Hostname: page-component-cd9895bd7-jn8rn Total loading time: 0 Render date: 2024-12-23T14:20:18.792Z Has data issue: false hasContentIssue false
  • English
  • Français

Identification and characterization of a surface-associated, subtilisin-like serine protease in Trichomonas vaginalis

Published online by Cambridge University Press:  06 July 2010

PABLO HERNÁNDEZ-ROMANO ,
ROBERTO HERNÁNDEZ ,
ROSSANA ARROYO ,
JOHN F. ALDERETE  and
IMELDA LÓPEZ-VILLASEÑOR
PABLO HERNÁNDEZ-ROMANO
Affiliation:
Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Tercer Circuito Exterior, Ciudad Universitaria, 04510 México D.F., México
ROBERTO HERNÁNDEZ
Affiliation:
Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Tercer Circuito Exterior, Ciudad Universitaria, 04510 México D.F., México
ROSSANA ARROYO
Affiliation:
Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados del IPN (CINVESTAV-IPN), México D.F., México
JOHN F. ALDERETE
Affiliation:
School of Molecular Biosciences, Washington State University, Pullman, WA, USA.
IMELDA LÓPEZ-VILLASEÑOR*
Affiliation:
Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Tercer Circuito Exterior, Ciudad Universitaria, 04510 México D.F., México
*
*Corresponding author: Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Tercer Circuito Exterior, Ciudad Universitaria, 04510 México D.F., México. Tel: +52 55-56228952. Fax: +52 55-56229212. E-mail: [email protected]
Get access Rights & Permissions [Opens in a new window]

Summary

Trichomonas vaginalis is a protozoan parasite causing trichomonosis, a sexually transmitted infection in humans. This parasite has numerous proteases, most of which are cysteine proteases that appear to be involved in adherence and cytotoxicity of host cells. In this report we identify and characterize a putative subtilisin-like serine protease (SUB1). The sub1 gene encodes a 101-kDa protein. In silico analyses predict signal and pro-peptides at the N-terminus, and a transmembrane helix at the carboxy-terminal region. The sub1 gene was found as single copy by Southern analysis, albeit additional serine protease related genes are annotated in the T. vaginalis genome. The expression of sub1 could only be detected by RT-PCR and Ribonuclease Protection Assays, suggesting a low abundant mRNA. The sub1 gene transcription start site was correctly assigned by RPA. The transcript abundance was found to be modulated by the availability of iron in the growth medium. Antibodies raised to a specific SUB1 peptide recognized a single protein band (~82 kDa) in Western blots, possibly representing the mature form of the protein. Immunofluorescence showed SUB1 on the trichomonad surface, and in dispersed vesicles throughout the cytoplasm. A bioinformatic analysis of genes annotated as serine proteases in the T. vaginalis genome is also presented. To our knowledge this is the first putative serine protease experimentally described for T. vaginalis.

Keywords

Trichomonas vaginalisserine proteasesurface proteincellular localizationsubtilisin
Type
Research Article
Information
Parasitology , Volume 137 , Issue 11 , September 2010 , pp. 1621 - 1635
Copyright
Copyright © Cambridge University Press 2010

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

REFERENCES

Alderete, J. F. and Neale, K. A. (1989). Relatedness of structures of a major immunogen in Trichomonas vaginalis isolates. Infection and Immunity 57, 1849–1853.CrossRefGoogle Scholar
Alderete, J. F., Provenzano, D. and Lehker, M. W. (1995). Iron mediates Trichomonas vaginalis resistance to complement lysis. Microbial Pathogenesis 19, 93–103.Google Scholar
