Trypanosoma rangeli expresses a β-galactofuranosyl transferase

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Experimental Parasitology 130 (2012) 246–252

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Trypanosoma rangeli expresses a b-galactofuranosyl transferase Patrícia Hermes Stoco a,⇑, Cassandra Aresi a, Débora Denardin Lückemeyer a, Maísa Michels Sperandio a, Thaís Cristine Marques Sincero a, Mário Steindel a, Luiz Claudio Miletti b, Edmundo Carlos Grisard a a b

Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de Santa Catarina, Florianópolis, SC 88040-970, Brazil Centro de Ciências Agroveterinárias, Universidade do Estado de Santa Catarina, Lages, SC, Brazil

a r t i c l e

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Article history: Received 17 October 2011 Received in revised form 12 December 2011 Accepted 13 December 2011 Available online 22 December 2011 Keywords: Trypanosoma rangeli GALFT Galactofuranosyl transferase Galactofuranose

a b s t r a c t Glycoconjugates play essential roles in cell recognition, infectivity and survival of protozoan parasites within their insect vectors and mammalian hosts. b-galactofuranose is a component of several glycoconjugates in many organisms, including a variety of trypanosomatids, but is absent in mammalian and African trypanosomes. Herein, we describe the presence of a b(1–3) galactofuranosyl transferase (GALFT), an important enzyme of the galactofuranose biosynthetic pathway, in Trypanosoma rangeli. The T. rangeli GALFT gene (TrGALFT) has an ORF of 1.2 Kb and is organized in two copies in the T. rangeli genome. Antibodies raised against an internal fragment of the transferase demonstrated a 45 kDa protein coded by TrGALFT was localized in the whole cytoplasm, mainly in the Golgi apparatus and equally expressed in epimastigotes and trypomastigotes from T. rangeli. Despite the high sequence similarity with Trypanosoma cruzi and Leishmania spp. orthologous TrGALFT showed a substitution of the metal-binding DXD motif, conserved amongst glycosyltransferases, for a DXE functionally analogous motif. Moreover, a reduced number of GALFT genes were present in T. rangeli when compared with other pathogenic kinetoplastid species. Ó 2011 Elsevier Inc. All rights reserved.

1. Introduction The carbohydrates present in glycoconjugates of the cell surface of trypanosomatids are essential to a variety of cell events, including infectivity and/or virulence in insect vectors and mammalian hosts. Among these, galactofuranose, a glycoside present in most pathogenic trypanosomatids, fungi and bacteria but absence in mammals, represents an interesting target for therapeutic approaches of several diseases related to these organisms (de Lederkremer and Colli, 1995; Oppenheimer et al., 2011; Pedersen and Turco, 2003; Shibata and Okawa, 2011). In Trypanosoma cruzi, galactofuranose is found in the lipopeptidophosphoglycan (LPPG) coat (de Lederkremer et al., 1980, 1985; Golgher et al., 1993), in mucins and other proteins, including the 80–90 kDa glycoproteins and glycopeptides involved in host cell adhesion and parasite internalization (De Arruda et al., 1989; Haynes et al., 1996; Serrano et al., 1995). Galactofuranose is also present in Leishmania spp. glycoconjugates related to parasite structure, such as lipophosphoglycans (LPG) (McConville et al., 1990; Previato et al., 1997; Turco and Descoteaux, 1992).

⇑ Corresponding author. E-mail addresses: [email protected] (P.H. Stoco), [email protected] com (C. Aresi), [email protected] (D.D. Lückemeyer), [email protected] com (M.M. Sperandio), [email protected] (T.C.M. Sincero), [email protected] (M. Steindel), [email protected] (L.C. Miletti), [email protected] (E.C. Grisard). 0014-4894/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2011.12.005

