ACTIVATION OF THE CELLULAR MITOGEN-ACTIVATED PROTEIN KINASE PATHWAYS ERK, P38 AND JNK DURING TOXOPLASMA GONDII INVASION

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ACTIVATION OF THE CELLULAR MITOGEN-ACTIVATED PROTEIN KINASE PATHWAYS ERK, P38 AND JNK DURING TOXOPLASMA GONDII INVASION

VALÈRE A.*, GARNOTEL R.**, VILLENA I.*, G U E N O U N O U M***, PINON J.M.* & AUBERT D.*

Summary:

Host cell invasion is essential for the pathogenicity of the obligate intracellular protozoan parasite Toxoplasma gondii. In the present study, we evaluated the ability of T. gondii tachyzoites to trigger phosphorylation of the different mitogen-activated protein kinases (MAPK| in human monocytic cells T H P 1 . Kinetic experiments show that the peak of extracellular-signal-regulated kinase (ERK 1 / 2 ] , P38 and cjun-NH2 terminal kinase (JNKs) phosphorylation occurs between 10 and 6 0 min. The use of specific inhibitors of E R K 1 / 2 , P38 and JNK1/2 phosphorylation indicates the specificity of MAPKs phosphorylation during invasion. Signaling through cellular and parasite mitogen-activated protein (MAP) kinase pathways appears to be critical for T. gondii invasion.

K E Y W O R D S : Toxoplasma gondii, human monocytic cell (THP1], MAP kinases, phosphorylation.

Resumé : CINÉTIQUE D'ACTIVATION DE TA VOIE DES M I T O G E N - ACTIVATED PROTEIN KINASES CELLULAIRES E R K , P 3 8 E T J N K DURANT L'INVASION PAR TOXOPLASMA GONDII

L'invasion d'une cellule hôte est essentielle pour la pathogénicité de Toxoplasma gondii, parasite protozoaire intra-cellulaire obligatoire. Dans cette étude, nous évaluons la capacité des tachyzoites de T. gondii à déclencher la phosphorylation des différentes mitogen-activated protein kinases (MAPK) dans une lignée de cellules myélomonocytaires humaines THP1. Le pic de phosphorylation des extracellular-signal-regulated kinase (ERK1/2), P38 et c-Jun-NH2 terminal kinase (JNKs) se produit entre 10 et 60 minutes et la spécificité de l'activation des MAPKs a pu être montrée par utilisation d'inhibiteurs spécifiques de ERKs, P38 et JNKs. Les voies de signalisation passant par les MAP kinases

cellulaires, mais aussi parasitaires paraissent jouer un rôle critique dans les processus d'invasion de T. gondii.

MOTS CLES : Toxoplasma gondii, cellules monocytaires humaines (THP1), MAP kinases, phosphorylation.

T g o n d i i , the etiologic agent o f t o x o p l a s m o s i s is an ubiquitous protozoan parasite. Infection is generally asymptomatic, but congenital t o x o - plasmosis can h a v e serious c o n s e q u e n c e s and the d i s e a s e can b e life-threatening in i m m u n o c o m p r o - mised patients. As an obligate intracellular parasite,

T. gondii invades host cell by an active process. Macro- p h a g e s play a k e y role in directing the host i m m u n e r e s p o n s e to infection. Recruitment and stimulation o f m a c r o p h a g e s by cytokine a n d / o r microbial products such as LPS result in the induction and release o f several k e y i m m u n e effector m o l e c u l e s . T h e y play a crucial role in the d e v e l o p m e n t o f immunity against a n u m b e r o f intracellular p a t h o g e n s including Toxo-

* L a b o r a t o i r e d e P a r a s i t o l o g i e - M y c o l o g i e , EA 2 0 7 0 , I F R 5 3 , U F R d e M é d e c i n e , U n i v e r s i t é d e R e i m s - C h a m p a g n e - A r d e n n e , 5 1 , r u e C o g n a c q - J a y . 5 1 0 9 5 R e i m s , F r a n c e .

** L a b o r a t o i r e d e B i o c h i m i e et d e B i o l o g i e M o l é c u l a i r e . CNRS ERE 2 5 3 4 , I F R 5 3 , U F R d e M é d e c i n e , U n i v e r s i t é d e R e i m s - C h a m p a g n e - A r d e n n e . 5 1 , r u e C o g n a c q - J a y , 5 1 0 9 5 R e i m s , F r a n c e .

*** L a b o r a t o i r e d ' I m m u n o l o g i e , EA 2 0 7 0 , IFR 5 3 , U F R d e P h a r m a c i e , U n i v e r s i t é d e R e i m s - C h a m p a g n e - A r d e n n e . 5 1 . r u e C o g n a c q - J a y , 5 1 0 9 5 R e i m s . F r a n c e .

