one object of the invention is an active molecule capable of modulating the activity of a native protein of a parasitic organism of the apicomplexa phylum , wherein said molecule is endowed with serine - threonine phosphatase activity , or a fragment thereof a fragment of said molecule is peptidic sequence capable of being recognized by a polyclonal serum obtained after immunization of a rabbit with a purified pp2c protein of t . gondii . in a preferred embodiment , the molecule endowed with serine - threonine phosphatase activity is a type 2c phosphatase ( pp2c ), and the parasitic organism of apicomplexa phylum is selected from the group comprising toxoplasma gondii , plasmodium falciparum and crystosporidium parvum . another object of the invention is a molecule for preventing or treating an infection due to a parasitic organism of the apicomplexa phylum wherein said molecule modulates the interaction between a protein of said parasitic organism endowed with serine - threonine phosphatase activity and toxofilin of said parasitic organism . in a preferred embodiment , the protein of said parasitic organism endowed with serine - threonine phosphatase activity is a type 2c phosphatase ( pp2c ), and the parasitic organism of apicomplexa phylum is selected from the group comprising toxoplasma gondii , plasmodium falciparum and crystosporidium parvum . the protein of said parasitic organism has a nucleic acid sequence and an amino acid sequence with sufficient identity compared to the sequence of fig1 for being endowed with serine - threonine phosphatase activity . the active site corresponding to the enzymatic activity is located from amino acid 18 to amino acid 325 ( included ). the molecular weight is 37 kda . another object of the invention is a method for screening molecules capable of modulating the activity of a native protein of a parasitic organism of the apicomplexa phylum endowed with serine - threonine phosphatase activity wherein said method comprises the steps of : a ) possibly fixing a native or a recombinant toxofilin of said parasitic organism to a matrix ; b ) phosphorylating said toxofilin with labeled atp using a parasite kinase fraction or a recombinant casein kinase ii ; d ) incubating the labeled toxofilin with or without the molecule to be tested and adding a native or a recombinant serine - threonine phosphatase of said parasitic organism ; wherein a variation of the labeling of the toxofilin incubated with the molecule to be tested compared with the labeling of the toxofilin incubated without the molecule to be tested is indicative of the capacity of the molecule to modulate said serine - threonine phosphatase activity of said protein . in a preferred embodiment , the protein of said parasitic organism endowed with serine - threonine phosphatase activity is a type 2c phosphatase ( pp2c ), and the parasitic organism of apicomplexa phylum is selected from the group comprising toxoplasma gondii , plasmodium falciparum and crystosporidium parvum . an other object of the invention in a molecule capable of modulating the activity of a native protein of a parasitic organism of the apicomplexa phylum endowed with serine - threonine phosphatase activity which is capable to be screened by said method . another object of the invention is a method for screening molecules for preventing or treating an infection due to a parasitic organism of the apicomplexa phylum wherein said method comprises the steps of : f ) possibly fixing a native or a recombinant toxofilin of said parasitic organism to a matrix ; g ) phosphorylating said toxofilin with labeled atp using a parasite kinase fraction or a recombinant casein kinase ii ; i ) incubating the labeled toxofilin with or without the molecule to be tested and adding a native or a recombinant serine - threonine phosphatase of said parasitic organism ; wherein a decrease of the labeling of the toxofilin incubated with the molecule to be tested compared with the labeling of the toxofilin incubated without the molecule to be tested is indicative of the capacity of the molecule to prevent or