Sex-partitioning of the Plasmodium falciparum Stage V Gametocyte Proteome Provides Insight into falciparum-specific Cell Biology
2014; Elsevier BV; Volume: 13; Issue: 10 Linguagem: Inglês
10.1074/mcp.m114.040956
ISSN1535-9484
AutoresDingyin Tao, Ceereena Ubaida‐Mohien, Derrick Mathias, Jonas G. King, Rebecca Pastrana‐Mena, Abhai K. Tripathi, Ilana Goldowitz, David R. Graham, Eli L. Moss, Matthias Marti, Rhoel R. Dinglasan,
Tópico(s)Mosquito-borne diseases and control
ResumoOne of the critical gaps in malaria transmission biology and surveillance is our lack of knowledge about Plasmodium falciparum gametocyte biology, especially sexual dimorphic development and how sex ratios that may influence transmission from the human to the mosquito. Dissecting this process has been hampered by the lack of sex-specific protein markers for the circulating, mature stage V gametocytes. The current evidence suggests a high degree of conservation in gametocyte gene complement across Plasmodium, and therefore presumably for sex-specific genes as well. To better our understanding of gametocyte development and subsequent infectiousness to mosquitoes, we undertook a Systematic Subtractive Bioinformatic analysis (filtering) approach to identify sex-specific P. falciparum NF54 protein markers based on a comparison with the Dd2 strain, which is defective in producing males, and with syntenic male and female proteins from the reanalyzed and updated P. berghei (related rodent malaria parasite) gametocyte proteomes. This produced a short list of 174 male- and 258 female-enriched P. falciparum stage V proteins, some of which appear to be under strong diversifying selection, suggesting ongoing adaptation to mosquito vector species. We generated antibodies against three putative female-specific gametocyte stage V proteins in P. falciparum and confirmed either conserved sex-specificity or the lack of cross-species sex-partitioning. Finally, our study provides not only an additional resource for mass spectrometry-derived evidence for gametocyte proteins but also lays down the foundation for rational screening and development of novel sex-partitioned protein biomarkers and transmission-blocking vaccine candidates. One of the critical gaps in malaria transmission biology and surveillance is our lack of knowledge about Plasmodium falciparum gametocyte biology, especially sexual dimorphic development and how sex ratios that may influence transmission from the human to the mosquito. Dissecting this process has been hampered by the lack of sex-specific protein markers for the circulating, mature stage V gametocytes. The current evidence suggests a high degree of conservation in gametocyte gene complement across Plasmodium, and therefore presumably for sex-specific genes as well. To better our understanding of gametocyte development and subsequent infectiousness to mosquitoes, we undertook a Systematic Subtractive Bioinformatic analysis (filtering) approach to identify sex-specific P. falciparum NF54 protein markers based on a comparison with the Dd2 strain, which is defective in producing males, and with syntenic male and female proteins from the reanalyzed and updated P. berghei (related rodent malaria parasite) gametocyte proteomes. This produced a short list of 174 male- and 258 female-enriched P. falciparum stage V proteins, some of which appear to be under strong diversifying selection, suggesting ongoing adaptation to mosquito vector species. We generated antibodies against three putative female-specific gametocyte stage V proteins in P. falciparum and confirmed either conserved sex-specificity or the lack of cross-species sex-partitioning. Finally, our study provides not only an additional resource for mass spectrometry-derived evidence for gametocyte proteins but also lays down the foundation for rational screening and development of novel sex-partitioned protein biomarkers and transmission-blocking vaccine candidates. Sexual stages represent only a small fraction of Plasmodium falciparum parasites that are present during human malaria infection, yet they alone are responsible for disease transmission (1.Babiker H.A. Schneider P. Reece S.E. Gametocytes: insights gained during a decade of molecular monitoring.Trends Parasitol. 