C o r r e s p o n d e n c e : D o m i n i q u e A u b e r t , L a b o r a t o i r e d e P a r a s i t o l o g i e - M y c o l o g i e , C H U H ô p i t a l M a i s o n - B l a n c h e , 4 5 , r u e C o g n a c q - J a y , 5 1 0 9 2 R e i m s , F r a n c e .

T e l . : 3 3 ( 0 ) 3 2 6 7 8 4 2 2 0 - F a x : 3 3 ( 0 ) 3 2 6 7 8 7 3 2 8 . E-mail: d a u b e r t @ c h u - r e i m s . f r

plasma gondii ( D e n k e r s & Gazzinelli, 1 9 9 8 ) . T h e m e c h a n i s m by w h i c h T. gondii initially stimulates m a c r o p h a g e s remains u n k n o w n . R e c e n t studies indi- cate that different protozoan products [glycosylphos- phatidylinositol ( G P I ) o f Trypanosoma cruzi, Plasmo- dium falciparum and Trypanosoma brucei (Ropert et al., 2 0 0 1 ; Schofield & Hackett, 1 9 9 3 ; T a c h a d o & S c h o - field, 1 9 9 4 ) ; l i p o p h o s p h o g l y c a n (LPG) o f Leishmania sp or soluble antigen extract o f T. gondii tachyzoites (Li et al., 1994)] h a v e an essential role in triggering various m a c r o p h a g e functions, similar to the importance o f LPS in infection with Gram negative bacteria (Ropert & Gazzinelli, 2 0 0 0 ) . LPS has b e e n reported to s t i m u l a t e signal t r a n s d u c t i o n t h r o u g h t h e MAPKs (Weinstein et a l . , 1992). B e l o n g i n g to the serine/threo- nine protein kinase family, MAP Kinases contribute to induce n u c l e a r r e s p o n s e s (Davis, 1 9 9 3 ) . T h e three main MAP kinases are extracellular-signal-regulated kinase (ERK), P 3 8 and cJun-NH2-terminal kinase (JNK).

T h e s e signaling pathways are important in protozoan such as Leishmania ( B e c k e r & Jaffe, 1 9 9 7 ) , Plasmo- dium or Theileria ( S h a w , 1 9 9 6 ) .

W e previously d e s c r i b e d a n e w effector m e c h a n i s m o f interferon g a m m a (IFN-y), the major cytokine o f host resistance to T. gondii infection, mediated b y p h o s - pholipase A2 ( P L A2) (Gomez-Marin et al., 1 9 9 6 ) . PLA2

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Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/2003101p59

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VALERE A., GARNOTEL R., VILLENA I. ETAL.

b e i n g a substrate for MAP kinases, w e also o b s e r v e d a rise in the MAP kinase activity o f infected T H P 1 cells, a n d s h o w e d that T. gondii p o s s e s s e d MAP kinase acti

vity ( G o m e z - M a r i n et al., 1 9 9 6 ) . In this study, w e c o m  pared the kinetics o f phosphorylation o f the different m e m b e r s o f the MAPK family. W e s h o w that T. gondii tachyzoites stimulate, with differential kinetics, the activation o f ERK, P 3 8 and JNK in THP1 h u m a n m o n o c y t i c c e l l s . T h e a v a i l a b i l i t y o f s p e c i f i c i n h i b i t o r s , allowed us to address the specificity o f t h e s e activa

tion during T. gondii invasion.

MATERIALS AND METHODS

IN VITRO INFECTION M O D E L

T

gondii tachyzoites o f the virulent, non cyst- forming RH strain w e r e maintained by inoculation in the peritoneal cavity o f female Swiss m i c e . Seventy-two hours after inoculation, tachyzoites w e r e r e c o v e r e d b y w a s h i n g the peritoneal cavity with 5 ml o f 0.9 % saline, penicillin 1 0 0 U / m l (Pharmacia, F r a n c e ) , streptomycin 4 0 µg/ml ( P h a r m a c i a , F r a n c e ) and amoxicillin 8 0 µg/ml ( E u r o b i o , F r a n c e ) . Isolated tachyzoites w e r e centrifuged at 2 , 0 0 0 g for 10 minutes and the pellets w e r e r e s u s p e n d e d in RPMI 1 6 4 0 cul

ture m e d i u m ( G i b c o Life T e c h n o l o g i e s , F r a n c e ) before being filtered through a 3 µm polycarbonate m e m b r a n e ( N u c l e o p o r e I n c . C a m b r i d g e , Mass., U S A ) yielding 9 9 . 9 % parasites free o f host cells. Parasite viability w a s a s s e s s e d by the trypan-blue e x c l u s i o n test ( 0 . 4 % solu

