponding to the relative decrease of cell infection, compared to control values (cell infection by untreated tachyzoites, i.e., 25.5 ± 6.9 % in (A), 20 ± 2.5 % in (B) and 34.5 ± 4.5 % in ((C)), arbitrarily brought to 100 %. Data are the means ± SE of three inde
pendent experiments.
98 Mémoire P a r a s i t e , 2 0 0 0 , 7, 9 5 - 1 0 1
Uracil incorporation (percentage Drug Drug of control, (15 min incubation) concentration mean ± SE)
5 uM 71 ± 8 %
RO31-8220 in U\I 6 8 ± 12.3 %
50 UM 58 ± 9.2 %
Protein kinase 100 UM 6.4 ± 2.1 %
inhibitors
1 UM 8 6 ± 4 %
Genistein 1 0 |XM 89.6 ± 2 %
2 0 |IM 8 8 ± 2 %
50 UM 84 ± 2 % 1 nM 134.5 ± 12.3 %
5 nM 136.7 ± 13.6 %
Bombesin 10 nM 150 ± 7.9 %
50 nM 143.5 ± 6.5 %
MAP kinase 100 nM 133.5 ± 1.5 %
and PKC activators
and PKC activators
PMA
100 nM 500 nM
1 0 0 0 nM
169.2 ± 13.8 % 108.5 ± 7.5 %
116.5 ± 9 %
Fig. 3- - Effects of MAP kinase and PKC activators on Toxoplasma infectivity.
Toxoplasma tachyzoites were preincubated with increasing concen
trations of bombesin for 15 min (A), or PMA for 15 min, or 30 min (B).
The efficacy of invasion was evaluated by microscopic counting of the percentage of infected cells after immunofluorescence staining.
Results are expressed as relative percentages, corresponding to the relative increase of cell infection, compared to control values (cell infection by untreated tachyzoites, i.e., 20.2 ± 5.5 % in (A), 18.5 ± 2.1 % in (B)), arbitrarily brought to 100 %. Data are means ± SE of three independent experiments.
by two different methodologies: I) by counting infected host cells after overnight culture, which directly reflects the efficacy o f invasion, and IT) b y [3H]-uracil i n c o r p o ration after 24 hours, which mostly reflects intracellular growth and m o r e undirectly invasion. Although these two techniques d o not measure exactly the s a m e event, w e present here the results obtained with both methods o f quantification, w h i c h w e r e c o m p a r a b l e for all the drugs tested.
Genistein, a tyrosine kinase inhibitor, had only a m o d e rate inhibitory effect o n parasite infectivity, suggesting that tyrosine kinases are not a major signalling pathway involved in cell invasion. O t h e r authors have reported
different results with another protozoan parasite, T. cruzi (Vieira etal, 1994; Favoreto et al, 1 9 9 8 ) . T h e y s h o w e d that genistein inhibited macrophages infection by T. cruzi b y up to 9 0 %. In o t h e r studies, genistein prevented proliferation o f T. brucei (Wheeler-Aim & Shapiro, 1 9 9 2 ) and Listeria ( V e l g e etal, 1 9 9 4 ) .
B y contrast, tachyzoite stimulation with b o m b e s i n , w h i c h activates MAP kinases in Swiss-3T3 cells (Pang
et al, 1993), had a m a r k e d effect o n both parasite infectivity and growth, as measured by both techniques o f quantification, suggesting that MAP kinases are a major crossing pathway in parasite signalling. In our experiments, tachyzoite activation with b o m b e s i n sti
mulated host cell invasion 2-fold. In addition, prein
cubation o f cell cultures with 5 0 nM b o m b e s i n led to a significant increase in Toxoplasma infectivity (not s h o w n ) , suggesting that signalling pathways involving MAP kinases in both the host cell and the parasite c o u l d b e involved in the invasion process. Similarly, prestimulation o f tachyzoites with PMA, a PKC activator (Kraft & Anderson, 1983), also induced a large increase in infectivity. As PKC is involved in MAP kinase acti
vation (Garrington & J o h n s o n , 1 9 9 9 ) , these results support a role o f MAP kinases in Toxoplasma invasion.
