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Transgenic, Fluorescent Leishmania mexicana Allow Direct Analysis of the Proteome of Intracellular Amastigotes

2008; Elsevier BV; Volume: 7; Issue: 9 Linguagem: Inglês

10.1074/mcp.m700343-mcp200

1535-9484

Daniel Paape, Christoph Lippuner, Monika Schmid, Renate Ackermann, Martin E. Barrios‐Llerena, Ursula Zimny‐Arndt, Volker Brinkmann, B. Arndt, Klaus Peter Pleissner, Peter R. Jungblut, Toni Aebischer,

Toxin Mechanisms and Immunotoxins

Investigating the proteome of intracellular pathogens is often hampered by inadequate methodologies to purify the pathogen free of host cell material. This has also precluded direct proteome analysis of the intracellular, amastigote form of Leishmania spp., protozoan parasites that cause a spectrum of diseases that affect some 12 million patients worldwide. Here a method is presented that combines classic, isopycnic density centrifugation with fluorescent particle sorting for purification by exploiting transgenic, fluorescent parasites to allow direct proteome analysis of the purified organisms. By this approach the proteome of intracellular Leishmania mexicana amastigotes was compared with that of extracellular promastigotes that are transmitted by insect vectors. In total, 509 different proteins were identified by mass spectrometry and database search. This number corresponds to ∼6% of gene products predicted from the reference genome of Leishmania major. Intracellular amastigotes synthesized significantly more proteins with basic pI and showed a greater abundance of enzymes of fatty acid catabolism, which may reflect their living in acidic habitats and metabolic adaptation to nutrient availability, respectively. Bioinformatics analyses of the genes corresponding to the protein data sets produced clear evidence for skewed codon usage and translational bias in these organisms. Moreover analysis of the subset of genes whose products were more abundant in amastigotes revealed characteristic sequence motifs in 3′-untranslated regions that have been linked to translational control elements. This suggests that proteome data sets may be used to identify regulatory elements in mRNAs. Last but not least, at 6% coverage the proteome identified all vaccine antigens tested to date. Thus, the present data set provides a valuable resource for selection of candidate vaccine antigens. Investigating the proteome of intracellular pathogens is often hampered by inadequate methodologies to purify the pathogen free of host cell material. This has also precluded direct proteome analysis of the intracellular, amastigote form of Leishmania spp., protozoan parasites that cause a spectrum of diseases that affect some 12 million patients worldwide. Here a method is presented that combines classic, isopycnic density centrifugation with fluorescent particle sorting for purification by exploiting transgenic, fluorescent parasites to allow direct proteome analysis of the purified organisms. By this approach the proteome of intracellular Leishmania mexicana amastigotes was compared with that of extracellular promastigotes that are transmitted by insect vectors. In total, 509 different proteins were identified by mass spectrometry and database search. This number corresponds to ∼6% of gene products predicted from the reference genome of Leishmania major. Intracellular amastigotes synthesized significantly more proteins with basic pI and showed a greater abundance of enzymes of fatty acid catabolism, which may reflect their living in acidic habitats and metabolic adaptation to nutrient availability, respectively. Bioinformatics analyses of the genes corresponding to the protein data sets produced clear evidence for skewed codon usage and translational bias in these organisms. Moreover analysis of the subset of genes whose products were more abundant in amastigotes revealed characteristic sequence motifs in 3′-untranslated regions that have been linked to translational control elements. This suggests that proteome data sets may be used to identify regulatory elements in mRNAs. Last but not least, at 6% coverage the proteome identified all vaccine antigens tested to date. Thus, the present data set provides a valuable resource for selection of candidate vaccine antigens. Leishmania spp. are unicellular parasites of the trypanosomatid family and the causative agents of a spectrum of diseases, the leishmaniases, that affect some 12 million people worldwide (1Herwaldt B.L. Leishmaniasis.Lancet. 