1887

Abstract

Leptospirosis, a worldwide zoonotic infection, is an important human and veterinary health problem. We have previously identified a leptospiral multipurpose adhesin, Lsa66, capable of binding extracellular matrix (ECM) components and plasminogen (PLG). In this work, we report the cloning, expression, purification and characterization of three fragments derived from the full-length Lsa66: N-terminal, intermediate and C-terminal regions. We employed BL21-SI as expression cells. The recombinant fragments tagged with N-terminal His were purified by metal-charged chromatography to major protein bands that were recognized by anti-His-tag mAbs. The recombinant fragments were evaluated for their capacity to attach to ECM components and to PLG. The intermediate region bound to laminin, plasma fibronectin and PLG. Laminin also bound to the C-terminal region. Antibodies in leptospirosis-positive serum samples recognized Lsa66, being the immune epitopes located at the N-terminal and intermediate fragments. The data confirm that Lsa66 is expressed during infection and that this protein might have a role in bacterial infection.

Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.078378-0
2014-09-01
2019-11-23
Loading full text...

Full text loading...

/deliver/fulltext/jmm/63/9/1119.html?itemId=/content/journal/jmm/10.1099/jmm.0.078378-0&mimeType=html&fmt=ahah

References

  1. Atzingen M. V. , Barbosa A. S. , De Brito T. , Vasconcellos S. A. , de Morais Z. M. , Lima D. M. , Abreu P. A. , Nascimento A. L. . ( 2008; ). Lsa21, a novel leptospiral protein binding adhesive matrix molecules and present during human infection. . BMC Microbiol 8:, 70. [CrossRef] [PubMed]
    [Google Scholar]
  2. Atzingen M. V. , Gómez R. M. , Schattner M. , Pretre G. , Gonçales A. P. , de Morais Z. M. , Vasconcellos S. A. , Nascimento A. L. . ( 2009; ). Lp95, a novel leptospiral protein that binds extracellular matrix components and activates e-selectin on endothelial cells. . J Infect 59:, 264–276. [CrossRef] [PubMed]
    [Google Scholar]
  3. Atzingen M. V. , Vieira M. L. , Oliveira R. , Domingos R. F. , Mendes R. S. , Barros A. T. , Gonçales A. P. , de Morais Z. M. , Vasconcellos S. A. , Nascimento A. L. . ( 2012; ). Evaluation of immunoprotective activity of six leptospiral proteins in the hamster model of leptospirosis. . Open Microbiol J 6:, 79–87. [CrossRef] [PubMed]
    [Google Scholar]
  4. Barbosa A. S. , Abreu P. A. , Neves F. O. , Atzingen M. V. , Watanabe M. M. , Vieira M. L. , Morais Z. M. , Vasconcellos S. A. , Nascimento A. L. . ( 2006; ). A newly identified leptospiral adhesin mediates attachment to laminin. . Infect Immun 74:, 6356–6364. [CrossRef] [PubMed]
    [Google Scholar]
  5. Bulach D. M. , Zuerner R. L. , Wilson P. , Seemann T. , McGrath A. , Cullen P. A. , Davis J. , Johnson M. , Kuczek E. . & other authors ( 2006; ). Genome reduction in Leptospira borgpetersenii reflects limited transmission potential. . Proc Natl Acad Sci U S A 103:, 14560–14565. [CrossRef] [PubMed]
    [Google Scholar]