2008; 24: 525-530Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). As such, the Malaria Eradication Research Agenda (malERA) has prioritized the need for studies that specifically address these transmission stages, with the hope of developing new transmission-blocking vaccines and drugs, as well as diagnostics that are specific for these sexual stages (2.Alonso P.L. Brown G. Arevalo-Herrera M. Binka F. Chitnis C. Collins F. Doumbo O.K. Greenwood B. Hall B.F. Levine M.M. Mendis K. Newman R.D. Plowe C.V. Rodriguez M.H. Sinden R. Slutsker L. Tanner M. A research agenda to underpin malaria eradication.PLoS Med. 2011; 8: e1000406Crossref PubMed Scopus (465) Google Scholar, 3.malERA Consultative Group on DrugsA research agenda for malaria eradication: drugs.PLoS Med. 2011; 8: e1000402Crossref PubMed Scopus (199) Google Scholar, 4.malERA Consultative Group on VaccinesA research agenda for malaria eradication: vaccines.PLoS Med. 2011; 8: e1000398Crossref PubMed Scopus (209) Google Scholar). In fact, one of the critical gaps in malaria transmission biology and surveillance centers on the lack of knowledge about the infectivity of symptomatic and asymptomatic gametocytemic individuals for mosquitoes. Many infected individuals harboring the Plasmodium falciparum sexual stage, or gametocyte, are asymptomatic carriers and they represent the primary reservoir for malaria transmission (5.Bousema T. Drakeley C. Epidemiology and infectivity of Plasmodium falciparum and Plasmodium vivax gametocytes in relation to malaria control and elimination.Clin. Microbiol. Rev. 2011; 24: 377-410Crossref PubMed Scopus (487) Google Scholar). Missing the opportunity to treat these carriers will increase the risk for epidemic malaria in regions that have approached the elimination phase. Thus, proper surveillance of gametocyte carriers is critical for evaluating ongoing malaria control and elimination programs. Surveillance is difficult, however, because gametocytes comprise only 0.1–2% of the total body parasite load during active infection (5.Bousema T. Drakeley C. Epidemiology and infectivity of Plasmodium falciparum and Plasmodium vivax gametocytes in relation to malaria control and elimination.Clin. Microbiol. Rev. 2011; 24: 377-410Crossref PubMed Scopus (487) Google Scholar), and are only observed in the bloodstream in their mature (Stage V) form, with the first four developing stages sequestered in tissues. Microscopy-based analysis for sex ratio determination and infectivity studies remains limited because of cost, training, and suitability for population-wide studies. Although light microscopy remains the gold standard for malaria diagnosis, the relatively low prevalence of circulating gametocytes makes it difficult to accurately detect much less quantify these stages. Moreover, because of variations in skill level of microscopists and inconsistency in method, exclusive use of light microscopy estimates of gametocyte carriage carries a high risk of error. Importantly, the presence of stage V gametocytes in the bloodstream alone, as determined by thick smear microscopy does not imply infectivity to mosquitoes. Ratios of male and female gametocytes in the blood circulation are skewed toward the female, but they can vary significantly based on co-infection, parasite and gametocyte density, and host environmental factors (6.Paul R.E. Brockman A. Price R.N. Luxemburger C. White N.J. Looareesuwan S. Nosten F. Day K.P. Genetic analysis of Plasmodium falciparum infections on the north-western border of Thailand.Trans. R. Soc. Trop. Med. Hyg. 1999; 93: 587-593Abstract Full Text PDF PubMed Scopus (32) Google Scholar), and it is therefore hypothesized that this variation in sex ratios will influence mosquito infectivity. For example, mature gametocyte sex ratios can change during the course of infection in response to host cues or especially following antimalarial treatment resulting in an increase in the number of males (6.Paul R.E. Brockman A. Price R.N. Luxemburger C. White N.J. Looareesuwan S. Nosten F. Day K.P. Genetic analysis of Plasmodium falciparum infections on the north-western border of Thailand.Trans. R. Soc. Trop. Med. Hyg. 1999; 93: 587-593Abstract Full Text PDF PubMed Scopus (32) Google Scholar, 7.Robert V. Read A.F. Essong J. Tchuinkam T. Mulder B. Verhave J.P. Carnevale P. Effect of gametocyte sex ratio on infectivity of Plasmodium falciparum to Anopheles gambiae.Trans. R. Soc. Trop. Med. Hyg. 1996; 90: 621-624Abstract Full Text PDF PubMed