1999; 354: 1191-1199Abstract Full Text Full Text PDF PubMed Scopus (1356) Google Scholar). They oscillate between free living, flagellated promastigotes transmitted by blood sucking insect vectors and intracellular, non-flagellated amastigotes. These dwell in vacuoles akin to late endosomes/early lysosomes primarily in phagocytic cells of vertebrate hosts (2Antoine J.C. Prina E. Lang T. Courret N. The biogenesis and properties of the parasitophorous vacuoles that harbour Leishmania in murine macrophages.Trends Microbiol. 1998; 6: 392-401Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). This life cycle brings about dramatic morphological and molecular changes provoked by, and evolved to cope with, the change in habitat (3Burchmore R.J. Barrett M.P. Life in vacuoles—nutrient acquisition by Leishmania amastigotes.Int. J. Parasitol. 2001; 31: 1311-1320Crossref PubMed Scopus (137) Google Scholar). Comprehensive molecular analysis of these changes in amastigotes may reveal targets for drug development and vaccine design, but our current understanding remains very rudimentary. The 32.8-Mbp genome sequence of Leishmania major was completed in 2005 and predicts ∼8300 ORFs organized in 133 polycistronic units of directional gene clusters spread over 36 chromosomes (4Ivens A.C. Peacock C.S. Worthey E.A. Murphy L. Aggarwal G. Berriman M. Sisk E. Rajandream M.A. Adlem E. Aert R. Anupama A. Apostolou Z. Attipoe P. Bason N. Bauser C. Beck A. Beverley S.M. Bianchettin G. Borzym K. Bothe G. Bruschi C.V. Collins M. Cadag E. Ciarloni L. Clayton C. Coulson R.M. Cronin A. Cruz A.K. Davies R.M. De Gaudenzi J. Dobson D.E. Duesterhoeft A. Fazelina G. Fosker N. Frasch A.C. Fraser A. Fuchs M. Gabel C. Goble A. Goffeau A. Harris D. Hertz-Fowler C. Hilbert H. Horn D. Huang Y. Klages S. Knights A. Kube M. Larke N. Litvin L. Lord A. Louie T. Marra M. Masuy D. Matthews K. Michaeli S. Mottram J.C. Muller-Auer S. Munden H. Nelson S. Norbertczak H. Oliver K. O'neil S. Pentony M. Pohl T.M. Price C. Purnelle B. Quail M.A. Rabbinowitsch E. Reinhardt R. Rieger M. Rinta J. Robben J. Robertson L. Ruiz J.C. Rutter S. Saunders D. Schafer M. Schein J. Schwartz D.C. Seeger K. Seyler A. Sharp S. Shin H. Sivam D. Squares R. Squares S. Tosato V. Vogt C. Volckaert G. Wambutt R. Warren T. Wedler H. Woodward J. Zhou S. Zimmermann W. Smith D.F. Blackwell J.M. Stuart K.D. Barrell B. Myler P.J. The genome of the kinetoplastid parasite, Leishmania major..Science. 2005; 309: 436-442Crossref PubMed Scopus (1118) Google Scholar) (GeneDB). Other species are being sequenced, and data suggest a very high degree of conservation and synteny of polycistronic units throughout the genus (5Peacock C.S. Seeger K. Harris D. Murphy L. Ruiz J.C. Quail M.A. Peters N. Adlem E. Tivey A. Aslett M. Kerhornou A. Ivens A. Fraser A. Rajandream M.A. Carver T. Norbertczak H. Chillingworth T. Hance Z. Jagels K. Moule S. Ormond D. Rutter S. Squares R. Whitehead S. Rabbinowitsch E. Arrowsmith C. White B. Thurston S. Bringaud F. Baldauf S.L. Faulconbridge A. Jeffares D. Depledge D.P. Oyola S.O. Hilley J.D. Brito L.O. Tosi L.R. Barrell B. Cruz A.K. Mottram J.C. Smith D.F. Berriman M. Comparative genomic analysis of three Leishmania species that cause diverse human disease.Nat. Genet. 2007; 39: 839-847Crossref PubMed Scopus (578) Google Scholar) (GeneDB). This information allowed the adaptation of highly processive methods such as microarray analyses (6Duncan R.C. Salotra P. Goyal N. Akopyants N.S. Beverley S.M. Nakhasi H.L. The application of gene expression microarray technology to kinetoplastid research.Curr. Mol. Med. 2004; 4: 611-621Crossref PubMed Scopus (36) Google Scholar, 7Akopyants N.S. Matlib R.S. Bukanova E.N. Smeds M.R. 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A comprehensive survey of the Plasmodium life cycle by genomic, transcriptomic, and proteomic analyses.Science. 