  6. Carvalho E. , Barbosa A. S. , Gómez R. M. , Cianciarullo A. M. , Hauk P. , Abreu P. A. , Fiorini L. C. , Oliveira M. L. , Romero E. C. . & other authors ( 2009; ). Leptospiral TlyC is an extracellular matrix-binding protein and does not present hemolysin activity. . FEBS Lett 583:, 1381–1385. [CrossRef] [PubMed]
    [Google Scholar]
  7. Chou L. F. , Chen Y. T. , Lu C. W. , Ko Y. C. , Tang C. Y. , Pan M. J. , Tian Y. C. , Chiu C. H. , Hung C. C. , Yang C.-W. . ( 2012; ). Sequence of Leptospira santarosai serovar Shermani genome and prediction of virulence-associated genes. . Gene 511:, 364–370. [CrossRef] [PubMed]
    [Google Scholar]
  8. Choy H. A. , Kelley M. M. , Chen T. L. , Møller A. K. , Matsunaga J. , Haake D. A. . ( 2007; ). Physiological osmotic induction of Leptospira interrogans adhesion: LigA and LigB bind extracellular matrix proteins and fibrinogen. . Infect Immun 75:, 2441–2450. [CrossRef] [PubMed]
    [Google Scholar]
  9. Coleman J. L. , Sellati T. J. , Testa J. E. , Kew R. R. , Furie M. B. , Benach J. L. . ( 1995; ). Borrelia burgdorferi binds plasminogen, resulting in enhanced penetration of endothelial monolayers. . Infect Immun 63:, 2478–2484.[PubMed]
    [Google Scholar]
  10. Domingos R. F. , Vieira M. L. , Romero E. C. , Gonçales A. P. , de Morais Z. M. , Vasconcellos S. A. , Nascimento A. L. . ( 2012; ). Features of two proteins of Leptospira interrogans with potential role in host–pathogen interactions. . BMC Microbiol 12:, 50. [CrossRef] [PubMed]
    [Google Scholar]
  11. Faine S. , Adler B. , Bolin C. , Perolat P. . (editors) ( 1999; ). Leptospira and Leptospirosis. Melbourne:: MedSci;.
    [Google Scholar]
  12. Fenno J. C. , Tamura M. , Hannam P. M. , Wong G. W. , Chan R. A. , McBride B. C. . ( 2000; ). Identification of a Treponema denticola OppA homologue that binds host proteins present in the subgingival environment. . Infect Immun 68:, 1884–1892. [CrossRef] [PubMed]
    [Google Scholar]
  13. Fernandes L. G. , Vieira M. L. , Kirchgatter K. , Alves I. J. , de Morais Z. M. , Vasconcellos S. A. , Romero E. C. , Nascimento A. L. . ( 2012; ). OmpL1 is an extracellular matrix- and plasminogen-interacting protein of Leptospira spp. . Infect Immun 80:, 3679–3692. [CrossRef] [PubMed]
    [Google Scholar]
  14. Hauk P. , Macedo F. , Romero E. C. , Vasconcellos S. A. , de Morais Z. M. , Barbosa A. S. , Ho P. L. . ( 2008; ). In LipL32, the major leptospiral lipoprotein, the C terminus is the primary immunogenic domain and mediates interaction with collagen IV and plasma fibronectin. . Infect Immun 76:, 2642–2650. [CrossRef] [PubMed]
    [Google Scholar]
  15. Hoke D. E. , Egan S. , Cullen P. A. , Adler B. . ( 2008; ). LipL32 is an extracellular matrix-interacting protein of Leptospira spp. and Pseudoalteromonas tunicata . . Infect Immun 76:, 2063–2069. [CrossRef] [PubMed]
    [Google Scholar]
  16. Koizumi N. , Watanabe H. . ( 2003; ). Molecular cloning and characterization of a novel leptospiral lipoprotein with OmpA domain. . FEMS Microbiol Lett 226:, 215–219. [CrossRef] [PubMed]
    [Google Scholar]
  17. Lima S. S. , Ching A. T. , Fávaro R. D. , Da Silva J. B. , Oliveira M. L. , Carvalho E. , Abreu P. A. , Vasconcellos S. A. , Ho P. L. . ( 2013; ). Adhesin activity of Leptospira interrogans lipoprotein identified by in vivo and in vitro shotgun phage display. . Biochem Biophys Res Commun 431:, 342–347. [CrossRef] [PubMed]