Alexander, P. A., Ruan, B. and Bryan, P. N. (2001). Cation-dependent stability of subtilisin. Biochemistry 40, 10634–10639.Google Scholar
Alvarez-Sanchez, M. E., Avila-Gonzalez, L., Becerril-Garcia, C., Fattel-Facenda, L. V., Ortega-Lopez, J. and Arroyo, R. (2000). A novel cysteine proteinase (CP65) of Trichomonas vaginalis involved in cytotoxicity. Microbial Pathogenesis 28, 193–202.Google Scholar
Alvarez-Sanchez, M. E., Carvajal-Gamez, B. I., Solano-Gonzalez, E., Martinez-Benitez, M., Garcia, A. F., Alderete, J. F. and Arroyo, R. (2008). Polyamine depletion down-regulates expression of the Trichomonas vaginalis cytotoxic CP65, a 65-kDa cysteine proteinase involved in cellular damage. The International Journal of Biochemistry & Cell Biology 40, 2442–2451.Google Scholar
Alvarez-Sanchez, M. E., Solano-Gonzalez, E., Yañez-Gomez, C. and Arroyo, R. (2007). Negative iron regulation of the CP65 cysteine proteinase cytotoxicity in Trichomonas vaginalis. Microbes and Infection/Institut Pasteur 9, 1597–1605.CrossRefGoogle ScholarPubMed
Allan, V. J. (2000). Protein Localization by Fluorescence Localization, Oxford University Press, New York, USA.Google Scholar
Anderson, E. T., Wetherell, M. G., Winter, L. A., Olmsted, S. B., Cleary, P. P. and Matsuka, Y. V. (2002). Processing, stability, and kinetic parameters of C5a peptidase from Streptococcus pyogenes. European Journal of Biochemistry 269, 4839–4851.CrossRefGoogle ScholarPubMed
Arroyo, R. and Alderete, J. F. (1989). Trichomonas vaginalis surface proteinase activity is necessary for parasite adherence to epithelial cells. Infection and Immunity 57, 2991–2997.CrossRefGoogle ScholarPubMed
Bagga, S., Hu, G., Screen, E. S. and St Leger, R. J. (2004). Reconstructing the diversification of subtilisins in the pathogenic fungus Metharzium anisopliae. Gene 324, 159–169.CrossRefGoogle ScholarPubMed
Bendtsen, J. D., Nielsen, H., Von, H. G. and Brunak, S. (2004). Improved prediction of signal peptides: SignalP 3.0. Journal of Molecular Biology 340, 783–795.CrossRefGoogle ScholarPubMed
Bennett-Lovsey, R. M., Herbert, A. D., Sternberg, M. J. and Kelley, L. A. (2008). Exploring the extremes of sequence/structure space with ensemble fold recognition in the program Phyre. Proteins 70, 611–625.CrossRefGoogle ScholarPubMed
Brown, G. D., Dave, J. A., Gey van Pittius, N. C., Stevens, L., Ehlers, M. R. W. and Beyers, A. D. (2000). The mycosins of Mycobacterium tuberculosis H37Rv: a family of subtilisin-like serine proteases. Gene 254, 147–155.CrossRefGoogle ScholarPubMed
Brown, M. T., Goldstone, H. M., Bastida-Corcuera, F., Delgadillo-Correa, M. G., McArthur, A. G. and Johnson, P. J. (2007). A functionally divergent hydrogenosomal peptidase with protomitochondrial ancestry. Molecular Microbiology 64, 1154–1163.CrossRefGoogle ScholarPubMed
Carlton, J. M., Hirt, R. P., Silva, J. C., Delcher, A. L., Schatz, M., Zhao, Q., Wortman, J. R., Bidwell, S. L., Alsmark, U. C., Besteiro, S., Sicheritz-Ponten, T., Noel, C. J., Dacks, J. B., Foster, P. G., Simillion, C., Van De, P. Y., Miranda-Saavedra, D., Barton, G. J., Westrop, G. D., Muller, S., Dessi, D., Fiori, P. L., Ren, Q., Paulsen, I., Zhang, H., Bastida-Corcuera, F. D., Simoes-Barbosa, A., Brown, M. T., Hayes, R. D., Mukherjee, M., Okumura, C. Y., Schneider, R., Smith, A. J., Vanacova, S., Villalvazo, M., Haas, B. J., Pertea, M., Feldblyum, T. V., Utterback, T. R., Shu, C. L., Osoegawa, K., De Jong, P. J., Hrdy, I., Horvathova, L., Zubacova, Z., Dolezal, P., Malik, S. B., Logsdon, J. M. JR., Henze, K., Gupta, A., Wang, C. C., Dunne, R. L., Upcroft, J. A., Upcroft, P., White, O., Salzberg, S. L., Tang, P., Chiu, C. H., Lee, Y. S., Embley, T. M., Coombs, G. H., Mottram, J. C., Tachezy, J., Fraser-Liggett, C. M. and Johnson, P. J. (2007). Draft genome sequence of the sexually transmitted pathogen Trichomonas vaginalis. Science 315, 207–212.Google Scholar