Scopus (99) Google Scholar). However, it remains unknown whether the transmission potential to mosquitoes of the individuals in these studies fluctuated because of the changes in sex ratio. There are currently no uncomplicated tools to distinguish male and female mature P. falciparum gametocytes (of which at least one of each is required for fertilization and ookinete development in the mosquito) at the molecular level. Although the proteome of Plasmodium gametocytes has been described (8.Silvestrini F. Lasonder E. Olivieri A. Camarda G. van Schaijk B. Sanchez M. Younis Younis S. Sauerwein R. Alano P. Protein export marks the early phase of gametocytogenesis of the human malaria parasite Plasmodium falciparum.Mol. Cell. Proteomics. 2010; 9: 1437-1448Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 9.Florens L. Washburn M.P. Raine J.D. Anthony R.M. Grainger M. Haynes J.D. Moch J.K. Muster N. Sacci J.B. Tabb D.L. Witney A.A. Wolters D. Wu Y. Gardner M.J. Holder A.A. Sinden R.E. Yates J.R. Carucci D.J. A proteomic view of the Plasmodium falciparum life cycle.Nature. 2002; 419: 520-526Crossref PubMed Scopus (1092) Google Scholar, 10.Hall N. Karras M. Raine J.D. Carlton J.M. Kooij T.W. Berriman M. Florens L. Janssen C.S. Pain A. Christophides G.K. James K. Rutherford K. Harris B. Harris D. Churcher C. Quail M.A. Ormond D. Doggett J. Trueman H.E. Mendoza J. Bidwell S.L. Rajandream M.A. Carucci D.J. Yates 3rd., J.R. Kafatos F.C. Janse C.J. Barrell B. Turner C.M. Waters A.P. Sinden R.E. A comprehensive survey of the Plasmodium life cycle by genomic, transcriptomic, and proteomic analyses.Science. 2005; 307: 82-86Crossref PubMed Scopus (663) Google Scholar, 11.Khan S.M. Franke-Fayard B. Mair G.R. Lasonder E. Janse C.J. Mann M. Waters A.P. Proteome analysis of separated male and female gametocytes reveals novel sex-specific Plasmodium biology.Cell. 2005; 121: 675-687Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar), these previous analyses fell just short of providing the partitioned male and female proteomes for P. falciparum. Moreover, the availability of the genomes of human, primate, and rodent malaria parasites and the acquisition of sequence information for recent field isolates of P. falciparum have created the opportunity to understand gene diversity and conservation in sexual stage development across Plasmodia. Identifying markers that differ between male and female P. falciparum stage V gametocytes is critical in informing transgenic approaches aimed at separating the two. It has been argued that the inherent evolutionary differences between rodent and human malaria parasites, especially for the sexual stages, limit the utility of the P. berghei gametocyte proteome (11.Khan S.M. Franke-Fayard B. Mair G.R. Lasonder E. Janse C.J. Mann M. Waters A.P. Proteome analysis of separated male and female gametocytes reveals novel sex-specific Plasmodium biology.Cell. 2005; 121: 675-687Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar) in providing a priori knowledge of these markers. Several iterations and improvements to the P. berghei genome have been made available since 2005, whereas MS search engines have improved commensurately, further compounding the issue. However, we would also argue that the current evidence suggests a high degree of conservation in gametocyte gene complement across Plasmodium (12.Sinden R.E. Malaria, sexual development, and transmission: retrospect and prospect.Parasitology. 2009; 136: 1427-1434Crossref PubMed Scopus (43) Google Scholar, 13.Sinden R.E. Carter R. Drakeley C. Leroy D. The biology of sexual development of Plasmodium: the design and implementation of transmission-blocking strategies.Malar. J. 2012; 11: 70Crossref PubMed Scopus (55) Google Scholar), and therefore presumably in sex-specific genes - despite key differences such as gametocyte sequestration and morphology. Here, we report on our effort to address these scientific gaps to a certain extent and to test our gametocyte gene conservation hypothesis through the use of comparative protein bioinformatics analyses of the mature stage V gametocyte proteomes of two distinct P. falciparum strains with our update of the bioinformatic analysis of the P. berghei male and female gametocyte proteomes. P. falciparum gametocytes were cultured in RPMI 1640 containing HEPES and glutamine and supplemented with 10% human