tion); s a m p l e s w e r e used w h e n the viability e x c e e d e d 9 5 % . H u m a n m o n o c y t i c THP1 cells (American T y p e Cell C o l l e c t i o n , R o c k e v i l l e , Maryland, U S A ) , w e r e grown in RPMI 1640 medium s u p p l e m e n t e d with 10 % d e c o m p l e m e n t e d fetal calf serum (FCS; B o e h r i n g e r , F r a n c e ) , 2 mM L-glutamine ( G i b c o , F r a n c e ) , penicillin ( 1 0 0 U / m l ) and streptomycin ( 5 0 µg/ml)[Gibco, France], at 3 7 ° C in an a t m o s p h e r e containing 5 % C O , . Non adherent cells w e r e distributed in 24-well plates (Nun- d o n , D e n m a r k ) and inoculated with 500,000 tachyzoites per well; the parasite/ THP1 cell ratio w a s 1 (final volume: 1 m l ) .

T O T A L PROTEIN ASSAY

After six hours incubation, cells a n d tachyzoites w e r e p l a c e d in lysis buffer ( 1 0 mM HEPES [Sigma, France], 150 mM NaCl, 1 mM sodium orthovanadate [Sigma, France], 10 % glycerol, 0.6 % Triton X 100, three anti- proteases: 0.2 m g / m l P e f a b l o c [Roth, Germany], 1 mM AEBSF [Uptima, France], and 10 µg/ml leupeptin [Sigma, France]) for 3 0 minutes, a n d w e r e then centrifuged at 4 0 0 g for 15 minutes. Aliquots o f supernatant ( 5 0 0 pi) containing the solubilized proteins w e r e then c o n c e n

trated b y precipitation in 4 ml o f absolute e t h a n o l at - 2 0 ° C overnight. After centrifugation at 2 0 0 g for 10 minutes, precipitated proteins w e r e determined a c c o r ding to the Bradford m e t h o d ( B i o - R a d Protein Micro Assay, F r a n c e ) .

KINETICS O F H O S T CELL M A P KINASE ACTIVATION ( W E S T E R N B L O T T I N G )

Host cell MAP kinase (ERK, P 3 8 a n d J N K ) activation in the p r e s e n c e o f viable T. gondii tachyzoites w a s d e t e c t e d in the total protein extract by W e s t e r n blot

ting. T h e following primary antibodies recognizing the activated ( p h o s p h o r y l a t e d ) forms o f the three MAP kinases were used: 1) murine monoclonal anti-phospho- p 4 4 / 4 2 MAP kinase antibodies [ ( T h r 2 0 2 / T y r 2 0 4 ) E 1 0 , (BioLabs, O z y m e s , F r a n c e ) specific for h u m a n E R K 1 / 2 , at a dilution o f 1/2,000]; 2 ) rabbit polyclonal antibodies against activated P 3 8 pAb p T G p Y [(Promega, F r a n c e ) specific for h u m a n P 3 8 ( a p p r o x i m a t e l y 4 8 k D a ) , at a dilution o f 1/2,000]; and 3 ) rabbit polyclonal antibodies against activated J N K pAb p T P p Y [(Promega, F r a n c e ) specific for h u m a n J N K ( d e t e c t i o n o f J N K 1 a n d J N K 2 at approximately 4 6 k D a a n d 5 4 k D a ) , at a dilution o f 1/2,000]. C o m p a r a b l e a m o u n t s o f MAP K i n a s e s are o b s e r v e d b y p r o b i n g parallel W e s t e r n b l o t s with anti

b o d i e s at a dilution o f 1 / 2 , 0 0 0 to u n p h o s p h o r y l a t e d MAP Kinases [rabbit polyclonal P 4 4 / 4 2 MAPK antibody (BioLabs, O z y m e s , F r a n c e ) ; goat polyclonal P 3 8 (Santa Cruz, T e b u , F r a n c e ) ; rabbit polyclonal J N K ( B i o L a b s , O z y m e s , France)]. According to the primary' antibodies, t w o types o f s e c o n d a r y antibodies w e r e used: p e r o x i - dase-conjugated polyvalent anti-immunoglobulin (goat a n t i m o u s e IgG, IgA and IgM), at a dilution o f 1 / 1 0 , 0 0 0 (Sigma, F r a n c e ) , or goat anti-rabbit IgG conjugated to alkaline p h o s p h a t a s e , at a dilution o f 1 / 1 0 , 0 0 0 (Sigma, F r a n c e ) . Non activated T H P 1 cells w e r e used as nega

tive control.