2005; 307: 82-86Crossref PubMed Scopus (651) Google Scholar). For this reason, cataloguing molecular changes when Leishmania transforms from promastigotes to amastigotes has been pursued by proteomics (11Walker J. Vasquez J.J. Gomez M.A. Drummelsmith J. Burchmore R. Girard I. Ouellette M. Identification of developmentally-regulated proteins in Leishmania panamensis by proteome profiling of promastigotes and axenic amastigotes.Mol. Biochem. Parasitol. 2006; 147: 64-73Crossref PubMed Scopus (77) Google Scholar, 12Dea-Ayuela M.A. Rama-Iniguez S. Bolas-Fernandez F. Proteomic analysis of antigens from Leishmania infantum promastigotes.Proteomics. 2006; 6: 4187-4194Crossref PubMed Scopus (37) Google Scholar, 13McNicoll F. Drummelsmith J. Muller M. Madore E. Boilard N. Ouellette M. Papadopoulou B. 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However, these axenic amastigotes can only be grown from a few species, and although they display a number of biochemical markers of the intracellular stage (3Burchmore R.J. Barrett M.P. Life in vacuoles—nutrient acquisition by Leishmania amastigotes.Int. J. Parasitol. 2001; 31: 1311-1320Crossref PubMed Scopus (137) Google Scholar), they fail to synthesize some major products of true amastigotes, e.g. the secreted amastigote-specific proteophosphoglycan in the case of Leishmania mexicana. Furthermore recent genome-wide mRNA profiling suggests that axenic amastigotes are more closely related to promastigotes than to ex vivo isolated amastigotes (8Holzer T.R. McMaster W.R. Forney J.D. Expression profiling by whole-genome interspecies microarray hybridization reveals differential gene expression in procyclic promastigotes, lesion-derived amastigotes, and axenic amastigotes in Leishmania mexicana.Mol. Biochem. Parasitol. 2006; 146: 198-218Crossref PubMed Scopus (134) Google Scholar). Thus, current data sets on amastigote proteomes likely underestimate differences and could miss important changes. Overcoming the technical hurdles to intracellular parasite purification therefore seems crucial. Here we report a novel purification protocol based on fluorescent parasites and fluorescent particle sorting to isolate amastigotes from their intracellular habitat in sufficient quantity and purity for direct proteome analysis. We applied this protocol to investigate the proteome of L. mexicana amastigotes and compared it with that of promastigotes. In total, 509 proteins (∼6% of predicted ORFs) were identified, and 34 were more abundant in amastigotes. Bioinformatics analysis of these data sets revealed a number of general characteristics of the parasite proteome and yielded novel insight into the biology of the parasite. L. mexicana mexicana (MNYC/BZ/62/M379) expressing DsRed (20Sorensen M. Lippuner C. Kaiser T. Misslitz A. Aebischer T. Bumann D. Rapidly maturing red fluorescent protein variants with strongly enhanced brightness in bacteria.FEBS Lett. 2003; 552: 110-114Crossref PubMed Scopus (89) Google Scholar) were maintained in selective medium as described previously (21Misslitz A. Mottram J.C. Overath P. Aebischer T. Targeted integration into a rRNA locus results in uniform and high level expression of transgenes in Leishmania amastigotes.Mol. Biochem. Parasitol. 2000; 107: 251-261Crossref PubMed Scopus (121) Google Scholar). Amastigotes were obtained and maintained in Schneider’s Drosophila medium (22Bates P.A. Complete developmental cycle of Leishmania mexicana in axenic culture.Parasitology. 1994; 108: 1-9Crossref PubMed Scopus (126) Google Scholar) supplemented with 20 μg/ml hygromycin B (Roche Applied Science). For proteome analyses, promastigotes were harvested in late logarithmic phase at a cell density of 6–7 × 107 parasites/ml. All animal experiments were approved by an ethics committee and licensed by the legal authority. BALB/c and C57BL/6 mice were purchased from Charles River, Sulzfeld, Germany and maintained in a conventional animal facility. Mice were infected with stationary phase promastigotes at the base of the tail where lesions developed. Mice with lesions were killed by cervical dislocation, and lesion tissue was excised for parasite isolation. Macrophages were differentiated from bone marrow of 6–8-week-old female mice as described previously (23Weinheber N. Wolfram M. Harbecke D. Aebischer T. Phagocytosis of Leishmania mexicana amastigotes by macrophages leads to a sustained suppression of IL-12 production.Eur. J. Immunol. 