treat an infection due to a parasitic organism of the apicomplexa phylum . in a preferred embodiment , the protein of said parasitic organism endowed with serine - threonine phosphatase activity is a type 2c phosphatase ( pp2c ), and the parasitic organism of apicomplexa phylum is selected from the group comprising toxoplasma gondii , plasmodium falciparum and crystosporidium parvum . another object of the invention is an active molecule for preventing or treating an infection due to a parasitic organism of the apicomplexa phylum which is capable to be screened by said method . another object of the invention is an antibody directed against a native protein of a parasitic organism of the apicomplexa phylum , said protein being endowed with serine - threonine phosphatase activity . another object of the invention is a method for preventing or treating an infection due to a parasite of the apicomplexa phylum wherein said method comprise administration of a molecule of the invention . in vivo 32 p orthophosphate labeling of tachyzoïte and toxofilin immunoprecipitation purified transiently extracellular parasites were rinsed in phosphate and serum - free buffer ( 10 mm tris - cl ph 7 . 4 , 150 mm nacl , 5 mm kcl , 5 mm mgcl 2 , 1 . 6 mm cacl 2 , 0 . 5 % glucose , 0 . 1 % bovine serum albumin ) and incubated at 10 8 per ml in the same medium supplemented with 500 μci per ml of orthophosphoric acid ( specific activity of 8 . 8 10 9 ci per mmole from nen life science products , inc ) ( 120 min , 37 ° c ., 5 % co 2 ). unincorporated radioactive phosphate was then washed out by rinsing three times the parasites in 50 ml of phosphate and serum - free buffer . 10 9 tachyzoïtes were lysed in 1 ml of [ 20 mm tris - cl ph 8 . 0 , 50 mm kcl , 0 . 1 mm ethyleneglycol - bis ( β - aminoethyl )- n , n , n , n ′- tetraacetic acid ( egta ), 0 . 1 mm ethylenediamine - tetraacetic acid ( edta )] supplemented with 0 . 5 % ( vol / vol ) protease inhibitor stocks by 5 liquid nitrogen freezing and defreezing cycles . lysates were centrifuged ( 10 min , 800 × g , 4 ° c .) and the corresponding supernatants were first clarified ( 20 min , 20 . 000 × g , 4 ° c . ), then precleared on sepharose cl - 4b ( pharmacia ) ( 1 hour , 4 ° c .). after removal of the sepharose - bound protein fractions , the soluble fractions were successively incubated with toxofilin antibodies ( overnight , 4 ° c .) and with protein g - sepharose ( 1 hour , 23 ° c .). after successive washes in buffer a ( 50 mm tris - hcl , ph 7 . 5 , 150 mm nacl ) supplemented with 0 . 1 % vol / vol tx - 100 and 0 . 5 % ( wt / vol ) serum albumin then supplemented only with 0 . 1 % vol / vol tx - 100 and a final wash in buffer a , the protein g - sepharose bound fraction was eluated in sds - page sample buffer . eluates were boiled prior to a 12 % acrylamide gel electrophoresis and radioactive scan of the dried gel . the inventors used the expression vector pgex6 - p3 ( pharmacia ) into which the full length toxofilin encoding cdna was cloned as described in poupel et al ( 2000 ) but to improve the yield of toxofilin production , the protocol was slightly modified as follows . an e . coli clone ( bl21 strain ) positive for the plasmid was grown up to od = 1 . 2 - 1 . 4 and induced with isopropylthio - β - d - galactoside ( 0 . 1 mm , 1 hour , 25 ° c .). at the end of the induction period the bacteria were pelleted and subsequently lysed in buffer pbs - and sonicated ( 30 seconds , 4 ° c .). the lysate was supplemented with tx - 100 ( 0 . 5 % vol / vol ) and n - tetradecyl - n , n - dimethyl - 3 ammonio - 1 - propanesulfonate ( 0 . 5 % wt / vol , sigma ) ( 15 min , 4 ° c .). the supernatant recovered after centrifugation ( 15 . 000 × g , 15 min , 4 ° c .) was incubated with sepharose cl - 4b ( 1 hour , 4 ° c .) and the unbound fraction was incubated with glutathione sepharose ( pharmacia ) ( 4 ° c ., overnight ). the beads were washed with 30 bead volumes of pbs - containing 0 . 1 % tx - 100 and with 10 volumes of prescission cleavage buffer ( 50 mm tris - hcl , ph 7 . 