Carruthers, V. B. (2006). Proteolysis and Toxoplasma invasion. International Journal for Parasitology 36, 595–600.CrossRefGoogle ScholarPubMed
Cohen, J. (2000). HIV transmission. AIDS researchers look to Africa for new insights. Science 287, 942–943.CrossRefGoogle Scholar
Conseil, V., Soete, M. and Dubremetz, J. F. (1999). Serine protease inhibitors block invasion of host cells by Toxoplasma gondii. Antimicrobial Agents and Chemotherapy 43, 1358–1361.CrossRefGoogle ScholarPubMed
Cotch, M. F., Pastorek, J. G., Nugent, R. P., Hillier, S. L., Gibbs, R. S., Martin, D. H., Eschenbach, D. A., Edelman, R., Carey, J. C., Regan, J. A., Krohn, M. A., Klebanoff, M. A., Rao, A. V. and Rhoads, G. G. (1997). Trichomonas vaginalis associated with low birth weight and preterm delivery. The Vaginal Infections and Prematurity Study Group. Sexually Transmitted Diseases 24, 353–360.CrossRefGoogle ScholarPubMed
Chang, Y. C., Kadokura, H., Yoda, K. and Yamasaki, M. (1996). Secretion of active subtilisin YaB by a simultaneous expression of separate pre-pro and pre-mature polypeptides in Bacillus subtilis. Biochemical and Biophysical Research Communications 219, 463–468.Google Scholar
Dailey, D. C., Chang, T. H. and Alderete, J. F. (1990). Characterization of Trichomonas vaginalis haemolysis. Parasitology 101, 171–175.CrossRefGoogle ScholarPubMed
Dejkriengkraikhul, P. and Wilairat, P. (1983). Requirement of malarial protease in the invasion of human red cells by merozoites of Plasmodium falciparum. Zeitschrift für Parasitenkunde 69, 313–317.CrossRefGoogle ScholarPubMed
Diamond, L. (1957). The establishment of various trichomonads of animals and man in axenic cultures. The Journal of Parasitology 43, 488–490.Google Scholar
Espinosa, N., Hernandez, R., Lopez-Griego, L. and Lopez-Villaseñor, I. (2002). Separable putative polyadenylation and cleavage motifs in Trichomonas vaginalis mRNAs. Gene 289, 81–86.CrossRefGoogle ScholarPubMed
Finn, R. D., Tate, J., Mistry, J., Coggill, P. C., Sammut, S. J., Hotz, H. R., Ceric, G., Forslund, K., Eddy, S. R., Sonnhammer, E. L. and Bateman, A. (2008). The Pfam protein families database. Nucleic Acids Research 36, D281–D288.Google Scholar
Fiori, P. L., Rappelli, P., Addis, M. F., Mannu, F. and Cappuccinelli, P. (1997). Contact-dependent disruption of the host cell membrane skeleton induced by Trichomonas vaginalis. Infection and Immunity 65, 5142–5148.CrossRefGoogle ScholarPubMed
Garcia, A. F., Benchimol, M. and Alderete, J. F. (2005). Trichomonas vaginalis polyamine metabolism is linked to host cell adherence and cytotoxicity. Infection and Immunity 73, 2602–2610.CrossRefGoogle ScholarPubMed
Garcia, A. F., Chang, T. H., Benchimol, M., Klumpp, D. J., Lehker, M. W. and Alderete, J. F. (2003). Iron and contact with host cells induce expression of adhesins on surface of Trichomonas vaginalis. Molecular Microbiology 47, 1207–1224.Google Scholar
Graycar, T. P., Ballinger, M. D. and Wells, J. A. (2004). Subtilisins. In Handbook of Proteolytic Enzymes Vol 2, 2nd Edn.(ed. Barrett, A. J., Rawlings, N. D. and Woessner, J. F.), pp. 1786–1792. Elsevier Academic Press, London, UK.Google Scholar