1998; 28: 2467-2477Crossref PubMed Scopus (80) Google Scholar) and infected with single cell axenic amastigotes at a multiplicity of infection of 7–10. Infected cells were incubated for 24 h at 34 °C and 5% CO2 before harvesting. Promastigotes in late logarithmic growth phase were harvested by centrifugation and processed according to Ref. 14Nugent P.G. Karsani S.A. Wait R. Tempero J. Smith D.F. Proteomic analysis of Leishmania mexicana differentiation.Mol. Biochem. Parasitol. 2004; 136: 51-62Crossref PubMed Scopus (88) Google Scholar. Briefly 2 × 108 parasites were washed once with PBS (Invitrogen, 14190-094) and three times with Tris/sucrose buffer (10 mm Tris-HCl, pH 7.4, 0.25 m sucrose). Parasites were resuspended in incomplete lysis buffer (2% N-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (SB3–10; Calbiochem), 2% CHAPS, 7 m urea, 2 m thiourea, 100 μg/ml leupeptin, 500 μg/ml Pefabloc (Roche Applied Science), 25 μl/ml Pefabloc protector (Roche Applied Science), 68.5 ng/ml pepstatin A, 174.2 μg/ml PMSF, 1 mm 1,10-phenanthroline, 1 mm each EDTA/EGTA, 25 μg/ml N-[N-(l-3-trans-carboxirane-2-carbonyl)-l-leucyl]-agmatine (E-64; Roche Applied Science); shock frozen in liquid nitrogen; and stored at −80 °C until 2-DE 1The abbreviations used are: 2-DE, two-dimensional gel electrophoresis; 1-D, one-dimensional; CAI, codon adaptation index; DAPI, 4′,6-diamidino-2-phenylindole; PMF, peptide mass fingerprint; UTR, untranslated mRNA region; nt, nucleotides; FACS, fluorescence-activated cell sorting; HSP, heat-shock protein; NCBI, National Center for Biotechnology Information. analysis. Infected bone marrow-derived macrophages were abraded in homogenization buffer (20 mm HEPES-KOH, pH 7.3, 0.25 m sucrose supplemented with “Complete Mini” (Roche Applied Science)). Cells were lysed by shear force in 1-ml portions of homogenization buffer using a 1-ml syringe fitted with a 26-gauge needle. Nuclei were pelleted for 2 min at 100 × g, and supernatants were loaded onto discontinuous sucrose gradients of 3 ml each: 60, 40, and 20% (w/w) sucrose in HEPES saline (30 mm HEPES-KOH, pH 7.3, 0.1 m NaCl, 0.5 mm CaCl2, 0.5 mm MgCl2) (24Chakraborty P. Sturgill-Koszycki S. Russell D.G. Isolation and characterization of pathogen-containing phagosomes.Methods Cell Biol. 1994; 45: 261-276Crossref PubMed Scopus (43) Google Scholar). Gradients were centrifuged for 25 min at 700 × g. Parasites were harvested from the 40/60% interphase, diluted in PBS, and centrifuged for 10 min at 1200 × g. The cells were resuspended in PBS supplemented with protease inhibitors (see above). 1 μg/ml 4′,6-diamidino-2-phenylindole (DAPI) was added to the sample before sorting on a FACSDiVa (BD Biosciences) for DsRed+ and DAPI− events. Sorted parasites were collected in 50-ml tubes containing 5 ml of PBS with protease inhibitors and centrifuged at 1200 × g for 10 min. Supernatant was removed completely, and parasite pellets were lysed in incomplete lysis buffer as described above for promastigotes. Excised lesion material was forced in PBS supplemented with Complete Mini through a 70-μm cell strainer (BD Biosciences). The material was centrifuged at 1200 × g at 4 °C. The resulting pellet was resuspended in an appropriate volume of PBS containing protease inhibitors and loaded onto the discontinuous sucrose gradient, and parasites were purified as above. DTT and Ampholytes were added to samples lysed in incomplete lysis buffer to a final concentration of 70 mm and 2%, respectively. Samples were mixed for 30 min at room temperature, and non-soluble material was cleared by centrifugation at room temperature (20,000 × g for 10 min). Supernatants were either used immediately or stored at −80 °C; the resulting pellet was kept and processed as described below. To resolve promastigote and amastigote samples via 2-DE, maximum volumes for preparative gels (corresponding to 240–300 μg of protein as determined by Lowry assay were applied to the anodic side of an IEF gel (diameter of 1.5 and 2.5 mm for promastigotes and amastigotes, respectively). The first dimension gel was loaded onto a 23 × 30-cm 2-DE gel system with a resolution power of about 5000 protein species (25Zimny-Arndt U. Schmid M. Ackermann R. Jungblut P.R. Classical proteomics: two-dimensional electrophoresis/MALDI mass spectrometry.in: Lipton M. Pasa-Tolic L. Methods in Molecular Biology: Mass Spectrometry of Proteins and Peptides. Humana Press Inc., Totowa, NJ2008Google Scholar). Gels were stained by Coomassie Brilliant Blue G-250 (26Doherty N.S. Littman B.H. Reilly K. Swindell A.C. Buss J.M. Anderson N.L. Analysis of changes in acute-phase plasma proteins in an acute inflammatory response and in rheumatoid arthritis using two-dimensional gel electrophoresis.Electrophoresis. 