serum and hypoxanthine as described earlier (14.Ponnudurai T. Meuwissen J.H. Leeuwenberg A.D. Verhave J.P. Lensen A.H. The production of mature gametocytes of Plasmodium falciparum in continuous cultures of different isolates infective to mosquitoes.Trans. R. Soc. Trop. Med. Hyg. 1982; 76: 242-250Abstract Full Text PDF PubMed Scopus (144) Google Scholar). P. falciparum NF54 strain was diluted to 0.5% mixed stage asexual parasites and 4% hematocrit in complete culture medium in six well plates. Plates were transferred to a 37 °C incubator and microaerophilic environment was created using desiccators candle jar. Media was exchanged every day without the addition of new blood from day one to day 17 (culture maturation). To remove asexual stage parasites 50 mm N-acetylglucosamine was added to the culture media from day eight (early stage gametocytes) until day 10. Blood smears were made every alternate day to monitor the progress of the culture and to determine gametocytemia on day 18. Stage V gametocytes were harvested from culture at day 17 postgametocytogenesis initiation and isolated by passage through a LS-25 Midi MACS column (CS Miltenyi). Synchronized trophozoite cultures that were used for Western blot analyses were generated from a recently thawed stabilate and harvested at 48 h post treatment of the mixed asexual culture with 5% sorbitol. The production of stage V gametocytes was performed using a modified version of a previously described protocol (15.Fivelman Q.L. McRobert L. Sharp S. Taylor C.J. Saeed M. Swales C.A. Sutherland C.J. Baker D.A. Improved synchronous production of Plasmodium falciparum gametocytes in vitro.Mol. Biochem. Parasitol. 2007; 154: 119-123Crossref PubMed Scopus (157) Google Scholar). Ten (10.Hall N. Karras M. Raine J.D. Carlton J.M. Kooij T.W. Berriman M. Florens L. Janssen C.S. Pain A. Christophides G.K. James K. Rutherford K. Harris B. Harris D. Churcher C. Quail M.A. Ormond D. Doggett J. Trueman H.E. Mendoza J. Bidwell S.L. Rajandream M.A. Carucci D.J. Yates 3rd., J.R. Kafatos F.C. Janse C.J. Barrell B. Turner C.M. Waters A.P. Sinden R.E. A comprehensive survey of the Plasmodium life cycle by genomic, transcriptomic, and proteomic analyses.Science. 2005; 307: 82-86Crossref PubMed Scopus (663) Google Scholar) ml cultures at 4% hematocrit and ∼5% ring parasitemia were sorbitol synchronized. After 24 h, trophozoite cultures were transferred to a T75 flask to which complete media and red blood cells were added to create a 30 ml culture with 2% hematocrit in each flask. After another 24 h, adding 50% old media and 50% new complete media stressed the newly reinvaded rings. Cultures were allowed to develop to late schizonts and then split into three T75 flasks evenly. Twenty (20) mLs of fresh media was then added to each flask. During sexual stage development, fresh media was added daily. At 48 h after invasion of a mixture of asexually and sexually committed merozoites, 1 ml of 1 m N-Acetyl-d-Glucosamine was added to all flasks in order to clear asexual parasites. Drug treatment was given during media changes for three consecutive days. On day nine of sexual development, the cultures were MACS column separated to purify late stage gametocytes. Purified cultures were washed in PBS and snap frozen. Preparation of the synchronized trophozoite protein lysate for Western blot analyses is as described above for NF54. The GiRBC 1The abbreviations used are:GiRBCgametocyte infected red blood cellGcVgametocyte stage VAsxasexual blood stagesSCXstrong cation exchangeppmparts per millionFAformic acidPSMAPepArML Search MASPECTRAS 2 AnalysisPbPlasmodium bergheiPfPlasmodium falciparumSSBSystematic Subtractive Protein Bioinformatic analysis. eluate from the MACS column was washed with cold PBS three times prior to protein extraction. The freeze-thaw method was applied to extract the soluble proteins by adding 120 μl 5 mm phosphate buffer containing 0.5 mm PMSF, 1 mm EDTA, and 1 mm protease inhibitors mixture (Sigma, St. Louis, MO) to 1 × 106 GiRBC pellets. A total of four freeze-thaw cycles were used. The supernatant was collected as the soluble protein fraction after centrifugation at 20,000 × g for 30 min at 4 °C. To get the membrane proteins, the pellet was washed with cold PBS for three times prior to being dissolved in 95 μl SDT-lysis buffer composed of 4% (w/v) SDS, 100 mm Tris/HCl, and 0.1 m