Background A family of hydrophilic acylated surface (HASP) proteins, containing extensive and variant amino acid repeats, is expressed at the plasma membrane in infective extracellular (metacyclic) and intracellular (amastigote) stages of Old World Leishmania species. While HASPs are antigenic in the host and can induce protective immune responses, the biological functions of these Leishmania-specific proteins remain unresolved. Previous genome analysis has suggested that parasites of the sub-genus Leishmania (Viannia) have lost HASP genes from their genomes. Methods/Principal Findings We have used molecular and cellular methods to analyse HASP expression in New World Leishmania mexicana complex species and show that, unlike in L. major, these proteins are expressed predominantly following differentiation into amastigotes within macrophages. Further genome analysis has revealed that the L. (Viannia) species, L. (V.) braziliensis, does express HASP-like proteins of low amino acid similarity but with similar biochemical characteristics, from genes present on a region of chromosome 23 that is syntenic with the HASP/SHERP locus in Old World Leishmania species and the L. (L.) mexicana complex. A related gene is also present in Leptomonas seymouri and this may represent the ancestral copy of these Leishmania-genus specific sequences. The L. braziliensis HASP-like proteins (named the orthologous (o) HASPs) are predominantly expressed on the plasma membrane in amastigotes and are recognised by immune sera taken from 4 out of 6 leishmaniasis patients tested in an endemic region of Brazil. Analysis of the repetitive domains of the oHASPs has shown considerable genetic variation in parasite isolates taken from the same patients, suggesting that antigenic change may play a role in immune recognition of this protein family. Conclusions/Significance These findings confirm that antigenic hydrophilic acylated proteins are expressed from genes in the same chromosomal region in species across the genus Leishmania. These proteins are surface-exposed on amastigotes (although L. (L.) major parasites also express HASPB on the metacyclic plasma membrane). The central repetitive domains of the HASPs are highly variant in their amino acid sequences, both within and between species, consistent with a role in immune recognition in the host.
References
[1]
Murray HW, Berman JD, Davies CR, Saravia NG (2005) Advances in leishmaniasis. Lancet 366: 1561–1577. doi: 10.1016/S0140-6736(05)67629-5
[2]
Ivens AC, Peacock CS, Worthey EA, Murphy L, Aggarwal G, et al. (2005) The genome of the kinetoplastid parasite, Leishmania major. Science 309: 436–442. doi: 10.1126/science.1112680
[3]
Peacock CS, Seeger K, Harris D, Murphy L, Ruiz JC, et al. (2007) Comparative genomic analysis of three Leishmania species that cause diverse human disease. Nat Genet 39: 839–847. doi: 10.1038/ng2053
[4]
Smith DF, Peacock CS, Cruz AK (2007) Comparative genomics: From genotype to disease phenotype in the leishmaniases. Int J Parasitol 37: 1173–1186. doi: 10.1016/j.ijpara.2007.05.015
[5]
Joshi PB, Kelly BL, Kamhawi S, Sacks DL, McMaster WR (2002) Targeted gene deletion in Leishmania major identifies leishmanolysin (GP63) as a virulence factor. Mol Biochem Parasitol 120: 33–40. doi: 10.1016/S0166-6851(01)00432-7
[6]
Yao C, Donelson JE, Wilson ME (2003) The major surface protease (MSP or GP63) of Leishmania sp. Biosynthesis, regulation of expression, and function. Mol Biochem Parasitol 132: 1–16. doi: 10.1016/S0166-6851(03)00211-1
[7]
McKean PG, Denny PW, Knuepfer E, Keen JK, Smith DF (2001) Phenotypic changes associated with deletion and overexpression of a stage-regulated gene family in Leishmania. Cell Microbiol 3: 511–523. doi: 10.1046/j.1462-5822.2001.00135.x
[8]
Flinn HM, Smith DF (1992) Genomic organisation and expression of a differentially-regulated gene family from Leishmania major. Nucleic Acids Res 20: 755–762. doi: 10.1093/nar/20.4.755
[9]
McKean PG, Delahay R, Pimenta PF, Smith DF (1997) Characterisation of a second protein encoded by the differentially regulated LmcDNA16 gene family of Leishmania major. Mol Biochem Parasitol 85: 221–231. doi: 10.1016/S0166-6851(97)02829-6
[10]
Flinn HM, Rangarajan D, Smith DF (1994) Expression of a hydrophilic surface protein in infective stages of Leishmania major. Mol Biochem Parasitol 65: 259–270. doi: 10.1016/0166-6851(94)90077-9
[11]
Alce TM, Gokool S, McGhie D, Stager S, Smith DF (1999) Expression of hydrophilic surface proteins in infective stages of Leishmania donovani. Mol Biochem Parasitol 102: 191–196. doi: 10.1016/S0166-6851(99)00074-2
[12]
Rangarajan D, Gokool S, McCrossan MV, Smith DF (1995) The gene B protein localises to the surface of Leishmania major parasites in the absence of metacyclic stage lipophosphoglycan. J Cell Sci 108: 3359–3366.