0 , 150 mm nacl , 1 mm edta , 1 mm dithiothreitol ( dtt )). the bound gst - polypeptide was cleaved with pre - scission protease to recover the recombinant toxofilin ( r - toxofilin ) without gst ( pharmacia ) ( 8 hours , 4 ° c .). soluble r - toxofilin was immunoprecipitated with anti - toxofilin antibodies ( overnight , 4 ° c .) and recovered on protein g - dynabeads ( 1 hour , 23 ° c .) ( dynal ) before the kinase / phosphatase assay . identification and cloning of t . gondii type 2c phosphatase ( tgpp2c ) native gel and peptide microsequencing : the native gel was performed as described in poupel et al . ( 2000 ). the gel slice containing the 36 kda actin - binding protein from the parasite was subjected to tryptic digestion ( 30 ° c ., 18 hours , 0 . 3 mg of trypsin in 0 . 1 m trishcl , ph 8 . 6 ; 0 . 01 % ( vol / vol ) tween 20 ). the tryptic peptides were recovered by hplc on a deae and a c18 columns . the sequencing of two peptides gave respectively svfdgtvgdfaqenv and nqsadnitamtvffk and the latter was found in one clone from the t . gondii database of expressed sequence tags ( est , washu - merk toxoplasma est project ). cdna library screening and dna sequencing : non - degenerate primers were synthesized for amplification of the target sequence from the clone identified as tgestzy48a06 . r1 . the oligonucleotide with the sequence : 5 ′- agtgcagacaacattactgcgatg - 3 ′ corresponding to part of one peptide microsequence ( sadnitam ) was used as the up stream primer , while 5 ′- agacacaccaagaatctcgtc - 3 ′ was chosen as the down stream primer in the tgest clone . the pcr conditions for amplification of the 207 bp dna product were as follows : a hot start of 2 min at 94 ° c . followed by 35 cycles ( 45 sec , 94 ° c . ; 30 sec , 53 ° c . ; 30 sec , 72 ° c .) and a final elongation step at 72 ° c . for 10 min . the 207 bp fragment recovered was 32 p - labeled using random priming ( megaprime kit , amersham ), purified on sephacryl s - 400 hr column ( pharmacia ) and used as a probe to screen a t . gondii tachyzoite cdna library ( kindly provided by j . w . ajioka , cambridge , uk ). after 2 rounds of screening , 12 independent overlapping clones were selected and their cdna was prepared for nucleotide sequencing performed by genset ( france ), using both vector and t . gondii sequence specific primers ( genset ). phosphatase assays were carried out using 10 μm 32 p - casein . briefly , the reaction mixture in a total of 30 μl , consisted of 10 μl containing 100 ng protein of tachyzoïte cytosolic fraction ( in 10 mm tris - cl ph 8 . 0 , 150 mm nacl , 0 . 1 % vol / vol of protease inhibitors , 4 ° c .) plus 10 μl of phosphatase assay buffer ( 50 mm tris - cl ph 7 . 4 , 0 . 5 % β - mercaptoethanol , 0 . 1 % bsa ) containing the different effectors . 10 μl of labeled substrate ( 32 p - casein ) was added to start the reactions ( 30 min , 30 ° c .) and 200 μl of 20 % trichloracetic acid to stop them . the mixtures were centrifuged ( 5 min , 15 . 000 × g ) and 180 μl of the supernatant was directly counted for 32 p radioactivity using a cerenkov counter . the fragment for expression of tgpp2c was prepared by pcr amplification of a full length tgpp2c encoding cdna , using primers introducing a ecori restriction site at position 5 ′ and a xbai restriction site at position 3 ′. for amplification of the upper strand : 5 ′- gccgaattcccatgaagtcctctgctgaaattag - 3 ′ and of the lower strand : 5 ′- gcctctagactaatcagtcttcttgaagaacactg - 3 ′. the amplified fragment was cloned into the expression vector pthiohis b ( invitrogen ) after digestion with ecori and xbai of both fragment and vector . for expression of the thiohis - tgpp2c , an e . coli clone ( top10 strain ) positive for the plasmid was grown up to od = 0 . 8 and induced with isopropylthio - β - d - galactoside ( 0 . 1 mm , 2 hours , 37 ° c .). at the end of the induction period , the bacteria were pelleted and subsequently lysed in buffer ( 20 mm nah 2 po 4 , 500 mm nacl , n - octylglucoside ( 0 . 5 % vol / vol ) supplemented with 0 . 