1998; 19: 355-363Crossref PubMed Scopus (159) Google Scholar). All visible protein spots were excised and processed for MALDI-TOF/TOF mass spectrometry as described previously (25Zimny-Arndt U. Schmid M. Ackermann R. Jungblut P.R. Classical proteomics: two-dimensional electrophoresis/MALDI mass spectrometry.in: Lipton M. Pasa-Tolic L. Methods in Molecular Biology: Mass Spectrometry of Proteins and Peptides. Humana Press Inc., Totowa, NJ2008Google Scholar). The residual insoluble material was washed twice with 9 m urea, 70 mm DTT, 2% CHAPS. After washing, it was centrifuged for 30 min at 100,000 × g at 20 °C. The pellet was resolved in Laemmli buffer, agitated at 37 °C for 10 min, then boiled for 5 min, and centrifuged again for 30 min at 100,000 × g at 20 °C. The supernatant was loaded on a large 15% SDS-PAGE gel. Sample lanes were cut in 96 slices, and every slice was divided in four pieces and transferred into a 96-well plate. Samples were washed three times with agitation for 30 min with 200 μl of destaining buffer and equilibrated in 200 μl of trypsin digestion buffer as described previously (25Zimny-Arndt U. Schmid M. Ackermann R. Jungblut P.R. Classical proteomics: two-dimensional electrophoresis/MALDI mass spectrometry.in: Lipton M. Pasa-Tolic L. Methods in Molecular Biology: Mass Spectrometry of Proteins and Peptides. Humana Press Inc., Totowa, NJ2008Google Scholar). The digested dried samples were resolved in 12 μl of 3% ACN, 0.1% formic acid. 6 μl were used for one LC-MS analysis. The remainder was stored at −80 °C. The peptides from excised spots from the 2-DE gels were analyzed by MALDI-TOF/TOF MS using a 4700 Proteomics Analyzer (Applied Biosystems, Foster City, CA) as described previously (25Zimny-Arndt U. Schmid M. Ackermann R. Jungblut P.R. Classical proteomics: two-dimensional electrophoresis/MALDI mass spectrometry.in: Lipton M. Pasa-Tolic L. Methods in Molecular Biology: Mass Spectrometry of Proteins and Peptides. Humana Press Inc., Totowa, NJ2008Google Scholar). After in-gel tryptic digestion peptides were dissolved in 1 μl of sample buffer (33% ACN, 0.1% TFA) of which 0.25 μl was mixed with 0.5 μl of matrix solution (α-cyano-4-hydroxycinnamic acid in 50% ACN, 0.3% TFA) and applied to a MALDI plate. The genome of L. mexicana is not yet available, therefore protein identities were assigned by searching the L. major protein database (version GeneDB_protein_database_090704 containing 8217 sequences) and NCBInr (version 20070908 containing 5,454,477 sequences). Proteins were identified using Mascot version 2.1 (Matrix Science) MS/MS Ion Search with combined peptide mass fingerprint (PMF) and MS/MS information, allowing a peptide mass tolerance of 30 ppm and ±0.3 Da for the fragment mass tolerance. A maximum of one missed cleavage was allowed; oxidation of methionine, N-terminal acetylation of the protein, propionamide at cysteine residues, and N-terminal pyroglutamic acid formation were defined as variable modifications in these searches. For identification of a protein within a 2-DE spot the following identification criteria were used (23Weinheber N. Wolfram M. Harbecke D. Aebischer T. Phagocytosis of Leishmania mexicana amastigotes by macrophages leads to a sustained suppression of IL-12 production.Eur. J. Immunol. 1998; 28: 2467-2477Crossref PubMed Scopus (80) Google Scholar). A protein was considered identified by the MS analysis if at least 30% sequence coverage was obtained, or in case sequence coverage was between 15 and 30%, at least one MS/MS spectrum with Mascot identity combined with mass loss information and hypercleavage sites (27Schmidt F. Krah A. Schmid M. Jungblut P.R. Thiede B. Distinctive mass losses of tryptic peptides generated by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight.Rapid Commun. Mass Spectrom. 