SPECIFICITY O F H O S T CELL M A P KINASE ACTIVATION DURING T. GONDII INVASION

Selective inhibitors o f the three MAP kinase families w e r e used. P D 0 9 8 0 5 9 ( 2 ' - a m i n o - 3 ' - m e t h o x y f l a v o n e , C a l b i o c h e m , F r a n c e ) , a n o n competitive inhibitor s p e cific for the ERK family (Alessi et al., 1 9 9 5 ) , inhibits the d e p h o s p h o r y l a t e d form o f M E K 1 / 2 and thereby prevents M E K 1 / 2 activation by Raf (without inhibiting the latter). S B 2 0 2 1 9 0 [4-(4-fluorophenyl)-2-(4-hydroxy- phenyl)-5-(4-pyridyl) lH-imidazole, Calbiochem, France]

directly inhibits the activation o f P 3 8 family m e m b e r s (Manthey, 1 9 9 8 ) . S B 2 0 3 5 8 0 [4-(4-fluorophenyl)-2-(4- methylsulfinylphenyl)-5-(4-pyridyl) l H - i m i d a z o l e , Cal

b i o c h e m , France], at the 5 0 µM concentration, inhibits J N K preferentially but also P 3 8 , as it only differs from the inhibitor S B 2 0 2 1 9 0 by a methylsulfinyl group.

TFIP1 cells were pretreated with a c h o s e n concentration

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М А Р К * A C T I V A T I O N I N T. GONDII I N V A S I O N

of the different inhibitors ( 5 0 µM) for 30 minutes before addition o f T. gondii. T h e concentration o f 5 0 pM, pre

serving cell viability o f at least 9 8 % (trypan blue exclu

sion test), was c h o s e n in a g r e e m e n t with other studies ( R o q u e s & Vidal, 1999; B o d e n et al, 2 0 0 0 ; Kettritz et al., 2 0 0 1 ) to evaluate the specificity o f MAPKs phos

phorylation.

RESULTS

ACTIVATION O F H O S T CELL E R K , J N K

AND P 3 8 PATHWAYS BY T. GONDII TACHYZOITES

W

e e x a m i n e d the kinetic o f activation o f THP1 cell ERK. J N K and P38 pathways by T. gondii tachyzoites. No variations o f unphosphory- lated MAP Kinase a m o u n t was o b s e r v e d (Figs 1-2-3), thus demonstrating that identical amounts o f proteins w e r e deposited. No basal level o f ERKs, P 3 8 or J N K s was detected in THP1 m o n o c y t i c cells. As s h o w n in Figures 1-2-3, tachyzoite infection leads to an earlier activation (five minutes) o f the three MAP Kinase (ERKs, JNKs and P38). W e o b s e r v e d that this effect was maxi

mal at 6 0 minutes with ERKs ( m o r e important for ERK2) (Fig. 1) and P38 (Fig. 2 ) . Both J N K isoforms were

Fig. 1. - T i m e - c o u r s e of c e l l u l a r u n p h o s p h o r y l a t e d o r p h o s p h o r y - lated Ip] E R K 1 a n d E R K 2 М А Р К d u r i n g T H P 1 cell i n v a s i o n b y T. gondii t a c h y z o i t e s . Total p r o t e i n ( 5 µg) w e r e a n a l y s e d b y W e s  tern b l o t t i n g u s i n g a p o l y c l o n a l P 4 2 / 4 4 М А Р К ( u p p e r p a n e l ) a n d a m o n o c l o n a l a n t i - p h o s p h o P 4 4 / 4 2 М А Р К ( l o w e r p a n e l . pF.RKs).

N e g a t i v e C o n t r o l ( N O : n o n a c t i v a t e d Т Н Р 1 c e l l s .

Fig. 2. - T i m e - c o u r s e of c e l l u l a r u n p h o s p h o r y l a t e d o r p h o s p h o r y - lated [p] P 3 8 M A P K d u r i n g T H P 1 cell i n v a s i o n b y T. gondii t a c h y  zoites. T o t a l protein ( 1 0 µg) w e r e a n a l y s e d b y W e s t e r n blotting using a p o l y c l o n a l P 3 8 MAPK ( u p p e r p a n e l ) a n d a p o l y c l o n a l a n t i - p h o s p h o P 3 8 M A P K ( l o w e r p a n e l , p P 3 8 ) . N e g a t i v e C o n t r o l ( N O : n o n a c t i v a t e d T H P 1 c e l l s .