1 % ( vol / vol ) protease inhibitor stocks by sonication ( 30 seconds , 4 ° c .) and one liquid nitrogen freezing and defreezing step ( 10 min , 4 ° c .). dnase was added to 2 μg / ml ( 30 min , 4 ° c .) followed by centrifugation ( 10 min , 14 . 000 × g , 4 ° c .). the supernatant was chromatographied on a nickel column ( probond , invitrogen ) and the imidazole eluate was dialyzed before being chromatographied on a phenylarsineoxide - agarose column ( thiobond , invitrogen ). mercaptoethanol eluates were dialyzed against 5 mm tris - hcl , ph 7 . 5 , 50 mm nacl and stored aliquoted in 5 % sucrose at 80 ° c . until use for testing the activity ( see above ). cytosol : frozen tachyzoites ( 10 9 ) were thawed on ice and lysed by 5 liquid nitrogen freezing and defreezing cycles in 500 μl of kinase buffer ( 10 mm tris - hcl , ph 7 . 5 , 150 mm nacl , 10 mm mgcl 2 , 1 mm dtt ) supplemented with 0 . 2 % ( vol / vol ) protease inhibitor stocks . the extract was centrifuged ( 15 min , 800 × g , 4 ° c .) to remove nuclei and unbroken cells . the supernatant was centrifuged ( 30 min , 100 . 000 × g , 4 ° c .) in a tl100 table top ultracentrifuge ( beckman ) using the tla 100 . 3 rotor . the resulting cytosol was stored frozen at − 80 ° c . in 100 μl aliquots until use . heparin chromatography : a cytosolic fraction from 10 9 parasites was pre - cleared on sepharose cl - 4b ( 1 hour , 4 ° c . ), and subsequently chromatographied on heparin sepharose ( pharmacia ) ( 1 hour , 4 ° c .). after several washes in 10 mm tris - hcl , ph 7 . 5 , 150 mm nacl supplemented with 0 . 5 % vol / vol tx - 100 , the heparin - bound proteins were recovered by a 0 . 5 m nacl elution in 10 mm tris - hcl , ph 7 . 5 . the eluate was dialyzed against kinase buffer ( overnight , 4 ° c .) prior to be used in kinase assay while the heparin unbound fraction ( i . e . : flow through ) was thoroughly recovered and stored at − 80 ° c . each lot was controlled for its activity on casein ( see above ). kinase reaction : 2 μg of immobilized rtoxofilin on protein g - dynabeads were washed in kinase buffer before to be incubated with a tachyzoïte cytosolic fraction prepared in kinase buffer ( μg of proteins in 100 μl ) and precleared on protein g - dynabeads . the reaction was started by adding 100 μm of na 2 atp and 10 μci of [ γ 32 p ] atp ( 3000 ci / mmol , nen life science product , inc ) ( 15 min , 30 ° c .). unbound materials and unincorporated [ γ 32 p ] atp were washed out with 200 volumes of kinase buffer containing with tx - 100 ( 0 . 5 % vol / vol ) followed by 100 volumes of kinase buffer . toxofilin and bound proteins were eluted in sds - page sample buffer prior to electrophoresis and radioactivity scan ( phosphoimager , molecular dynamics ). toxofilin phosphorylation was quantified using nih image quant software . to characterize the kinase activity responsible for toxofilin phosphorylation , three types of experiments were carried out : 1 ) pharmacological inhibitors such as heparin ( 20 μg per ml , sigma ), gtp ( 200 μm , sigma ), 5 , 6 - dichloro - 1 - b - d - ribofuranosylbenzimidazole ( 100 μm , calbiochem ) or staurosporine ( 1 μm , calbiochem ) were added 15 min before starting the kinase reaction . 2 ) a fraction eluted after heparin chromatography of the cytosol and the corresponding unbound fraction ( see above ) were assayed for their respective kinase activity towards rtoxofilin . phosphatase reaction : the purified recombinant tgpp2c dialyzed against kinase buffer was added ( doses activité ) either before to start the kinase assay or after the last wash in kinase buffer . in the latter case , control and test samples were incubated for 15 additional minutes ( 30 ° c .) before a final wash in kinase buffer . in some control experiments , one unit of a recombinant fragment of rabbit catalytic type 1 phosphatase ( up state biotechnology ) which is known to dephosphorylate several t . gondii tachyzoïte proteins was replacing tgpp2c . eluates were treated as described for the kinase assay . a rabbit polyclonal antibody raised against the gst - partial pp2c was prepared and absorbed on gst to get only the pp2c reactive immunoglobulins . it has been initially