MAP kinase activation through PMA is a transient and reversible event, w h i c h is b o t h time- and dose-depen
dent. For this reason, the effect o f this drug on tachy
zoite invasion was o b s e r v a b l e only w h e n a given concentration was applied for a given time period, i.e;
5 0 0 nM for 3 0 min, as assessed by m i c r o s c o p i c c o u n ting. Conversely, tachyzoite treatment with R 0 3 1 - 8 2 2 0 , a PKC inhibitor, significantly reduced both parasite infec
tivity and growth, as s h o w n by the near-abolition o f uracil incorporation. T a k e n together, these results could confirm a role o f MAP kinases via PKC activation. It was reported that PMA can increase host cell infection by
T. cruzi (Vieira etal, 1994), while staurosporine, a PKC inhibitor, has a profound effect on the division and mor
phology o f Leishmania promastigotes ( B e c k e r & Jaffe, 1997) and inhibits erythrocyte invasion by P. knowlesi (Ward et al, 1994), both effects suggesting a role o f PKC in kinetoplastid survival and infectivity.
T o confirm the increase in infectivity occurring via MAP kinase activation, w e preincubated Toxoplasma tachy
zoites with P D 0 9 8 0 5 9 , a specific inhibitor o f MEK. MEK is an upstream dual-specific MAP kinase kinase that activates the MAP kinases ERK1 and ERK2 by phos
phorylation o f Thr-183 and Tyr-185 residues (Her etal., 1993). Host cell infection by tachyzoites pretreated with P D 0 9 8 0 5 9 was markedly inhibited, pointing to activa
tion o f ERK1- or ERK2-like proteins in T. gondii inva
sion p r o c e s s . Similar results have b e e n obtained using another in vitro m o d e l (Gomez-Marin etal, 1 9 9 8 ) . T a k e n t o g e t h e r , o u r results s u g g e s t that ERK-like kinases may b e involved in Toxoplasma infectivity
P a r a s i t e , 2 0 0 0 , 7, 9 5 - 1 0 1
Mémoire - 9 9
MAP
KINASES ANDTOXOPLASMA INFECTIVTTY
a n d growth, at least in vitro, through PKC activation.
In o t h e r p a t h o g e n s , such as Listeria a n d Salmonella typhimurium, the ERK pathway is required for host cell invasion ( P a c e et al, 1993; H o b b i e et ai, 1997; T a n g
& Sutherland, 1998). T h e involvement o f MAP k i n a s e pathways in the invasion p r o c e s s c o u l d b e related to various roles o f MAP k i n a s e s in eukaryotic cells, s u c h as: i) c y t o s k e l e t o n regulation a n d formation o f actin stress fibers through the small GTP-binding protein R h o (Ridley & Hall, 1994), it) activation o f PLA2 (Hirasawa et al., 1995; X i n g et al, 1996), a n d Ht) e x o c y t o s i s o f secretory vesicles ( C o x & Parsons, 1 9 9 7 ) . In particular, the role o f MAP k i n a s e s in Toxoplasma infectivity c o u l d b e e x p l a i n e d in several ways. First, a PLA2 from the parasite is k n o w n to b e required for host cell p e n e tration (Saffer & Schwartzman, 1991), an e n z y m e which c o u l d b e activated b y parasite MAP kinases. T h e inva
sion p r o c e s s takes p l a c e in several stages, including cell-surface recognition, parasite attachment, a n d e x o cytosis o f specific s e c r e t o r y o r g a n e l l e s (rhoptries a n d m i c r o n e m e s ) , that c o u l d d e p e n d o n MAP kinase acti
vation. Finally, t h e parasite actin c y t o s k e l e t o n is essen
tial for its motility a n d e n t i y into the host cell, as demonstrated b y the inhibition o f invasion b y cytocha- lasin D ( D o b r o w o l s k i & Sibley, 1996). As MAP kinases (also k n o w n as microtubule-associated protein kinases) are involved in the polymerization o f actin stress fibers through small G T P - b i n d i n g protein, they m a y also contribute to parasite motility a n d invasiveness.
In c o n c l u s i o n , o u r results point to t h e involvement o f MAP k i n a s e signalling pathways in the host cell inva
sion b y Toxoplasma gondii, possibly via PKC activa
tion.
A C K N O W L E D G E M E N T S
T
his w o r k w a s s u p p o r t e d b y Université Paris 5, F a c u l t é d e M é d e c i n e C o c h i n - P o r t Royal a n d ADERMEPT. W e thank Odile Puijalon (Pasteur Institute, Paris) for helpful d i s c u s s i o n s , a n d David Y o u n g for t h e English revision o f the manuscript.REFERENCES
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Recu le 17 Janvier 2000 Accepte le 3 avril 2000
Mémoire 101
MAP KINASES AND TOXOPLASMA INFECTIVITY
Parasite, 2 0 0 0 , 7, 9 5 - 1 0 1