Fig. 3- - T i m e - c o u r s e of c e l l u l a r u n p h o s p h o r y l a t e d o r p h o s p h o r y - l a t e d [p] J N K 1 a n d J N K 2 M A P K d u r i n g T H P 1 cell i n v a s i o n b y T. gondii t a c h y z o i t e s . T o t a l p r o t e i n ( 1 0 µ g ) w e r e a n a l y s e d b y W e s  tern blotting using a p o l y c l o n a l J N K 1 / J N K 2 MAPK ( u p p e r p a n e l ) a n d a p o l y c l o n a l a n t i - p h o s p h o J N K 1 / J N K 2 M A P K ( l o w e r p a n e l . p J N K s ) . N e g a t i v e C o n t r o l ( N O : n o n a c t i v a t e d T H P 1 c e l l s .

weakly activated with a maximum at 10 minutes (JNK1) and a d e c r e a s e toward basal level after 6 0 minutes (Fig. 3). T h e s e results s h o w that invasion by T. gondii stimulates all three MAPKs in human m o n o c y t i c T H P I cells.

SPECIFIC EFFECT O F M A P K INHIBITORS ON E R K 1 / E R K 2 , P 3 8 AND J N K 1 / J N K 2

PHOSPHORYLATION

W e determined the blocking effect o f the specific inhi

bitors PD 0 9 8 0 5 9 , S B 2 0 2 1 9 0 and S B 2 0 3 5 8 0 on the three MAPKs activation in human m o n o c y t i c cells acti

vated by T. gondii. Host cell MAPKs activation in pre

s e n c e o f viable T. gondii tachyzoites was detected in the total protein extract at 3 0 minutes (JNKs) or o n e hour (ERKs and P 3 8 ) by Western blotting. W e demons

trated that the three inhibitors prevent activation o f host cell MAPKs during parasite infection (Fig. 4 ) . Using spe

cific inhibitors allowed us to address the specificity o f these activations during T. gondii invasion.

DISCUSSION

W

hile the different morphological steps o f T. gondii invasion into host cells (recogni

tion, attachment and internalization) are n o w well studied, the molecular mechanisms are still poorly described. Our present data h a v e s h o w n that cellular MAP Kinases (ERKs. P 3 8 , J N K s ) are activated during the parasite invasion. Previous reports have s h o w n that soluble antigen extract o f T. gondii tachyzoites induced a pattern o f protein-tyrosine phosphorylation similar to those with LPS (Li et al., 1 9 9 4 ) and that protein tyro

sine phosphorylation and dephosphorylation are intra

c e l l u l a r s i g n a l i n g m e c h a n i s m s b y w h i c h LPS c a n mediate s o m e responses in m a c r o p h a g e s (Weinstein et al, 199.3). Our findings correlate recent studies (Robert- G a n g n e u x et al, 2000; Roisin et al. 2 0 0 0 ) , which also detected MAP kinase activity in T. gondii and charac-

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Fig. 4. - S p e c i f i c e f f e c t s o f M A P K i n h i b i t o r s o n cell M A P K a c t i v a tion b y T. gondii u s i n g W e s t e r n b l o t t i n g . A s i n g l e c o n c e n t r a t i o n o f 5 0 uM w a s u s e d for e a c h drug: P D 0 9 8 0 5 9 [1]. S B 2 0 2 1 9 0 [2] a n d S B 2 0 3 5 8 0 [3]. Five m i c r o g r a m s ( E R K 1 / E R K 2 ) o r ten m i c r o g r a m s ( P 3 8 , J N K 1 / J N K 2 ) w e r e a n a l y s e d w i t h o u t ( - ) o r w i t h ( + ) i n h i b i t o r .

terized three MAP kinases in T. gondii lysates. Their molecular weights are respectively 47 k D a and 4 3 k D a ( w h i c h w e r e putatively identified as ERK1 and ERK2 h o m o l o g s ) and 8 0 kDa. Our studies support the impor

tance o f phosphorylation in the activation o f signaling pathways in this parasite.

W e demonstrated the ability o f T. gondii tachyzoites to trigger phosphorylation o f E R K f / 2 , P 3 8 as J N K 1 / 2 in human m o n o c y t i c cells. All three classes o f MAPKs are simultaneously activated b y tachyzoites in THP1 cells with a m a x i m u m activation at 10 minutes (JNK) or 6 0 minutes (ERKs, P 3 8 ) postcontact. However, the individual subtypes exhibit differential kinetics o f acti

vation in r e s p o n s e to parasite. For e x a m p l e , while the activation o f JNK1 and J N K 2 rapidly d e c r e a s e s after 6 0 minutes, tachyzoite stimulation o f E R K 1 / 2 or P 3 8 acti

vation is longer sustained. W e also demonstrated that the three inhibitors prevent activation o f host cell MAPKs during parasite infection, s h o w i n g the specifi

city o f MAPKs phosphorylation during T. gondii inva

sion.