The metabolic pathways involved in the removal or attachment of galactofuranose from trypanosomatids glycoconjugates have been reported, including a T. cruzi exo b-D-galactofuranosidase (Miletti et al., 2003). Genes encoding b(1–3) galactofuranosyl transferase (GALFT) enzymes have been described for T. cruzi (El-Sayed et al., 2005) and Leishmania spp. (Ivens et al., 2005; Spãth et al., 2000; Zhang et al., 2004), but expression and protein characterization was achieved only for Leishmania sp. On the other hand, the absence of GALFT in African trypanosomes remains an intriguing question (Berriman et al., 2005). Considering the importance of galactofuranose glycoconjugates for mammalian pathogenic trypanosomatids, in this work, we described for the first time the presence of GALFT in the protozoan parasite Trypanosoma rangeli. This parasite infects a variety of mammalian species, including humans in Central and South America, and probably has an impact on the misdiagnosis of Chagas disease (Grisard et al., 1999a, 2010). After colonization of the triatomine vector gut, the parasites reach the hemocoel, where the epimastigotes multiplicate in the hemolymph, invade the salivary glands and transform into metacyclic trypomastigotes (D’Alessandro, 1976; Grisard et al., 1999b; Guhl and Vallejo, 2003). Transmission to the mammalian host occurs by the bite of infected triatomines, in particular those from the genus Rhodnius. Although T. rangeli is considered pathogenic to the invertebrate host, this parasite is completely harmless to the mammalian host, where its biology, including intracellular multiplication ability, remains controversial.

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While several aspects of the T. rangeli life cycle within triatomine bugs are well known (Grisard et al., 1999a, 2010; Guhl and Vallejo, 2003), the parasite development in the mammalian host remains unknown. This lack of information emphasizes the need for further studies into the parasite–host cell interaction, where galactofuranose glycoconjugates might play an important role. As several glycoconjugates have important roles in cell–parasite interactions in pathogenic protozoa, the expression of beta galactofuranosyl transferase, a key enzyme in the galactofuranose pathway, was investigated in the non-pathogenic parasite T. rangeli. 2. Materials and methods 2.1. Parasites and culture conditions Epimastigotes of T. rangeli Choachí strain and T. cruzi Y strain were maintained in liver infusion tryptose (LIT) medium supplemented with 10% FCS at 27 °C after cyclic passages in micetriatomine-mice. Differentiation of T. rangeli epimastigotes to trypomastigotes was accomplished in vitro under chemically defined conditions, as described previously (Koerich et al., 2002). T. cruzi trypomastigotes were obtained from the supernatant of Vero cells (ATCC-CCL81) cultures infected with bloodstream trypomastigote forms and maintained in Dulbecco’s modified Eagle’s Medium – DMEM (Sigma–Aldrich), pH 7.4, supplemented with 5% FCS (Carvalho and De Souza, 1983; Eger-Mangrich et al., 2001). 2.2. T. rangeli DNA and RNA isolation Total DNA was purified using 2  108 T. rangeli epimastigotes by standard phenol–chloroform procedures. Total RNA was obtained using the TrizolÒ reagent (Invitrogen). Messenger RNA (mRNA) was purified using the lMACs mRNA Isolation (Miltenyi Biotec). Assessment of purity and quality was performed spectrophotometrically (260/280 nm) and by ethidium bromide-stained agarose gel electrophoresis. 2.3. Cloning and sequencing of TrGALFT gene Using T. rangeli transcriptome data (Grisard et al., 2010), two primers (GalF 50 -GAG CTT GAG AAG ATT TAT GGG TGG-30 /GalR 50 -GTT CTC GTC AAA ATA TCC CAC CG-30 ) were designed to obtain the entire 1.2 Kb of the T. rangeli b-galactofuranosyl transferase gene sequence by RT-PCR using SuperScript IIÒ reverse transcriptase (RT) (Invitrogen). Amplification of the 50 and 30 ends of TrGALFT cDNA was performed using the specific primers and a primer directed to the spliced leader sequence (50 -CCC GAA TTC TGT ACT ATA TTG GT-30 ) or an oligo(dT)35 primer (Invitrogen). All amplification products were cloned in pGEM-T-Easy vector (Promega) and sequenced on both strands in a Megabace 1000Ò DNA Analysis System using the DYEnamic ET terminators kit (GE Healthcare) according to the manufacturer’s conditions. 2.4. Sequence assembling and analysis All high quality DNA sequences (Phred P20) were assembled and analyzed using the Phred/Phrap/Consed package (Ewing and Green, 1998) and then compared with public databases using the BLAST algorithm (Altschul et al., 1997). Analysis of deduced protein sequences was carried out using the Proteomic Tools provided by the ExPASy (http://www.expasy.org). Amino acid sequences of other kinetoplastid species GALFT’s were retrieved from the TriTrypDB (Aslett et al., 2010) for comparative and phylogenetic analysis (Supplementary material). Multiple alignments of amino acid sequences were performed using CLUSTAL W package (Thompson