DTT, pH 7.6, and then boiled at 95 °C for 5 min. The supernatant was collected as the membrane protein fraction after centrifugation at 20,000 × g for 5 min at 4 °C. gametocyte infected red blood cell gametocyte stage V asexual blood stages strong cation exchange parts per million formic acid PepArML Search MASPECTRAS 2 Analysis Plasmodium berghei Plasmodium falciparum Systematic Subtractive Protein Bioinformatic analysis. We used a Multi-Lane Combined In-gel Digestion (MLCID) strategy to reduce the impact of nonspecific absorption during the process of in-gel tryptic digestion and to avoid losing SDS-PAGE separation power. For NF54 parasites, we used three lanes for the soluble protein fraction and four lanes for the membrane fraction, respectively, and each lane was loaded with 20 μl of sample under reducing conditions. After resolving on a 4–20% precast gradient gel (BioRad, Hercules, CA), the proteins were stained with Coomassie. GiRBC soluble and membrane fractions were cut into 14 slices by combining three lanes (soluble) and 16 slices by combining four lanes (membrane). Both the soluble and membrane fractions from Dd2 were cut into 14 slices by combing three lanes. Gel slices were cut into 1 × 1 mm pieces prior to de-staining, reduction and alkylation, tryptic digestion and peptide extraction. The extracted peptides were lyophilized and then resuspended in 2% acetonitrile, 97.9% water, and 0.1% formic acid buffer for LC-MS/MS analysis. Biological in-gel digestion replicates were analyzed independently as follows. One third of the MLCID sample of all the fractions, were injected onto an Agilent LC-MS system comprised of a 1200 LC system coupled to a 6520 Q-TOF via an HPLC Chip Cube interface. The only exception to this process was made for the first low molecular weight fraction, which consisted primarily of hemoglobin, and only 1/50th of this fraction was injected. The sample was trapped and analyzed using an Agilent Polaris-HR-Chip-3C18 chip (360 nL, 180 Å C18 trap with a 75 μm i.d., 150 mm length, 180 Å C18 analytical column). Peptides were loaded onto the enrichment column automatically by autosampler using 97% solvent A (0.1% formic acid in water) and 3% solvent B (0.1% formic acid in 90% acetonitrile) at a flow rate of 2 μl/min. Elution of peptides from the analytical column was performed using a gradient starting at 97% A at 300 nL/min. The mobile phase was 3–10% B for 4 min, 10–35% B for 56 min, 35–99% for 2 min, and maintained at 99% B for 6 min, followed by re-equilibration of the column with 3% B for 10 min. Data dependent (autoMS2) mode was used for MS acquisition by Agilent 6520 Q-TOF at 2 GHz. Precursor MS spectra were acquired from m/z 315 to 1700 and the top 4 peaks were selected for MS/MS analysis. Product scans were acquired from m/z 50 to 1700 at a scan rate of 1.5/second. A medium isolation width (∼4 amu) was used, and a collision energy of slope 3.9 V/100 Da with a 2.9 V offset was applied for fragmentation. A dynamic exclusion list was applied, with precursors excluded of 0.50 min after two MS/MS spectrum was acquired. Each sample was further fractionated into 14 membrane and 14 soluble fractions. Raw data from Dd2 sample runs (two biological replicates, 217,165 MS/MS total spectra) and NF54 GiRBC sample runs (three biological replicates, 497,006 MS/MS total spectra) was converted to mzXML format using Trapper (Institute for Systems Biology, Seattle, Washington). A merged search was performed on the mzXML data for each fraction using the PepArML metasearch engine (16.Edwards N. Wu X. Tseng C. An unsupervised, model-free, machine-learning combiner for peptide identifications from tandem mass spectra.Clin. Proteomics. 2009; 5: 23-36Crossref Scopus (47) Google Scholar), which automatically conducts target and decoy searches using the following: Mascot (17.Perkins D.N. Pappin D.J. Creasy D.M. Cottrell J.S. Probability-based protein identification by searching sequence databases using mass spectrometry data.Electrophoresis. 1999; 20: 3551-3567Crossref PubMed Scopus (6772) Google Scholar), OMSSA (18.Geer L.Y. Markey S.P. Kowalak J.A. Wagner L. Xu M. Maynard D.M. Yang X. Shi W. 