[13]
Denny PW, Gokool S, Russell DG, Field MC, Smith DF (2000) Acylation-dependent protein export in Leishmania. J Biol Chem 275: 11017–11025. doi: 10.1074/jbc.275.15.11017
[14]
Knuepfer E, Stierhof YD, McKean PG, Smith DF (2001) Characterization of a differentially expressed protein that shows an unusual localization to intracellular membranes in Leishmania major. Biochem J 356: 335–344. doi: 10.1042/0264-6021:3560335
[15]
McKean PG, Trenholme KR, Rangarajan D, Keen JK, Smith DF (1997) Diversity in repeat-containing surface proteins of Leishmania major. Mol Biochem Parasitol 86: 225–235. doi: 10.1016/S0166-6851(97)00035-2
[16]
Jensen AT, Gaafar A, Ismail A, Christensen CB, Kemp M, et al. (1996) Serodiagnosis of cutaneous leishmaniasis: assessment of an enzyme-linked immunosorbent assay using a peptide sequence from gene B protein. Am J Trop Med Hyg 55: 490–495.
[17]
Jensen AT, Gasim S, Moller T, Ismail A, Gaafar A, et al. (1999) Serodiagnosis of Leishmania donovani infections: assessment of enzyme-linked immunosorbent assays using recombinant L. donovani gene B protein (GBP) and a peptide sequence of L. donovani GBP. Trans R Soc Trop Med Hyg 93: 157–160. doi: 10.1016/S0035-9203(99)90291-2
[18]
Stager S, Smith DF, Kaye PM (2000) Immunization with a recombinant stage-regulated surface protein from Leishmania donovani induces protection against visceral leishmaniasis. J Immunol 165: 7064–7071.
[19]
Stager S, Alexander J, Kirby AC, Botto M, Rooijen NV, et al. (2003) Natural antibodies and complement are endogenous adjuvants for vaccine-induced CD8(+) T-cell responses. Nat Med 9: 1287–1292. doi: 10.1038/nm933
[20]
Moreno J, Nieto J, Masina S, Canavate C, Cruz I, et al. (2007) Immunization with H1, HASPB1 and MML Leishmania proteins in a vaccine trial against experimental canine leishmaniasis. Vaccine 25: 5290–5300. Epub 2007 Jun 5294. doi: 10.1016/j.vaccine.2007.05.010
[21]
Peacock CS, Seeger K, Harris D, Murphy L, Ruiz JC, et al. (2007) Comparative genomic analysis of three Leishmania species that cause diverse human disease. Nat Genet 39: 839–847. doi: 10.1038/ng2053
[22]
El-Sayed NM, Myler PJ, Blandin G, Berriman M, Crabtree J, et al. (2005) Comparative genomics of trypanosomatid parasitic protozoa. Science 309: 404–409. doi: 10.1126/science.1112181
[23]
Hertz-Fowler C, Peacock CS, Wood V, Aslett M, Kerhornou A, et al. (2004) GeneDB: a resource for prokaryotic and eukaryotic organisms. Nucleic Acids Res 32: D339–343. doi: 10.1093/nar/gkh007
[24]
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215: 403–410. doi: 10.1016/S0022-2836(05)80360-2
[25]
Carver TJ, Rutherford KM, Berriman M, Rajandream MA, Barrell BG, et al. (2005) ACT: the Artemis Comparison Tool. Bioinformatics 21: 3422–3423. doi: 10.1093/bioinformatics/bti553
[26]
Eisenhaber B, Bork P, Yuan Y, Loffler G, Eisenhaber F (2000) Automated annotation of GPI anchor sites: case study C. elegans. Trends Biochem Sci 25: 340–341. doi: 10.1016/S0968-0004(00)01601-7
[27]
Maurer-Stroh S, Eisenhaber B, Eisenhaber F (2002) N-terminal N-myristoylation of proteins: prediction of substrate proteins from amino acid sequence. J Mol Biol 317: 541–557. doi: 10.1006/jmbi.2002.5426
[28]
Zhou F, Xue Y, Yao X, Xu Y (2006) CSS-Palm: palmitoylation site prediction with a clustering and scoring strategy (CSS). Bioinformatics 22: 894–896. doi: 10.1093/bioinformatics/btl013
