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A cyanobacterial serine protease of Plasmodium falciparum is targeted to the apicoplast and plays an important role in its growth and development

A cyanobacterial serine protease of Plasmodium falciparum is targeted to the apicoplast and plays an important role in its growth and development
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  A cyanobacterial serine protease of  Plasmodium falciparum is targeted to the apicoplast and plays an important role inits growth and development mmi_7251 873..890 Sumit Rathore, 1† Dipto Sinha, 1† Mohd Asad, 1† Thomas Böttcher, 2† Farhat Afrin, 3 Virander S. Chauhan, 1 Dinesh Gupta, 1 Stephan A. Sieber 2 and Asif Mohmmed 1 * 1 International Centre for Genetic Engineering and Biotechnology, New Delhi 110 067, India. 2 Department of Chemistry, Technical University of Munich, Lichtenbergstr. 4, D-85747 Garching, Germany. 3 Department of Biotechnology, Jamia Hamdard University, New Delhi 110 062, India. Summary The prokaryotic ATP-dependent protease machiner-ies such as ClpQY and ClpAP in the malaria parasitemay represent potential drug targets. In the presentstudy, we show that the orthologue of cyanobacterialClpP protease in  Plasmodium falciparum   (PfClpP) isexpressed in the asexual blood stages and pos-sesses serine protease activity. The PfClpP waslocalized in the apicoplast using a GFP-targetingapproach, immunoelectron microscopy and by immu-nofluorescence assays. A set of cell permeable b -lactones, which specifically bind with the active siteof prokaryotic ClpP, were screened using an  in vitro  protease assay of PfClpP. A PfClpP-specific proteaseinhibitor was identified in the screen, labelled asU1-lactone.  In vitro   growth of the asexual stage para-sites was significantly inhibited by U1-lactonetreatment. The U1-treated parasites showed develop-mental arrest at the late-schizont stage. We furthershow that the U1-lactone treatment resulted in forma-tion of abnormal apicoplasts which were not able togrow and segregate in the parasite progeny; theseeffects were also evident by blockage in the replica-tion of the apicoplast genome. Overall, our data showthat the PfClpP protease has confirmed localization inthe apicoplast and it plays important role in develop-ment of functional apicoplasts. Introduction Malaria remains a major health problem in the tropical andsubtropical countries causing 300–500 million cases and1–2 million deaths globally every year (Snow  et al  ., 2005;Hay  et al  ., 2009). The widespread development of drug-resistant parasite strains against commonly used drugscreates a necessity to identify new drug targets anddevelop new pharmacaphores.Availability of  Plasmodium  genome and proteome data has provided new opportunityto identify novel drug targets. The metabolic pathways inthe mitochondrion and the apicoplast, two parasiteorganelles of prokaryotic srcin, may represent suitabledrug targets in the parasite. Selected antibiotics such asdoxycycline and clindamycin which target some of theseprokaryotic metabolic pathways have already been shownto possess antiparasitic efficacies and are used in malariatreatments (Waller and McFadden, 2005; Goodman  et al  .,2007; Schlitzer, 2007; Dahl and Rosenthal, 2008). Theapicoplast is a reduced cyanobacterial plastid in the para-site; it plays an important role in biosynthesis of haem,isopentenyl diphophate and fatty acids (Ralph  et al  .,2004), thus the apicoplast is considered to be crucial forparasite survival. Antibacterial agents such as ciprofloxa-cin, rifampicin and thiostrepton that target DNA replica-tion, transcription and translation of the apicoplast,respectively, have been also shown to kill the parasite(McConkey  et al  ., 1997; Lin  et al  ., 2002; Williamson  et al  .,2002; Chaubey  et al  ., 2005). Majority of other apicoplastfunctions are carried out by nuclear-encoded proteins thatare targeted to the apicoplast through a bipartiteN-terminal leader sequence (Waller  et al  ., 2000). Detailed in silico   analyses predicated that about 545 nuclear-encoded parasite proteins are targeted to the apicoplast(Foth  et al  ., 2003; Ralph  et al  ., 2004). A number of theseproteins are fundamentally different from their host coun-terpart due to their cyanobacterial srcin and thus can beconsidered as potential drug targets.Parasite proteases have been considered as potentialdrug targets for malaria as they play crucial roles in dif-ferent metabolic pathways and can be inhibited by spe-cific inhibitors (Blackman, 2000; Rosenthal  et al  ., 2002).A total of 93 proteases have been identified in the Accepted 30 May, 2010. *For correspondence.; Tel. ( + 91) 11 2674 1358; Fax ( + 91) 11 26742316.  † These authors contributed equally to this work. Molecular Microbiology  (2010)  77 (4), 873–890    doi:10.1111/j.1365-2958.2010.07251.xFirst published online 22 June 2010  © 2010 Blackwell Publishing Ltd  Plasmodium falciparum   genome sequence (Wu  et al  .,2003; Ramasamy  et al  ., 2007) and a number of those arebeing characterized functionally (Shenai  et al  ., 2000;Dasaradhi  et al  ., 2005; Koussis  et al  ., 2009; Moura  et al  .,2009; Russo  et al  ., 2009). However, no protein degrada-tion machinery is characterized from any of the twoprokaryotic organelles in the parasite so far. ATPase-dependent protease machineries including the eukaryotic26S proteasome and the prokaryotic casenolytic pro-teases (Clp) systems are large protein degradation com-plexes that play essential role in cell cycle regulation (DeMot  et al  ., 1999; Ciechanover, 2005). We earlier charac-terized the ClpQ/HslV threonine protease in  P. falciparum  and showed that it is functional in the parasite cytosol(Ramasamy  et al  ., 2007). The  P. falciparum   genome alsoharbours cynobacterial Clp protease as well as its puta-tive ATPase partner. These ATPases form large multi-subunit complexes with the respective Clp proteases andact as chaperons to unfold the substrate proteins whichsubsequently get degraded by the protease component.The  P. falciparum   homologue of cynobacterial ClpP pro-tease, PfClpP, was selected as a promising target as itshares low homology with its counterpart in the host. Thecrystal structure of PfClpP shows presence of theconserved active site of serine proteases (PDB: 2F6I;Vedadi  et al  ., 2007). In the present study, we have carriedout detailed characterization of PfClpP, including itsbiochemical properties and its localization in the parasite,we also developed an  in vitro   protease assays for PfClpPand identified a PfClpP-specific inhibitor. Based on thisoptimized inhibitor, we developed and synthesized anactivity-based probe and validated the target specificity.Further, by blocking PfClpP protease activity in the para-site using the specific inhibitor, we show that PfClpP playsan essential role in the development of the parasite api-coplast and progression of the asexual stages of theparasite. Results Sequence analysis of PfClpP  The  P. falciparum   ClpP protein (PfClpP; PFC0310c) is a370-aa-long protein with a putative N-terminal hydro-phobic signal sequence (1–21 aa) and a  CLP protease  domain (179–359 aa) (Pfam Accession No. PF00574)(Fig. 1A). A  BLAST  search analysis showed that the pro-tease domain of PfClpP has high homology with ClpP ofsome of the primitive cyanobacteria such as  Isochrysis galbana   (47% homology),  Nostoc   sp. PCC 7120 (47%homology),  Nodularia spumigena   CCY9414 (47%homology),  Synechococcus   sp. RS9916 (51% homol-ogy) and with proteobacteria such as  Burkholderia mul- tivorans   ATCC 17616 (51% homology) and  Ralstonia eutropha   JMP134 (48% homology). A sequence align-ment of the PfClpP protein with homologues of ClpP inprokaryotes and cyanobacteria showed that it containsthe conserved active-site triad residues (Ser 264 –Asp 338 –His 289 ), in addition, most of the conserved residues inthese ClpP homologue are also present in PfClpP(Fig. S1A). Homologues of PfClpP proteases arealso identified from  P. berghei   (PB001115.03.0),  P. cha- baudi   (PC001282.02.0),  P. vivax   strain SaI-1(PVX_119490),  P. knowlesi   (PKH_083260) and  P. yoelii yoelii   strain 17XNL (PY06630) using the genomedatabase. An alignment of the predicted proteinssequences of these genes showed that the ClpP pro-tease is highly conserved among these  Plasmodium  species (Fig. S1B) Analyses of transcription and translation of PfClpP in the asexual blood-stage parasites  To study the expression pattern of  pfclpP   in differentdevelopmental stages of the asexual blood parasites,quantitative real-time PCR was carried out using totalRNA samples prepared from tightly synchronized  P. falci- parum   3D7 parasite cultures at 8, 16, 30, 40 and 48 hafter invasion. Quantitative real-time PCR analysis usinggene-specific primers also showed maximum transcrip-tion of  pfclpP   in late-trophozoite- and early-schizont-stageparasites (36 and 40 h after invasion) whereas there wasno detectable transcription in the early ring, and late ring(8 and 16 h after invasion respectively) (Fig. S2A). Ascontrols, quantitative PCR from the same set of cDNAsamples were also carried out for two other  P. falciparum  genes, erythrocyte binding antigen-175 ( eba-175  ) and thecysteine protease  falcipain-2  . As expected, maximumtranscript of  eba-175   gene was also found in cDNAsamples