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et al., 1994), leading to construction of phylogenetic trees using neighbor joining method and bootstrap analysis by the MEGA 4.1 software (Tamura et al., 2007). 2.5. Southern blot T. rangeli genomic DNA (15 lg/lane) was digested with TaqI, EcoRI and PstI restriction endonucleases, separated by electrophoresis, and transferred onto nylon membranes (Sigma–Aldrich). A peroxidase-labeled probe was prepared using a 519 bp PCR fragment (nucleotides 304–807) and the ECL Direct Nucleic Acid Labeling and Detection System (GE Healthcare). The membrane was hybridized and washed using a standard protocol and developed using the ECL kit according to the manufacturer’s directions. 2.6. Heterologous expression and antibody production For expression, a TrGALFT gene fragment (aa 96–269) (Fig. 1) was amplified by PCR using gene specific primers ExpGal-F (50 CTC GAG GAG CTT GAG AAG ATT TAT GGG TGG-30 ) and ExpGal-R (50 -GGA TCC GTT CTC GTC AAA ATA TCC CAC CGT-30 ) modified with appropriate restriction sites (the underlined nucleotides correspond to XhoI and BamHI restriction sites, respectively). The 519 bp amplicon pre-digested with XhoI and BamHI, was cloned into a pET14b expression vector (Novagen) and used to transform Escherichia coli BL21 (DE3) competent cells. The His6-tagged TrGALFT recombinant protein was obtained by induction of the cells at OD600 of 0.6 with 1 mM of isopropyl-b-Dthiogalactopyranoside (IPTG) at 27 °C for 3 h. Cells were then harvested by centrifugation (6000g for 15 min at 4 °C) and lysed using a denaturing buffer (8 M Urea; 10 mM Tris; 100 mM NaH2PO4, pH 8.0). The samples were then incubated at 65 °C to dissolve inclusion bodies and centrifuged (10,000g for 30 min at 4 °C). The supernatants were submitted to purification using Ni–NTA affinity chromatography columns (Qiagen) and the purity of the obtained protein was assessed by 12% SDS–PAGE stained with Coomassie brilliant blue R-250. Protein concentrations were determined by the Bradford method using bovine serum albumin as standard. The purified 22 kDa recombinant TrGALFT (rTrGALFT) was subcutaneously inoculated in Balb/C mice (50 lg) using Freund’s complete adjuvant (Sigma–Aldrich), with two consecutive inoculations at 10 days intervals using Alu-Gel (Serva). Mouse serum was collected 10 days after the third injection and tested for anti-TrGALFT antibodies. The UFSC Ethics Committee for Animal Care approved all procedures involving experimental animals (Protocol 23080.025618/ 2009-81). 2.7. Western blot TrGALFT recombinant protein and total epimastigotes and trypomastigotes protein extracts (30 lg) from T. rangeli and T. cruzi were mixed with Laemmli sample buffer, boiled for 5 min and resolved on 12% SDS–PAGE gels. Protein separations were transferred onto nitrocellulose membranes (GE Healthcare) in appropriate buffer (25 mM Tris; 192 mM glycine; 20% v/v methanol, pH 8.3) in a TE 70 Semi-Dry Transfer Unit (GE Healthcare). Membranes were then blocked with 5% non-fat milk in PBS (pH 7.4) with 0.1% Tween-20 (PBS-T) for 1 h at room temperature. After blocking, membranes were incubated for 1 h with anti-TrGALFT polyclonal serum (1:500), anti-His-Tag monoclonal antibody (1:10,000) or anti-a tubulin monoclonal antibody (1:10,000) used as loading control. After four washes in PBS-T, membranes were incubated for 1 h with anti-mouse IgG antibody conjugated with peroxidase (1:10,000) (Sigma–Aldrich). Membranes were developed using ECL reagent (GE Healthcare) and detection was achieved in radiographic films.

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Fig. 1. Alignment of amino acid sequences of the Trypanosoma rangeli (TrGALFT) and Trypanosoma cruzi b-galactofuranosyl transferase genes. Black bar indicates the region used for heterologous expression of TrGALFT. Identical residues are in gray background. Black arrows represent start and end points of the COG1216 conserved domain related to predicted glycosyltransferases. Box indicates the DxE motif on TrGALFT.