raised using gst - partial pp2c separated in a polyacrylamide gel slice directly injected to rabbits ( according to standard protocol of eurogentec , 4 immunizations on day 0 , 14 , 28 and 56 ). each immunization performed with a composition containing from 20 to 100 μg of pp2c which is a polypeptide comprising 265 amino acids from v64 to k328 included as referred in fig2 . it is possible to covalently fix the recombinant toxofilin to a matrix ( resin or membrane ), to phosphorylate it with 32 p adenosine tri phosphateusing either an enriched parasite kinase fraction which is already available or even a recombinant casein kinase ii from other source ( see materiel and methods ). human casein kinase ii works well at phosphorylating toxofilin . in addition , the inventors are presently cloning the toxoplasma casein kinase ii . once phosphorylation has been controlled by radioactive counting , it is easy to incubate the sample ( 32 p - labelled toxofilin ) with or without ( control ) putative inhibitors and add recombinant pp2c . the criteria to analyze will be the radioactive counts and to observe if those counts have or have not decreased . an efficient phosphatase hydrolyses the 32 p phosphate which is then lost in the washes and consequently induces a decrease in radio - active counts . if the phosphatase activity is blocked by an inhibitor ( either towards the catalytic site or affecting the 3d structure of the catalytic site ), the 32 p phosphate will not be hydrolyzed . such assay also allows quantitative analysis of the inhibitory effect towards the phosphatase activity . some flurogenic substrates have been recently developed as an alternative to radio - activity for several phosphatase activity dosages . one might think about incorporating such fluorochrome to toxofilin . additionally , other substrate such as casein are commonly used to assay phosphatase activity including pp2c activity . protocol to screen for inhibitors of the host cell invasion by toxoplasma gondii . one feature of toxoplasma gondii tachyzoïte is that it can enter virtually any kind of cells , making in vitro invasion assay quite simple to realize . it is also feasible to incubate tachyzoïtes with orthophosphate ( see materials and methods ) and at the same time expose or not them to different putative inhibitors ( different doses . . . ). in that case , it is possible to check if this / these inhibitor ( s ) affect the phosphorylation of toxofilin ( preparation of cytosol , immunoprecipitation of toxofilin , electrophoresis and radioactive scanning to detect if toxofilin has or not incorporated 32p ). for invasion assay , the tachyzoïtes can be resuspended in 2 ml of dulbecco &# 39 ; s mem ( usually 5 × 10 7 ) supplemented with 2 % of heat - inactivated foetal calf serum and expose to the putative inhibitors ( different doses , duration . . . ) before being incubated with 70 - 80 % confluent human foetal fibroblasts previously plated on glass coverslips ( 20 min , 37 ° c ., 5 % co 2 ). it will be interesting to leave the inhibitor during the invasion assay ( in case it is reversible ) or to wash it off before the assay and finally to check any affect on the host cell . after a short contact between parasites and host cell ( 15 to 30 min ), both will be fixed in 2 % paraformaldehyde in pbs - ( 15 min , 23 ° c .). extracellular parasites will be stained with a monoclonal anti - p30 surface protein of t . gondii ( 40 μg / ml , euromedex ) and revealed using the alexa488 anti - mouse igg conjugate ( molecular probes ) while both internalized and extracellular parasite will be vizualized by 4 ′, 6 diamidino - 2 - phenylindole staining ( dapi , 5 μg / ml ) under microscope . the number of cells containing parasites out of 100 cells randomly selected will be reported in triplicate for each treatment . in addition , for each coverslip , the number of internalized parasites per cell will be counted on 4 samples of 25 infected cells .
the present invention relates to a serine - threonine phosphatase protein of a parasitic organism of the apixomplexa phylum and uses thereof .