W e haved s h o w e d that activation o f b o t h cellular and parasitic MAP k i n a s e s w a s essential for parasite inva

sion into m o n o c y t i c cell ( G o m e z - M a r i n et al, f 9 9 8 ) . With other host cells m o d e l (murine fibroblastic cell line NIH-3T3), R o b e r t - G a n g n e u x et al. ( 2 0 0 0 ) d e m o n s  trated that treatments with several c o m p o u n d s as PD 0 9 8 0 5 9 led to a reduced tachyzoite invasion. MAP kinases p h o s p h o r y l a t i o n linked to tachyzoite p e n e tration o r s i m p l e parasite-host cell c o n t a c t , c o u l d trigger the PLA2 activation c a s c a d e in the host cell.

This activation pathway, previously d e s c r i b e d in Sal

monella typhimurium ( P a c e et al, 1 9 9 3 ) , is very rapid.

Moreover, our study o f the kinetics o f host cell MAP k i n a s e s d e m o n s t r a t e d early activation (within five m i n u t e s ) o f ERKs and J N K s , in the p r e s e n c e o f the parasite.

In other protozoan parasites, the importance o f MAP kinases was recently emphasized. Thus, in Leishmania

mexicana, a MAP kinase h o m o l o g u e (LMPK) has b e e n described as necessary for the survival and d e v e l o p ment o f the parasite within host cells, and involved in m o r p h o l o g i c a l differentiation during p r o m a s t i g o t e - amastigote transformation ( W i e s e , 1 9 9 8 ) . Leishmania lipophosphoglycans, w h i c h p r o m o t e parasite survival, act b y stimulating ERK MAPKs to inhibit m a c r o p h a g e IL-12 production ( F e n g et al, 1 9 9 9 ) . In Plasmodium falciparum ( D o e r i g et al, 1 9 9 6 ) , a MAP kinase desi

gnated as Pf MAP has b e e n characterized. Moreover, protein phosphorylation in Plasmodium knowlesi mero- zoites played an important role in their internalization into the erythrocytes (Ward et al, 1 9 9 4 ) . T h e m o d u  lation o f host cell protein kinase C activity has b e e n o b s e r v e d in a n u m b e r o f parasitic infections, including Leishmania donovani ( D e s c o t e a u x et al, 1 9 9 2 ) , Plas

modium falciparum (Hall et al, 1 9 9 7 ) and Toxoplasma gondii (Grunvald et al, 1 9 9 6 ) .

Our results imply the participation o f the three studied MAP kinase pathways, ERKs, P 3 8 and JNKs. T h e invol

vement o f these three signaling pathways has b e e n pre

viously described in bacteria infection such as Salmo

nella typhimurium ( H o b b i e et al, 1 9 9 7 ) and Listeria monocytogenes (Tang et al, 1 9 9 8 ) . T h e r e is growing e v i d e n c e that modulation o f host cell MAPK pathways during infection p r o c e s s m a y b e part o f o p p o s e d viru

l e n c e strategies used by several pathogens. Thus, Leish

mania donovani e v a d e the activation o f P 3 8 , J N K and E R K 1 / 2 during infection o f naive m a c r o p h a g e s (Prive

& D e s c o t e a u x , 2 0 0 0 ) . With Listeria monocytogenes ( T a n g et al, 1 9 9 8 ) , the induction o f MAPK pathways may b e required for uptake by host epithelial cells. For Salmonella typhimurium, the activation o f MAPK path

w a y s may contribute to the acute inflammatory res

p o n s e s induced ( H o b b i e et al, 1 9 9 7 ) .

In this study, w e s h o w e d the kinetic o f activation o f THP1 cell MAPKs pathways by T. gondii tachyzoites.

W e demonstrated the ability o f T. gondii to trigger an early and specific phosphorylation o f cellular ERKs, P38 and JNKs.

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MAPKs A C T I V A T I O N IN T. GONDII I N V A S I O N

ACKNOWLEDGEMENTS

T

his work was supported by Université de Reims Champagne-Ardenne, France. A. Valère was the recipient o f a grant from Fondation B a y e r Santé.

W e thank David Y o u n g for c h e c k i n g our English.

REFERENCES

A L E S S I D.R., C U E N D A A., C O H E N P., D U D L E Y D.T. & S A L T I E L A.R.