2.8. Immunofluorescence assays Cells were harvested from media by centrifugation (3000g, 10 min), washed twice in PBS, adjusted to a concentration of 1  106 cells ml1 and deposited on microscope coverslips previously treated with 0.1% of poly-L-lysine. Then, cells were fixed with 4% (w/v) p-formaldehyde in PBS and permeabilized with 0.1% NP40. The coverslips were saturated with PBS containing 0.1% Tween20 and 5% dry milk powder and incubated (1 h, 37 °C) with antiTrGALFT serum (1:50) in PBS containing 0.5% BSA. The coverslips were washed with PBS and incubated (15 min, 37 °C) with Alexafluor 488-conjugated goat-anti-mouse IgG (1:1000) (Molecular Probes). After three washes in PBS, the parasites were incubated for 5 min with PBS containing 4,6-diamidino-2-phenylindole (DAPI) 1 lg ml1. The coverslips were mounted on microscopy slides using an anti-fading solution (Invitrogen) and inspected by confocal microscopy (Leica DMI6000 B). 3. Results 3.1. Cloning and sequence analysis of TrGALFT Based on Orestes profiles showing significant similarity to a T. cruzi b-galactofuranosyl transferase gene, RT-PCR allowed the isolation of DNA fragment, which corresponded to the complete TrGALFT gene sequence, including the 50 and 30 UTR regions. The TrGALFT 1.2 Kb ORF encodes a protein of 400 amino acids with a calculated molecular mass of 45 kDa (GenBank ID JN135044). Comparative analysis of the amino acid TrGALFT sequence revealed identities between 73% and 55% with T. cruzi orthologs. The predicted glycosyltransferase conserved domain (COG 1216) was found, including the region between amino acids 111–325. In this region the amino acid motif ‘‘FDENFYPAYFEDVEY’’ likely represents the catalytic site, although TrGALFT contains a DxE instead of a DxD motif, the glycosyltransferases-conserved metal binding motif (Fig. 1). Sequence search using the TriTrypDB database revealed 79 amino acid sequences corresponding to b-galactofuranosyl transferase

genes. The majority of sequences were present in the T. cruzi genome (37 genes and 29 pseudogenes) that showed a high intra-specific conservancy. The same analysis against Leishmania spp. revealed three distinct groups of orthologous genes that were found in L. major, L. mexicana, L. infantum and L. braziliensis genomes with variable similarities. In contrast, only two gene copies were detected in the T. rangeli genome by Southern blot assay using a specific probe for the TrGALFT (Fig. 2A). Phylogenetic analysis based on amino acid sequences of bgalactofuranosyl transferases from distinct species, excluding T. cruzi pseudogenes, grouped T. rangeli sequence closely to T. cruzi orthologs (Fig. 2B). 3.2. Expression and purification of TrGALFT fragment The His-tagged rTrGALFT protein fragment was expressed in Escherichia coli. Since rTrGALFT was largely insoluble, purification from bacterial inclusion bodies was performed by standard methods. After successful purification and refolding, a protein band with a relative molecular mass of 22 kDa was evidenced in SDS–PAGE (Fig. 3A) as also confirmed by Western blot (Fig. 3B). An anti-TrGALFT antiserum produced in mice was able to specifically recognize a 45 kDa polypeptide in total protein extracts from both T. rangeli epimastigote and trypomastigote forms (Fig. 3C). Pre-immune serum was assayed with purified rTrGALFT and T. rangeli epimastigotes lysate as negative controls (data not shown). This result proves the expression of a GALFT by T. rangeli as initially pointed out by a transcriptome analysis (Grisard et al., 2010). Based on the sequence difference and the rationale for heterologous expression, no recognition was observed for total protein extracts of both T. cruzi biological forms (Fig. 3C). 3.3. Immunofluorescence assays (IFA) Assessment of the subcellular localization of TrGALFT in T. rangeli epimastigotes and trypomastigotes by IFA showed a dispersed cytoplasmatic distribution for both forms of the parasite (Fig. 4).

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Fig. 2. (A) Genomic organization of TrGALFT gene. Schematic representation of the gene arrangement and Southern blot analysis of genomic DNA using specific probe for TrGALFT. (B) Phylogenetic analysis of deduced amino acid sequences of b-galactofuranosyl transferase from T. rangeli and other kinetoplastid species by the Neighbor joining method using MEGA 4.1 software. For sequence details please refer to Supplementary material.