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The data was searched by a combined database of SwissProt Human and Plasmodium falciparum sequences from GeneDB (2013.02), which consists of 28,960 entries with the following parameters; fixed modification: carbamidomethyl cysteine and variable modification: oxidized methionine; mass tolerance: 30 ppm and 20 ppm respectively for precursor and fragment ions; one missed cleavage. The results from the metasearch were combined and the results were parsed into the MASPECTRAS 2 data analysis system (25.Ubaida Mohien C. Hartler J. Breitwieser F. Rix U. Remsing Rix L. Winter G.E. Thallinger G.G. Bennett K.L. Superti-Furga G. Trajanoski Z. Colinge J. MASPECTRAS 2: an integration and analysis platform for proteomic data.Proteomics. 2010; 10: 2719-2722Crossref PubMed Scopus (19) Google Scholar) with data filters of 1% spectra FDR and 5% peptide FDR, and protein identifications were then clustered to remove redundancy. Proteins were clustered together if there was a peptide identification shared between them, because this indicates substantial sequence similarity, and the protein with the greatest number of peptides identified was considered the unique protein identification from that group. Throughout this paper we report only the unique identifications. Proteins identified by single peptides were manually validated. The data analysis pipeline meets all MIAPE standards (26.Taylor C.F. Paton N.W. Lilley K.S. Binz P.A. Julian Jr., R.K. Jones A.R. Zhu W. Apweiler R. Aebersold R. Deutsch E.W. Dunn M.J. Heck A.J. Leitner A. Macht M. Mann M. Martens L. Neubert T.A. Patterson S.D. Ping P. Seymour S.L. Souda P. Tsugita A. Vandekerckhove J. Vondriska T.M. Whitelegge J.P. Wilkins M.R. Xenarios I. Yates 3rd, J.R. Hermjakob H. The minimum information about a proteomics experiment (MIAPE).Nat. 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Proteome analysis of separated male and female gametocytes reveals novel sex-specific Plasmodium biology.Cell. 2005; 121: 675-687Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar), the individual MS raw files from Male (113,213 total MS/MS spectra) and Female data set (243,468 total MS/MS spectra) were searched against a combined database of SwissProt Human, Mouse, and P. berghei. Using these results we determined the male/female partitioned proteomes for P. falciparum gametocytes through a subtractive bioinformatics proteomics approach. Briefly, in our approach, we take protein identification lists and use set comparisons to generate protein lists that are enriched for biological states, with those protein lists clustered to remove redundancy. Therefore, we took the NF54 and Dd2 gametocyte-infected red blood cell lysate proteome and subtracted out all host proteins, generating the NF54 and Dd2 gametocyte proteomes. Putative male-specific, female-specific, and sex-unspecific proteomes were generated by taking protein identifications unique to NF54 and Dd2, respectively. These putative proteomes were then BLAST searched against the two previous data sets of Khan et al. and Silvestrini et al. (8.Silvestrini F. Lasonder E. Olivieri A. Camarda G. van Schaijk B. Sanchez M. Younis Younis S. Sauerwein R. Alano P. Protein export marks the early phase of gametocytogenesis of the human malaria parasite Plasmodium falciparum.Mol. Cell. Proteomics. 2010; 9: 1437-1448Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 11.Khan S.M. Franke-Fayard B. Mair G.R. Lasonder E. Janse C.J. Mann M. Waters A.P. Proteome analysis of separated male and female gametocytes reveals novel sex-specific Plasmodium biology.Cell. 2005; 121: 675-687Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar). Identified proteins were annotated by GeneDB (02, 2013); specifically, the Gene Ontology database was searched by BLAST homology for annotations. The surface expressed (S.E.) proteins were predicted by searching for canonical signal peptides with the SignalP 4.1 Server (28.Petersen T.N. Brunak S. von Heijne G. Nielsen H. SignalP 4.0: discriminating signal peptides from transmembrane regions.Nat. Methods. 2011; 8: 785-786Crossref PubMed Scopus (7117) Google Scholar). Transmembrane domain information was obtained on all identified proteins by the transmembrane protein prediction tool TMHMM Server v. 2.0 (29.Krogh A. Larsson B. von Heijne G. Sonnhammer E.L. Predicting transmembrane protein topology with a hidden markov model: application to complete genomes.J. Mol. Biol. 2001; 305: 567-580Crossref PubMed Scopus (9139) Google Scholar). 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