PD 098059 is a specific inhibitor of the activation of mitogen- activated protein kinase kinase in vitro and in vivo. The Journal of Biological Chemistry, 1995. 270. 27489-27494.

B E C K E R S. & JAFFE C.L. Effect of protein kinase inhibitors on the growth, morphology, and infectivity of Leishmania promastigotes. Parasitoly Research, 1997, 83, 273-280.

B O D E N S.E., B E R T S C H E T., A M M O N H.P. & S A F A Y H I H. MEK-1/2 inhibition prevents 5-lipoxygenase translocation in N-for- mylpeptide-challenged human neutrophils. The Iinterna- tional Journal of Biochemistry and Cell Biology, 2000, 32, 1069-1074.

D A V I S R.J. The mitogen-activated-protein kinase signal trans- duction pathway. The Journal of Biological Chemistry, 1993, 268. 14553-14556.

D E N K E R S E.Y. & G A Z Z I N E L L I R.T. Regulation and function of T-cell-mediated immunity during Toxoplasma gondii infec- tion. Clinical Microbiology Reviews, 1998, 11, 569-588.

D E S C O T E A I x A., M A T L A S H E W S K I G . & T U R C O S . J . Inhibition of macrophage protein kinase C-mediated protein phosphory- lation by Leishmania donovani lipophosphoglycan. Jour- nal of Immunology, 1992, 149, 3008-3015.

D O E R I G CM.. P A R Z Y D., L A N G S L E Y G . , H O R R O C K S P., C A R T E R R.

& DOERIG C D . A MAP kinase homologue from the human malaria parasite. Plasmodium falciparum. Gene, 1996.

117, 1-6.

F E N G G . J . , G O O D R I D G E U . S . . H A R N E T T M.M., W E I X . Q . , N I K O -

LAEV A.V., HIGSON A.P. & L I E W F Y . Extracellular signal- related kinase (ERK) and P38 mitogen-activated protein (MAP) kinases differentially regulate the lipopolysaccharide-mediated induction of inducible nitric oxide synthase and IL-12 in macrophages: Leismania phosphoglycans subvert macrophage IL-12 production by targeting MAP kinase. Journal of Immunology, 1999, 163, 6403-6412.

G R U N V A L D E., C H I A R A M O N T E M., H I E X Y S., W Y S O K A M., T R I N - C H I E R I G . , V O G E L S.N., G A Z Z I N E L L I R.T. & SHER A. Bioche- mical characterization and protein kinase C dependency of monokine-inducing activities of Toxoplasma gondii.

Infection and Immunity 1996, 64, 2010-2018.

G O M E Z - M A R I N J . E . , B O N H O M M E A., G U E N O U N O U M. & P I N O N J.M.

Role of Interferon-y against invasion by Toxoplasma gondii in a human monocytic cell line (THP1): involvement of the parasite's secretory phospholipase A2. Cellular Immu-

nology. 1996, 169, 218-225.

G O M E Z - M A R I N J . E . , V A L E R E A.. B O N H O M M E A., EL' B T A O U R I H., A N T O N T C E L L I F . , B U R L E T H., A L B E R T D., V I L L E N A I.. G U E N O U - N O U M., H A Y E B. & P I N O N J . M . Interferon-g signal transduction during parasite infection: modulation of MAP kinases

Parasite, 2003. 10. 59-64

in the infection of human monocytes cells (THP1) by Toxoplasma gondii. Parasite Immunology. 1998, 20, 6 3 1 - 635.

H A L L B . S . , D A R A M O L A O.O., B A R D E X G . & TARGETT G . A . Modu- lation of protein kinase C activity in Plasmodium falcipa- rum-infected erythrocytes. Blood. 1997. 89. 1770-1778.

H O B B I E S., C H E N L.M.. D A V I S R.J. & GALAN J.E. Involvement of mitogen-activated protein kinase pathways in the nuclear responses and cytokine production induced by Salmonella typhimurium in cultured intestinal epithelial cells. Journal of Immunology, 1997, 159, 5550-5559.

K E T T R I T Z R., S C H R E I B E R A., LUFT F . C . & H A L L E R H. Role of mitogen-activated protein kinases in activation of human neu- trophils by antineutrophil cytoplasmic antibodies. Journal of American Society of Nephrology, 2001, 12, 37-46.

LI Z . Y . , M A N T H E Y C.L., P E R E R A P . Y . , S H E R A. & V O G E L S.N. Toxo- plasma gondii soluble antigen induces a subset of lipo- polysaccharide-inducible genes and tyrosine phosphopro- teins in peritoneal macrophages. Infection and Immunity, 1994, 62, 3434-3440.