Fig. 3. Expression of T. rangeli b-galactofuranosyl transferase (TrGALFT). (A) Heterologous expression and purification of a recombinant fragment of TrGALFT (rTrGALFT) by affinity chromatography as revealed by 12% SDS–PAGE stained with Coomassie Blue R-250. M = molecular weight marker, 1 = total bacterial extract, 2 = flow through, 3 and 4 = column washes, 5, 6 and 7 = protein elutions (250 mM imidazole). (B) Western blot of purified rTrGALFT using anti-His tag monoclonal antibody. N = Negative control (Non-transformed bacterial extract), PGal = Purified rTrGALFT. (C) Western blot analysis of T. rangeli and T. cruzi epimastigotes (Epi) and trypomastigotes (Trypo) lysates revealed with anti-TrGALFT serum. Alpha-tubulin was used as loading control.

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High level of fluorescence was observed in a small region near the kinetoplast, suggesting the predominant presence of this enzyme in the Golgi apparatus.

4. Discussion Considering the crucial importance of galactofuranose-containing molecules on the host cell–parasite interface for pathogenic trypanosomatids, the present study addresses the expression of b-galactofuranosyl transferase by T. rangeli, a non-virulent parasite, whose life cycle in mammals is unknown. Although several galactofuranose-containing glycoconjugates have been found in different organisms, including pathogenic protozoa (Richards and Lowary, 2009), the presence of galactofuranose residues in GPI-anchored proteins (Añez-Rojas et al., 2006) or mucins has not been demonstrated in T. rangeli, and no studies on the galactofuranose metabolism enzymes in this parasite have been reported. In this paper, the expression of a galactofuranose metabolism-related enzyme by T. rangeli, a b-galactofuranosyl transferase is reported for the first time. The TrGALFT gene predicts a protein showing high similarity to the active LPG-specific GALFT encoded by LPG1 from L. major and other GALFTs found in other kinetoplastid species. Different from L. major, where a transmembrane protein domain is found in all GALFTs (Zhang et al., 2004), this domain was intriguingly absent in the T. rangeli GALFT. Furthermore, TrGALFT sequence contains a modification of the luminal C-terminal domain with the presence of a DxE motif instead of the canonical DxD motif found in glycosyltransferases. As pointed out by Ku et al. (2007), both motifs are functionally analogous and are involved in coordinating the nucleotide sugar donor-Mn(II) complex, which is important for the transferase activity.

The simultaneous expression of different GALFTs is a common phenomenon among some species, where different transferases are required for distinct linkage types. For example, LPG-1, the most studied GALFT from L. major, is responsible for adding galactofuranose residues to the lipophosphoglycan (LPG) core structure, but LPG1-mutants continued to synthesize the galactofuranose-containing glycosylinositolphospholipids (GIPLs) (Spãth et al., 2000). In T. cruzi, structural studies of mucins have discovered novel linkages and new patterns of glycosylation, suggesting differences in the expression and activity of specific glycosyltransferases (Mendonça-Previato et al., 2005). The divergence of GALFT gene copy number between T. cruzi, which has a higher number of GALFT than T. rangeli (two copies), is probably related to the diversity of galactofuranose-containing glycoconjugates in each species. Comparing the variability of putative glyconconjugates, transcriptomic data of T. rangeli suggests a reduced number of possibly glycosylated proteins when compared with T. cruzi, including sialidases and mucins gene families, that could be related to the non-pathogenic characteristic of T. rangeli (Grisard et al., 2010). Indeed, the reduced number of gene copies per gene families in T. rangeli has been suggested by EST mapping of both parasites forms (Grisard et al., 2010). The TrGALFT is expressed as a 45 kDa protein in epimastigote and trypomastigote forms of the parasite. In IFA assays, the TrGALFT was found dispersed in the cytoplasm, contrasting with the LPG1 (GALFT from L. major) localization, which is exclusively in the Golgi apparatus. The presence of TrGALFT in the Golgi apparatus was not excluded, since high fluorescence levels were observed close to the kinetoplast, coinciding with the localization of the Golgi apparatus in this species (Zhang et al., 2004). Although TrGALFT is similar to T. cruzi GALFTs, antibodies raised against TrGALFT did not recognize any protein in T. cruzi extracts. This lack of

Fig. 4. Immunolocalization of b-galactofuranosyl transferase in T. rangeli epimastigotes and trypomastigotes using anti-TrGALFT serum in IFA assays. (1) Light microscopy, (2) Detection of anti-TrGALFT antibodies by alexa fluor 488-labeled conjugate, (3) DAPI staining and (4) merged images.