    [Google Scholar]
  18. Lin Y. P. , Lee D. W. , McDonough S. P. , Nicholson L. K. , Sharma Y. , Chang Y. F. . ( 2009a; ). Repeated domains of leptospira immunoglobulin-like proteins interact with elastin and tropoelastin. . J Biol Chem 284:, 19380–19391. [CrossRef] [PubMed]
    [Google Scholar]
  19. Lin Y. P. , Greenwood A. , Yan W. , Nicholson L. K. , Sharma Y. , McDonough S. P. , Chang Y. F. . ( 2009b; ). A novel fibronectin type III module binding motif identified on C-terminus of Leptospira immunoglobulin-like protein, LigB. . Biochem Biophys Res Commun 389:, 57–62. [CrossRef] [PubMed]
    [Google Scholar]
  20. Longhi M. T. , Oliveira T. R. , Romero E. C. , Gonçales A. P. , de Morais Z. M. , Vasconcellos S. A. , Nascimento A. L. . ( 2009; ). A newly identified protein of Leptospira interrogans mediates binding to laminin. . J Med Microbiol 58:, 1275–1282. [CrossRef] [PubMed]
    [Google Scholar]
  21. Mendes R. S. , Von Atzingen M. , de Morais Z. M. , Gonçales A. P. , Serrano S. M. , Asega A. F. , Romero E. C. , Vasconcellos S. A. , Nascimento A. L. . ( 2011; ). The novel leptospiral surface adhesin Lsa20 binds laminin and human plasminogen and is probably expressed during infection. . Infect Immun 79:, 4657–4667. [CrossRef] [PubMed]
    [Google Scholar]
  22. Merien F. , Truccolo J. , Baranton G. , Perolat P. . ( 2000; ). Identification of a 36-kDa fibronectin-binding protein expressed by a virulent variant of Leptospira interrogans serovar icterohaemorrhagiae. . FEMS Microbiol Lett 185:, 17–22. [CrossRef] [PubMed]
    [Google Scholar]
  23. Nascimento A. L. , Verjovski-Almeida S. , Van Sluys M. A. , Monteiro-Vitorello C. B. , Camargo L. E. , Digiampietri L. A. , Harstkeerl R. A. , Ho P. L. , Marques M. V. . & other authors ( 2004a; ). Genome features of Leptospira interrogans serovar Copenhageni. . Braz J Med Biol Res 37:, 459–477. [CrossRef] [PubMed]
    [Google Scholar]
  24. Nascimento A. L. , Ko A. I. , Martins E. A. , Monteiro-Vitorello C. B. , Ho P. L. , Haake D. A. , Verjovski-Almeida S. , Hartskeerl R. A. , Marques M. V. . & other authors ( 2004b; ). Comparative genomics of two Leptospira interrogans serovars reveals novel insights into physiology and pathogenesis. . J Bacteriol 186:, 2164–2172. [CrossRef] [PubMed]
    [Google Scholar]
  25. Oliveira R. , Longhi M. T. , Gonçales A. P. , de Morais Z. M. , Vasconcellos S. A. , Nascimento A. L. . ( 2010; ). LipL53, a temperature regulated protein from Leptospira interrogans that binds to extracellular matrix molecules. . Microbes Infect 12:, 207–217. [CrossRef] [PubMed]
    [Google Scholar]
  26. Oliveira R. , de Morais Z. M. , Gonçales A. P. , Romero E. C. , Vasconcellos S. A. , Nascimento A. L. . ( 2011; ). Characterization of novel OmpA-like protein of Leptospira interrogans that binds extracellular matrix molecules and plasminogen. . PLoS ONE 6:, e21962. [CrossRef] [PubMed]
    [Google Scholar]