[29]
Schonian G, Nasereddin A, Dinse N, Schweynoch C, Schallig HD, et al. (2003) PCR diagnosis and characterization of Leishmania in local and imported clinical samples. Diagn Microbiol Infect Dis 47: 349–358. doi: 10.1016/S0732-8893(03)00093-2
[30]
Uliana SR, Nelson K, Beverley SM, Camargo EP, Floeter-Winter LM (1994) Discrimination amongst Leishmania by polymerase chain reaction and hybridization with small subunit ribosomal DNA derived oligonucleotides. J Eukaryot Microbiol 41: 324–330. doi: 10.1111/j.1550-7408.1994.tb06085.x
[31]
Castilho MS, Pavao F, Oliva G, Ladame S, Willson M, et al. (2003) Evidence for the two phosphate binding sites of an analogue of the thioacyl intermediate for the Trypanosoma cruzi glyceraldehyde-3-phosphate dehydrogenase-catalyzed reaction, from its crystal structure. Biochemistry 42: 7143–7151. doi: 10.1021/bi0206107
[32]
Zauli-Nascimento RC, Miguel DC, Yokoyama-Yasunaka JK, Pereira LI, Pelli de Oliveira MA, et al. (2010) In vitro sensitivity of Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonensis Brazilian isolates to meglumine antimoniate and amphotericin B. Trop Med Int Health 15: 68–76. doi: 10.1111/j.1365-3156.2009.02414.x
[33]
Depledge DP, Evans KJ, Ivens AC, Aziz N, Maroof A, et al. (2009) Comparative Expression Profiling of Leishmania: Modulation in Gene Expression between Species and in Different Host Genetic Backgrounds. PLoS Negl Trop Dis 3: e476. doi: 10.1371/journal.pntd.0000476
[34]
Bates PA (1994) Complete developmental cycle of Leishmania mexicana in axenic culture. Parasitology 108: 1–9. doi: 10.1017/S0031182000078458
[35]
Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132: 365–386. doi: 10.1385/1-59259-192-2:365
[36]
Gamboa D, Van Eys G, Victoir K, Torres K, Adaui V, et al. (2007) Putative markers of infective life stages in Leishmania (Viannia) braziliensis. Parasitology 134: 1689–1698. doi: 10.1017/S003118200700306X
[37]
Studier FW (2005) Protein production by auto-induction in high density shaking cultures. Protein Expr Purif 41: 207–234. doi: 10.1016/j.pep.2005.01.016
[38]
Zhang WW, Matlashewski G (2000) Analysis of antisense and double stranded RNA downregulation of A2 protein expression in Leishmania donovani. Mol Biochem Parasitol 107: 315–319. doi: 10.1016/S0166-6851(99)00236-4
[39]
Ma S (2000) Identification and characterisation of HASPB homologues in New World Leishmania species. University of London. PhD thesis.
[40]
Pimenta PF, Pinto da Silva P, Rangarajan D, Smith DF, Sacks DL (1994) Leishmania major: association of the differentially expressed gene B protein and the surface lipophosphoglycan as revealed by membrane capping. Exp Parasitol 79: 468–479. doi: 10.1006/expr.1994.1108
[41]
Sadlova J, Price HP, Smith BA, Votypka J, Volf P, et al. (2010) The stage-regulated. HASP and SHERP proteins are essential for differentiation of the protozoan parasite, Leishmania major, in its sand fly vector, Phlebotomus papatasi. Cellular Microbiology, Jul 16: doi: 10.1111/j.1462-5822.2010.01507.x
[42]
Requena JM, Soto M, Quijada L, Alonso C (1997) Genes and chromosomes of Leishmania infantum. Mem Inst Oswaldo Cruz 92: 853–858. doi: 10.1590/S0074-02761997000600022
[43]
Smith M, Blanchette M, Papadopoulou B (2008) Improving the prediction of mRNA extremities in the parasitic protozoan Leishmania. BMC Bioinformatics 9: 158. doi: 10.1186/1471-2105-9-158