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recognition may be due to a low expression level of this protein in T. cruzi or distinct epitopes between both parasites. The expression of the TrGALFT by both epimastigotes and trypomastigotes corroborates with the results formerly described for T. cruzi (Golgher et al., 1993). In T. rangeli, expression of GALFT by epimastigotes could be related to the attachment to the midgut surface of triatomine vector as observed for T. cruzi (Nogueira et al., 2007), or the adherence of epimastigote forms to the gland cell microvilli by their flagella (Meirelles et al., 2005). However, in the case of trypomastigotes, its function is unknown and may be related to the recognition of receptors on host cells that have intelectin-like receptors (Tsuji et al., 2001), although no evidence has been found related to the attachment of T. rangeli in any mammalian cells. Since the T. rangeli life cycle within mammalian hosts is still unknown and the host–cell parasite interplay is barely studied, but indicating that T. rangeli is probably not an intracellular parasite, expression of molecules involved in parasite–host cell interaction remains an intriguing question. Since other enzymes of the galactofuranose metabolism may be present in T. rangeli, the ongoing effort to sequence the entire parasite genome combined with functional genomics should reveal important aspects of the functional role of this enzyme in the biology of the parasite. 5. Conflict of interest The authors declare no conflict of interest. 6. Financial support PHS, DDL, CA, MMS and TCMS were recipients of CAPES or CNPq scholarships (Brazilian Government Agencies). This work was funded by FINEP, Brazil. Funders have no role on study design, data generation and analysis, decision to publish, or preparation of the manuscript. Acknowledgments To Dr. Maria Julia Manso Alves and Ibeth Cristina Romero Calderón M.Sc. for critical reading and suggestions on the manuscript. To Mariel Asbury Marlow M.Sc. for the English revision. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.exppara.2011.12.005. References Altschul, S.F., Madden, T.L., Schäffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402. Añez-Rojas, N., García-Lugo, P., Crisante, G., Rojas, A., Añez, N., 2006. Isolation, purification and characterization of GPI-anchored membrane proteins from Trypanosoma rangeli and Trypanosoma cruzi. Acta Tropica 97, 140–145. Aslett, M., Aurrecoechea, C., Berriman, M., Brestelli, J., Brunk, B.P., Carrington, M., Depledge, D.P., Fischer, S., Gajria, B., Gao, X., Gardner, M.J., Gingle, A., Grant, G., Harb, O.S., Heiges, M., Hertz-Fowler, C., Houston, R., Innamorato, F., Iodice, J., Kissinger, J.C., Kraemer, E., Li, W., Logan, F.J., Miller, J.A., Mitra, S., Myler, P.J., Nayak, V., Pennington, C., Phan, I., Pinney, D.F., Ramasamy, G., Bogers, M.B., Roos, D.S., Ross, C., Sivam, D., Smith, D.F., Srinivasamoorthy, G., Stoeckert, C.J.J., Subramanian, S., Thibodeau, R., Tivey, A., Treatman, C., Velarde, G., Wang, H., 2010. TriTrypDB: a functional genomic resource for the Trypanosomatidae. Nucleic Acids Res. 38, D457–462. Berriman, M., Ghedin, E., Hertz-Fowler, C., Blandin, G., Renauld, H., Bartholomeu, D.C., Lennard, N.J., Caler, E., Hamlin, N.E., Haas, B., Böhme, U., Hannick, L., Aslett, M.A., Shallom, J., Marcello, L., Hou, L., Wickstead, B., Alsmark, U.C., Arrowsmith, C., Atkin, R.J., Barron, A.J., Bringaud, F., Brooks, K., Carrington, M., Cherevach, I., Chillingworth, T.J., Churcher, C., Clark, L.N., Corton, C.H., Cronin, A., Davies, R.M.D., Djikeng, A., Feldblyum, T., Field, M.C., Fraser, A., Goodhead, I., Hance, Z.,

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