from schizont-stage parasites whereas falcipain-2   showed maximum transcript levels introphozoite-stage parasites (Fig. S2A).Western blot analysis of total parasite lysates fromculture at different time points using antibodies againstPfClpP detected a band of  ~ 23 kDa mainly in thetrophozoite-stage parasites (Fig. S2B). The calculatedmolecular mass of PfClpP without the putative signalsequence and pro-domain region is 22.02 kDa; thus the ~ 23 kDa band may represent the PfClpP protein afterN-terminal processing which removes the pro-domainregion as shown for other parasite proteases (Shenai et al  ., 2000; Mordmüller  et al  ., 2006; Ramasamy  et al  .,2007). No band was detected using pre-immune sera; inaddition, the anti-PfClpP antibodies did not react with thelysate of uninfected RBCs. Our results of transcriptionand translation analyses suggest that PfClpP isexpressed in blood-stage parasites at trophozoite andschizont stages. 874  S. Rathore  et al .    © 2010 Blackwell Publishing Ltd,  Molecular Microbiology  ,  77 , 873–890  Fig. 1.  Expression and localization of the PfClpP fusion protein with GFP in transgenic parasites.A. Schematic representation of the domain structure of PfClpP (Gene ID PFC310c) showing location of signal sequence (SS), pro-domain andprotease domain, respective amino acid positions are also indicated.B. Schematic diagram showing the PfClpP-N-terminal region and GFP fusion, labelled as PfClpP-N–GFP, the fusion gene was cloned invector pARL1a +  and transgene was expressed in the parasite driven by the promoter of chloroquine resistant transporter gene (crt 5 ′  UTR)and  P. berghei dhfr   terminator (3 ′  UTR).C. Immunoblot analysis using GFP-specific antibodies and trophozoite-stage wild-type (WT) and transgenic parasites expressingPfClpP-N–GFP. A band of  ~ 40 kDa, representing the GFP fusion protein, is recognized by GFP-specific antibodies in the transgenics, but notin the wild-type parasite lines.D. Blot ran in parallel and probed with anti-PfClpP antibodies detected native PfClpP protein ( ~ 23 kDa) in both parasite lines.E. Parallel blot was probed with anti-HRPII antibodies to show equal loading.F. Fluorescent microscopic images of live transgenic parasites at trophozoite, early-schizont and late-schizont stages, expressingPfClpP-N–GFP fusion protein. The parasite nuclei were stained with DAPI and slides were visualized by fluorescence microscope.G. Fluorescent microscopic images of transgenic parasites co-stained for mitochondria (Mt) showing GFP fluorescence pattern in closeassociation but distinct from mitochondrial staining. Cyanobacterial serine protease in  P. falciparum  apicoplast   875  © 2010 Blackwell Publishing Ltd,  Molecular Microbiology  ,  77 , 873–890  Localization of PfClpP in the transgenic parasites by GFP targeting  The PfClpP sequence was analysed using bioinformaticsprediction software PATS (Zuegge  et al  ., 2001), whichidentified it to be an apicoplast-targeted protein with thefirst 101 residues harbouring the hydrophobic N-terminalsignal sequence and the apicoplast-targeting transitpeptide sequence (score 0.923). To ascertain the local-ization of PfClpP in the parasite, a GFP-targetingapproach was employed. The N-terminal region of ClpPthat contains the signal sequence and the transit peptidesequence was expressed in fusion with GFP in the trans-genic parasites (Fig. 1B–E). These transgenic parasiteswere studied for localization of the PfClpP–GFP fusionprotein. Fluorescence of the GFP fusion protein waslocalized in a cellular organelle that showed characteris-tic shape, structure and division pattern of the parasiteapicoplast during the asexual blood-stage cycle. Inyoung stages of the parasite, the apicoplast is present asa crescent to round-shaped structure close to thenucleus, in late-trophozoite- and early-schizont-stageparasites the apicoplast elongates and takes a multi-branched shape, which then divides at the schizontstage and each merozoite has one apicoplast (Fig. 1F).To ascertain that the GFP fluorescence is not associatedwith mitochondria, we also carried out co-staining ofthese transgenic parasites using MitoTracker, themitochondria-specific live stain. The GFP fluorescencepattern was in close association but distinct from mito-chondrial staining (Fig. 1G). The mitochondria showedtypical elongated structure in the trophozoite stages, inearly schizonts it showed branched morphology and thendivided in the late-schizont-stage parasites. To furtherascertain the localization of PfClpP–GFP fusion proteinin the apicoplast, a colocalization study was carried outfor the fusion protein with the apicoplast resident protein,acyl carrier protein (ACP), by an immunofluorescenceassay. The anti-ACP antibody staining was found to becolocalized with the GFP fluorescence in these parasites,suggesting clearly that the PfClpP–GFP fusion protein islocalized in the parasite apicoplast (Fig. 2A). In addition,the anti-PfClpP antibody staining also showed colocal-ization with the GFP fluorescence (Fig. 2B) confirmingthat the native PfClpP protein is also localized in theparasite apicoplast. Immunoelectron microscopic studieswith the transgenic parasite using anti-GFP antibodyshowed specific labelling in the lumen of the apicoplast;the apicoplasts are clearly identified as characteristicmulti-membrane structures in these parasites (Fig. 2C).In some sections the staining was also observed in themulti-membranes of the apicoplast that may representthe protein in transit to the lumen. No staining wasobserved with secondary antibody alone omitting theprimary antibody or using pre-immune mice sera asprimary antibodies. Expression of recombinant PfClpP and protease activity assays  We established and characterized the  in vitro   proteaseactivity assays for the PfClpP proteases, with a view touse this assay to identify a specific inhibitor of PfClpPprotease which can be used to assess the functionalsignificance of the protease in the parasite. A fragment ofPfClpP (168 aa–370 aa) containing the protease domainwas expressed in  Escherichia coli  . The correspondingrecombinant PfClpP protease ( ~ 23 kDa) was expressedas a soluble protein in cytosol of the  E. coli   BL21(DE3)cells and was purified by affinity chromatography(Fig. 3A). The purified recombinant protein eluted asa single peak on the C-8 column in the RP-HPLC(Fig. S3A).The purified recombinant PfClpP protease wasassessed for its protease activity using an  in vitro   pro-tease assay. The PfClpP protease showed chymotrypsin-like serine protease activity using the synthetic peptidesubstrate Suc-LLVY-AMC in these assays with a  K  mvalue of 34.3  m M (Fig. 3B and Fig. S4C); however, PfClpPdid not cleave other model peptide substrates forchymotrypsin-like proteases, N-Suc-AAPF-AMC andN-Suc-F-AMC. Similarly, the enzyme displayed no activitytowards peptide substrate having basic residues at the P1position, N-Suc-AFK-AMC or towards a peptide substrateZ-FR-AMC, a known substrate for cysteine protease. Inthese assay conditions the peptide hydrolysis of Suc-LLVY-AMC was found to be optimal at neutral pH. Theactivity was markedly affected by reducing agent concen-trations and optimum concentration of DTT was found tobe 3 mM (Fig. S4A and B). The peptidase activity ofPfClpP was inhibited by serine protease inhibitors, chy-mostatin and PMSF; however, its activity was not inhibitedby inhibitors of other proteases class including E-64 andleupeptin (cysteine proteases inhibitors) and pepstatin(asparticproteaseinhibitor)(Fig.S4D).Usingchymostatinas a specific inhibitor and DMSO as a negative control ina 96-well plate format, a Z ′  factor of 0.75 was repeatedlyobtained for the  in vitro   protease assay. The Z ′  factorvalue shows robustness of the assay and its suitability toidentify specific inhibitor. PfClpP forms a multi-subunit complex  To understand the multimerization status of the recombi-nant PfClpP protein, we analysed the recombinant proteinby a gel filtration chromatography using Sepharose-6.Thefractions containing recombinant PfClpP overlapped withfractions containing molecular weight standard aldolase 876  S. Rathore  et al .    © 2010 Blackwell Publishing Ltd,  Molecular Microbiology  ,  77 , 873–890  Fig. 2.  Immunofluorescence assay and immunoelectron microscopy to localize PfClpP.A. Transgenic parasites expressing PfClpP-N–GFP were immunostained with antibodies specific to the apicoplast localized acyl carrier protein(ACP). The parasite nuclei were stained with DAPI and slides were visualized by confocal laser scanning microscope. The PfClpP-N–GFPfusion protein and ACP were colocalized in the parasite apicoplast. T, trophozoite stage; ES, early-schizont stage.B. Transgenic parasite expressing PfClpP-N–GFP were immunostained with anti-PfClpP antibodies. The PfClpP staining was overlapping withthe GFP fluorescence.C. Localization of PfClpP by immunoelectron microscopy. Ultra-thin sections of transgenic  P. falciparum   parasites expressing PfClpP-N–GFPwere labelled with anti-GFP antibody and gold labelled secondary antibody. Labelling was observed in the apicoplast having characteristic fourmembranes. Scale bar  =  250 nm. Cyanobacterial serine protease in  P. falciparum  apicoplast   877  © 2010 Blackwell Publishing Ltd,  Molecular Microbiology  ,  77 , 873–890
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