Harris, P. K., Yeoh, S., Dluzewski, A. R., O′Donell, R. A., Whiters-Martinez, C., Hackett, F., Bannister, L. H., Mitchell, G. H. and Blackman, M. J. (2005). Molecular identification of a malaria merozoite surface sheddase. Plos Pathogens 1, 0241–0251.Google Scholar
Hernandez-Gutierrez, R., Avila-Gonzalez, L., Ortega-Lopez, J., Cruz-Talonia, F., Gomez-Gutierrez, G. and Arroyo, R. (2004). Trichomonas vaginalis: characterization of a 39-kDa cysteine proteinase found in patient vaginal secretions. Experimental Parasitology 107, 125–135.Google Scholar
Hernandez-Gutierrez, R., Ortega-Lopez, J. and Arroyo, R. (2003). A 39-kDa cysteine proteinase CP39 from Trichomonas vaginalis, which is negatively affected by iron may be involved in trichomonal cytotoxicity. The Journal of Eukaryotic Microbiology 50 (Suppl. ) 696–698.CrossRefGoogle ScholarPubMed
Hirt, R. P., Noel, C. J., Sicheritz-Ponten, T., Tachezy, J. and Fiori, P. L. (2007). Trichomonas vaginalis surface proteins: a view from the genome. Trends in Parasitology 23, 540–547.Google Scholar
Hong, Y. C., Kong, H. H., Ock, M. S., Kim, I. S. and Chung, D. I. (2000). Isolation and characterization of a cDNA encoding a subtilisin-like serine proteinase (ahSUB) from Acanthamoeba healyi. Molecular and Biochemical Parasitology 111, 441–446.Google Scholar
Hunter, S., Apweiler, R., Attwood, T. K., Bairoch, A., Bateman, A., Binns, D., Bork, P., Das, U., Daugherty, L., Duquenne, L., Finn, R. D., Gough, J., Haft, D., Hulo, N., Kahn, D., Kelly, E., Laugraud, A., Letunic, I., Lonsdale, D., Lopez, R., Madera, M., Maslen, J., McAnulla, C., McDowall, J., Mistry, J., Mitchell, A., Mulder, N., Natale, D., Orengo, C., Quinn, A. F., Selengut, J. D., Sigrist, C. J., Thimma, M., Thomas, P. D., Valentin, F., Wilson, D., Wu, C. H. and Yeats, C. (2009). InterPro: the integrative protein signature database. Nucleic Acids Research 37, D211–D215.Google Scholar
Inouye, M. (1991). Intramolecular chaperone: the role of the pro-peptide in protein folding. Enzyme 45, 314–321.Google Scholar
Kall, L., Krogh, A. and Sonnerhammer, E. L. (2004). A combined transmembrane topology and signal peptide prediction method. The Journal of Molecular Biology 338, 1027–1036.Google Scholar
Kall, L., Krogh, A. and Sonnerhammer, E. L. (2007). Advantages of combined transmembrane topology and signal peptide prediction--the Phobius web server. Nucleic Acids Research 35, W429–W432.Google Scholar
Kelley, L. A., MacCallum, R. M. and Sternberg, M. J. (2000). Enhanced genome annotation using structural profiles in the program 3D-PSSM. The Journal of Molecular Biology 299, 499–520.Google Scholar
Kissinger, P., Amedee, A., Clark, R. A., Dumestre, J., Theall, K. P., Myers, L., Hagensee, M. E., Farley, T. A. and Martin, D. H. (2009). Trichomonas vaginalis treatment reduces vaginal HIV-1 shedding. Sexually Transmitted Diseases 36, 11–16.Google Scholar
Kissinger, P., Secor, W. E., Leichliter, J. S., Clark, R. A., Schmidt, N., Curtin, E. and Martin, D. H. (2008). Early repeated infections with Trichomonas vaginalis among HIV-positive and HIV-negative women. Clinical Infectious Diseases 46, 994–999.Google Scholar
Klemba, M. and Goldberg, D. E. (2002). Biological roles of proteases in parasitic protozoa. Annual Review of Biochemistry 71, 275–305.Google Scholar
Kyte, J. and Doolittle, R. F. (1982). A simple method for displaying the hydropathic character of a protein. The Journal of Molecular Biology 157, 105–132.CrossRefGoogle ScholarPubMed
Laga, M., Manoka, A., Kivuvu, M., Malele, B., Tuliza, M., Nzila, N., Goeman, J., Behets, F., Batter, V. and Alary, M. (1993). Non-ulcerative sexually transmitted diseases as risk factors for HIV-1 transmission in women: results from a cohort study. AIDS 7, 95–102.Google Scholar