2006; 20: 933-936Crossref PubMed Scopus (10) Google Scholar) was necessary to reach identification status. If sequence coverage was below 15%, two MS/MS spectra had to fit with the protein, or one MS/MS spectrum and additional information from 2-DE, e.g. from neighboring spots or pI or protein Mr, was necessary. Processed slices of the 1-D gel were analyzed by nano-LC ESI-MS/MS. Peptides were separated on an Agilent 1100 series chromatography unit (Agilent Technologies, Palo Alto, CA). Samples were loaded onto a trap column (Zorbax 300SB, C18, 5 × 0.3 mm, Agilent Technologies) and washed for 5 min with a flow rate of 0.02 ml/min and buffer A (3% ACN, 0.1% formic acid). Peptides were eluted for 75 min using a gradient of 5 min of 3% buffer B (99.9% ACN, 0.1% formic acid) increasing to 15% buffer B within 3 min and over a 42-min period increasing to 45% buffer B increasing for 5 min to 90% buffer B onto a separation column (Zorbax 300SB, C18, 3.5 μm/150 × 0.075 mm, Agilent Technologies) with a flow rate of 0.3 μl/min. The column outlet was coupled on line to an LC/MSD Trap XCT mass spectrometer (Agilent Technologies). Spectra were recorded between 10 and 68 min of the run in standard/enhanced mode. Precursor masses with m/z ratio of 200–2200 were allowed. Database search was performed using Mascot (version 2.1) MS/MS Ion Search. A peptide mass tolerance of 0.4 Da and a fragment mass tolerance of ±0.4 Da were accepted. A maximum of one missed cleavage and the same variable modifications as defined above were allowed. For ESI-MS/MS data analysis, the ion score cutoff was 26, and proteins were classified as identified either if at least two peptides with a Mascot score above the statistically relevant threshold (p < 0.05) were found or if only one peptide achieved the required Mascot score that at least four consecutive y- or b-ions with a significant signal to background ratio could be determined. Parasite samples were fixed in 25% electron microscopy grade glutaraldehyde fixative and processed for transmission electron microscopy as described previously (28Al Younes H.M. Rudel T. Brinkmann V. Szczepek A.J. Meyer T.F. Low iron availability modulates the course of Chlamydia pneumoniae infection.Cell. Microbiol. 2001; 3: 427-437Crossref PubMed Scopus (89) Google Scholar). 3′-Untranslated mRNA regions (3′-UTR) analysis was performed using the oligo-analysis tool (29van Helden J. Andre B. Collado-Vides J. A web site for the computational analysis of yeast regulatory sequences.Yeast. 2000; 16: 177-187Crossref PubMed Scopus (150) Google Scholar). The tool identifies oligomers ranging in length from 4 to 8 nt that are more frequent within the UTR sequence of a group of co-regulated genes compared with a set of non-regulated genes. In our case, 51 intergenic sequences from the L. major genome were chosen for analysis of regulated loci. This test set was compared against a random selection of intergenic sequences from annotated protein-coding genes on L. major chromosomes 4, 7, 12, 18, 21, 25, 28, 30, and 36, totaling 2677 sequences. Statistics and significance values were determined according to Ref. 30van Helden J. del Olmo M. Perez-Ortin J.E. Statistical analysis of yeast genomic downstream sequences reveals putative polyadenylation signals.Nucleic Acids Res. 2000; 28: 1000-1010Crossref PubMed Google Scholar. The 51 regulated test loci (see supplemental Table 3) represented the 3′-intergenic regions of 35 ORFs for which the respective proteins were solely identified in amastigote samples by the present proteome analysis and 16 additional 3′-intergenic regions of ORFs encoding proteins found to be more abundant in amastigotes by isotope-coded protein labeling (71Freiwald A. Establishing of ICPL labeling for proteome studies of the micro-organism Leishmania mexicana. Technische Fachhochschule, Berlin2006Google Scholar). The cysteine proteinase B ORF array was excluded as synthesis of the homologous L. major ORFs appears not to be stage-specifically regulated (31Sakanari J.A. Nadler S.A. Chan V.J. Engel J.C. Leptak C. Bouvier J. Leishmania major: comparison of the cathepsin L- and B-like cysteine protease genes with those of other trypanosomatids.Exp. Parasitol. 1997;