M A N T H E Y C.L., W A N G S.W.. K I N N E Y S . D . & Y A O Z . SB 202190, a selective inhibitor of P38 mitogen-activated protein kinase, is a powerful regulator of LPS-induced mRNAs in mono- cytes. Journal of Leukocyte Biology, 1998, 64, 409-417.

P A C E J . , H A Y M A N M.J. & G A I A X J.E. Signal transduction and inva- sion of epithelial cells by Salmonella typhimurium. Cell.

1993, 72. 505-514.

P R I V E C. & D E S C O T E A U X A. Leishmania donovani promastigotes evade the activation of mitogen-activated protein kinases P38, C-jun N-terminal kinase, and extracellular signal-regulated kinase-1/2 during infection of naive macrophage.

European Journal of Immunology, 2000, 30, 2235-2244.

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 - C A M E T J . & Roisix M.P. Involvement of the mitogen-activated protein (MAP) kinase signaling pathway in host cell invasion by Toxo- plasma gondii. Parasite. 2000. 7, 95-101.

R O I S I X M.P., R O B F . R T - G A X G X E I x F „ C R E U Z E T C. & DUPOUY-

CAET J . Biochemical characterization of mitogen-activated protein (MAP) kinase activity in Toxoplasma gondii. Para- sitology Research. 2000. 86. 588-598.

R O P E R T C. & G A Z Z I N E L L I R.T. Signaling in immune system cells by glycosylphosphatidylinositol anchor and related struc- tures derived from parasitic protozoa. Current Opinion in Microbiology. 2000, 3. 395-403.

R O P E R T C . A L M E I D A I.C., C I . O S E L M., T R A V A S S O S L.R., F E R G U S S O N

M.A.J., C O H E N P. & G A Z Z I N E L L I R.T. Requirement of mitogen- activated protein kinases and IKB phosphorylation for induction of proinflammatory cytokines synthesis by macrophages indicates functionnal similarity of receptors triggered by glycosylphosphatidylinositol anchors from parasitic protozoa and bacteria lipopolysaccharide. Journal of Immunology. 2001. 166, 3423-3431.

R O Q U E S M. & V I D A L H. A phosphatidylinositol 3-Kinase/p70 ribosomal S6 protein kinase pathway is required for the regulation by insulin of the p85 alpha regulatory subunit of phosphatidylinositol 3-kinase gene expression in human muscle cells. The Journal of Biological Chemistiy, 1999, 26, 34005-34010.

Note de recherche ⁶³

(6)

VA1ÈRE A.. GARNOTEL R , VILLENA I. ETAL.

S C H O F I E L D L. & HACKETT F . Signal transduction in host cells by glycosylphosphatidylinositol toxin of malaria parasites.

The Journal of Experimental Medicine, 1993, 177, 145-153.

S H A W M.K. Theileria parva sporozoite entry into bovine lym- phocytes involves both parasite and host cell signal trans- duction processes. Experimental Parasitology, 1996, 84, 344-354.

T A C H A D O S.D. & S C H O F I E L D L. Glycosylphosphatidylinositol toxin o f Trypanosoma brucei regulates I L - l α and T N F - α expression in macrophages by protein tyrosine kinase mediated signal transduction. Biochemical and Biophysical Research Communications, 1994, 205, 984-991.

T A N G P., S U T H E R L A N D C.L., GOLD M.R. & F I N L A Y B.B. Listeria monocytogenes invasion o f epithelial cells requires the MEK-l/ERK-2 mitogen-activated protein kinase pathway.

Infection and Immunity, 1998, 66, 1106-1112.

W A R D G.E., F U J I O K A H.. A I K A W A M. & M I L L E R L.H. Staurospo- rine inhibits invasion of erythrocytes by malarial mero- zoites. Experimental Parasitology. 1994, 79, 480-487.

W E I N S T E I N S.L., S A N G H E R A J.S., L E M K E K . , D E F R A N C O A.L. &

P E L E C H S.L. Bacterial lipopolysaccharide induces tyrosine phosphorylation and activation of mitogen-activated pro

tein kinases in macrophages. The Journal of Biological Che

mistry, 1992, 267, 14955-14962.

W E I N S T E I N S.L., JUNE C H . & D E F R A N C O A.L. Lipopolysaccha- ride-induced protein tyrosine phosphorylation in human macrophage is mediated by CD14. Journal of Immunology.

1993, 151, 3829-3838.

W I E S E M. A mitogen-activated protein (MAP) kinase homo

logue of Leishmania mexicana is essential for parasite sur

vival in the infected host. The EMBO Journal. 1998, 7 7, 2619-2628.

Reçu le 26 mars 2002 Accepté le 16 décembre 2002

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