Leher, H., Silvany, R., Alizadeh, H., Huang, J. and Niederkorn, J. Y. (1998). Mannose induces the release of cytopathic factors from Acanthamoeba castellanii. Infection and Immunity 66, 5–10.Google Scholar
Lehker, M. W., Arroyo, R. and Alderete, J. F. (1991). The regulation by iron of the synthesis of adhesins and cytoadherence levels in the protozoan Trichomonas vaginalis. The Journal of Experimental Medicine 174, 311–318.CrossRefGoogle ScholarPubMed
Liang, M. P., Banatao, D. R., Klein, T. E., Brutlag, D. L. and Altman, R. B. (2003). WebFEATURE: An interactive web tool for identifying and visualizing functional sites on macromolecular structures. Nucleic Acids Research 31, 3324–3327.Google Scholar
Liston, D. R. and Johnson, P. J. (1999). Analysis of a ubiquitous promoter element in a primitive eukaryote: early evolution of the initiator element. Molecular and Cellular Biology 19, 2380–2388.Google Scholar
Lopez-Villaseñor, I., Contreras, A. P., Lopez-Griego, L., Alvarez-Sanchez, E. and Hernandez, R. (2004). Trichomonas vaginalis ribosomal DNA: analysis of the intergenic region and mapping of the transcription start point. Molecular and Biochemical Parasitology 137, 175–179.Google Scholar
McClelland, R. S., Sangare, L., Hassan, W. M., Lavreys, L., Mandaliya, K., Kiarie, J., Ndinya-Achola, J., Jaoko, W. and Baeten, J. M. (2007). Infection with Trichomonas vaginalis increases the risk of HIV-1 acquisition. The Journal of Infectious Diseases 195, 698–702.Google Scholar
Mendoza-Lopez, M. R., Becerril-Garcia, C., Fattel-Facenda, L. V., Avila-Gonzalez, L., Ruiz-Tachiquin, M. E., Ortega-Lopez, J. and Arroyo, R. (2000). CP30, a cysteine proteinase involved in Trichomonas vaginalis cytoadherence. Infection and Immunity 68, 4907–4912.Google Scholar
Miller, S. A., Binder, E. M., Blackman, M. J., Carruthers, V. B. and Kim, K. (2001). A conserved subtilisin-like protein TgSUB1 in microneme organelles of Toxoplasma gondii. The Journal of Biological Chemistry 276, 45341–45348.Google Scholar
Miller, S. A., Thathy, V., Ajioka, J. W., Blackman, M. J. and Kim, K. (2003). TgSUB2 is a Toxoplasma gondii rhoptry organelle processing proteinase. Molecular Microbiology 49, 883–894.CrossRefGoogle ScholarPubMed
Min, D. Y., Hyun, K. H., Ryu, J. S., Ahn, M. H. and Cho, M. H. (1998). Degradations of human immunoglobulins and hemoglobin by a 60 kDa cysteine proteinase of Trichomonas vaginalis. The Korean Journal of Parasitology 36, 261–268.Google Scholar
Moon, E. K., Lee, S. T., Chung, D. I. and Kong, H. H. (2006). Intracellular localization and trafficking of serine proteinase AhSub and cysteine proteinase AhCP of Acanthamoeba healyi. Eukaryotic Cell 5, 125–131.Google Scholar
Mundodi, V., Kucknoor, A. S. and Alderete, J. F. (2008). Immunogenic and plasminogen-binding surface-associated alpha-enolase of Trichomonas vaginalis. Infection and Immunity 76, 523–531.CrossRefGoogle ScholarPubMed
Nielsen, H., Engelbrecht, J., Brunak, S. and Von, H. G. (1997). Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Engineering 10, 1–6.Google Scholar
Nonaka, T., Fujihashi, M., Kita, A., Saeki, K., Ito, S., Horikoshi, K. and Miki, K. (2004). The crystal structure of an oxidatively stable subtilisin-like alkaline serine protease, KP-43, with a C-terminal beta-barrel domain. The Journal of Biological Chemistry 279, 47344–47351.Google Scholar
Ong, S. J., Hsu, H. M., Liu, H. W., Chu, C. H. and Tai, J. H. (2006). Multifarious transcriptional regulation of adhesion protein gene ap65-1 by a novel Myb1 protein in the protozoan parasite Trichomonas vaginalis. Eukaryotic Cell 5, 391–399.Google Scholar
Ong, S. J., Hsu, H. M., Liu, H. W., Chu, C. H. and Tai, J. H. (2007). Activation of multifarious transcription of an adhesion protein ap65-1 gene by a novel Myb2 protein in the protozoan parasite Trichomonas vaginalis. The Journal of Biological Chemistry 282, 6716–6725.CrossRefGoogle ScholarPubMed
Ong, S. J., Huang, S. C., Liu, H. W. and Tai, J. H. (2004). Involvement of multiple DNA elements in iron-inducible transcription of the ap65-1 gene in the protozoan parasite Trichomonas vaginalis. Molecular Microbiology 52, 1721–1730.CrossRefGoogle ScholarPubMed
Provenzano, D. and Alderete, J. F. (1995). Analysis of human immunoglobulin-degrading cysteine proteinases of Trichomonas vaginalis. Infection and Immunity 63, 3388–3395.Google Scholar
Rholam, M. and Fahy, C. (2009). Processing of peptide and hormone precursos at the dibasic cleavage sites. Cellular and Molecular Life Sciences 66, 2075–2091.Google Scholar
Sajid, M., Withers-Martinez, C. and Blackman, M. J. (2000). Maturation and specificity of Plasmodium falciparum subtilisin-like protease-1, a malaria merozoite subtilisin-like serine protease. The Journal of Biological Chemistry 275, 631–641.Google Scholar
Siezen, R. J. and Leunissen, J. A. (1997). Subtilases: the superfamily of subtilisin-like serine proteases. Protein Science 6, 501–523.Google Scholar
Sippl, M. J. (1993). Recognition of errors in three-dimensional structures of proteins. Proteins 17, 355–362.CrossRefGoogle ScholarPubMed
Sommer, U., Costello, C. E., Hayes, G. R., Beach, D. H., Gilbert, R. O., Lucas, J. J. and Singh, B. N. (2005). Identification of Trichomonas vaginalis cysteine proteases that induce apoptosis in human vaginal epithelial cells. The Journal of Biological Chemistry 280, 23853–23860.Google Scholar
St Leger, R. J., Joshi, L., Roberts, D. W. (1997). Adaptation of proteases and carbohydrases of saprophytic, phytopatogenic fungi to the requeriments of their ecological niches. Microbiology 143, 1983–1992.CrossRefGoogle Scholar
Subbian, E., Yabuta, Y. and Shinde, U. (2004). Positive selection dictates the choice between kinetic and thermodynamic protein folding and stability in subtilases. Biochemistry 43, 14348–14360.CrossRefGoogle ScholarPubMed
van der Hoorn, R. A. L. (2008). Plant Proteases: From Phenotypes to Molecular Mechanisms. Annual Review of Plant Biology 59, 191–223.CrossRefGoogle ScholarPubMed
Van Der Pol, B., Kwok, C., Pierre-Louis, B., Rinaldi, A., Salata, R. A., Chen, P. L., Van De Wijgert, J., Mmiro, F., Mugerwa, R., Chipato, T. and Morrison, C. S. (2008). Trichomonas vaginalis infection and human immunodeficiency virus acquisition in African women. The Journal of Infectious Diseases 197, 548–554.Google Scholar
Viikki, M., Pukkala, E., Nieminen, P. and Hakama, M. (2000). Gynaecological infections as risk determinants of subsequent cervical neoplasia. Acta Oncologica 39, 71–75.Google Scholar
Wiederstein, M. and Sippl, M. J. (2007). ProSA-web: interactive web service for the recognition of errors in three-dimensional structures of proteins. Nucleic Acids Research 35, W407–W410.CrossRefGoogle ScholarPubMed
Wilson, M. E. and Britigan, B. E. (1998). Iron acquisition by parasitic protozoa. Parasitology Today 14, 348–353.Google Scholar
Withers-Martinez, C., Jean, L. and Blackman, M. J. (2004). Subtilisin-like proteases of the malaria parasite. Molecular Microbiology 53, 55–63.CrossRefGoogle ScholarPubMed