  27. Picardeau M. , Bulach D. M. , Bouchier C. , Zuerner R. L. , Zidane N. , Wilson P. J. , Creno S. , Kuczek E. S. , Bommezzadri S. . & other authors ( 2008; ). Genome sequence of the saprophyte Leptospira biflexa provides insights into the evolution of Leptospira and the pathogenesis of leptospirosis. . PLoS ONE 3:, e1607. [CrossRef] [PubMed]
    [Google Scholar]
  28. Pinne M. , Choy H. A. , Haake D. A. . ( 2010; ). The OmpL37 surface-exposed protein is expressed by pathogenic Leptospira during infection and binds skin and vascular elastin. . PLoS Negl Trop Dis 4:, e815. [CrossRef] [PubMed]
    [Google Scholar]
  29. Pinne M. , Matsunaga J. , Haake D. A. . ( 2012; ). Leptospiral outer membrane protein microarray, a novel approach to identification of host ligand-binding proteins. . J Bacteriol 194:, 6074–6087. [CrossRef] [PubMed]
    [Google Scholar]
  30. Ramos C. R. , Abreu P. A. , Nascimento A. L. , Ho P. L. . ( 2004; ). A high-copy T7 Escherichia coli expression vector for the production of recombinant proteins with a minimal N-terminal His-tagged fusion peptide. . Braz J Med Biol Res 37:, 1103–1109. [CrossRef] [PubMed]
    [Google Scholar]
  31. Ren S. X. , Fu G. , Jiang X. G. , Zeng R. , Miao Y. G. , Xu H. , Zhang Y. X. , Xiong H. , Lu G. . & other authors ( 2003; ). Unique physiological and pathogenic features of Leptospira interrogans revealed by whole-genome sequencing. . Nature 422:, 888–893. [CrossRef] [PubMed]
    [Google Scholar]
  32. Ricaldi J. N. , Fouts D. E. , Selengut J. D. , Harkins D. M. , Patra K. P. , Moreno A. , Lehmann J. S. , Purushe J. , Sanka R. . & other authors ( 2012; ). Whole genome analysis of Leptospira licerasiae provides insight into leptospiral evolution and pathogenicity. . PLoS Negl Trop Dis 6:, e1853. [CrossRef] [PubMed]
    [Google Scholar]
  33. Ristow P. , Bourhy P. , da Cruz McBride F. W. , Figueira C. P. , Huerre M. , Ave P. , Girons I. S. , Ko A. I. , Picardeau M. . ( 2007; ). The OmpA-like protein Loa22 is essential for leptospiral virulence. . PLoS Pathog 3:, e97. [CrossRef] [PubMed]
    [Google Scholar]
  34. Siemens N. , Patenge N. , Otto J. , Fiedler T. , Kreikemeyer B. . ( 2011; ). Streptococcus pyogenes M49 plasminogen/plasmin binding facilitates keratinocyte invasion via integrin-integrin-linked kinase (ILK) pathways and protects from macrophage killing. . J Biol Chem 286:, 21612–21622. [CrossRef] [PubMed]
    [Google Scholar]
  35. Smith S. G. , Mahon V. , Lambert M. A. , Fagan R. P. . ( 2007; ). A molecular Swiss army knife: OmpA structure, function and expression. . FEMS Microbiol Lett 273:, 1–11. [CrossRef] [PubMed]
    [Google Scholar]
  36. Souza N. M. , Vieira M. L. , Alves I. J. , de Morais Z. M. , Vasconcellos S. A. , Nascimento A. L. . ( 2012; ). Lsa30, a novel adhesin of Leptospira interrogans binds human plasminogen and the complement regulator C4bp. . Microb Pathog 53:, 125–134. [CrossRef] [PubMed]
    [Google Scholar]
  37. Stevenson B. , Choy H. A. , Pinne M. , Rotondi M. L. , Miller M. C. , Demoll E. , Kraiczy P. , Cooley A. E. , Creamer T. P. . & other authors ( 2007; ). Leptospira interrogans endostatin-like outer membrane proteins bind host fibronectin, laminin and regulators of complement. . PLoS ONE 2:, e1188. [CrossRef] [PubMed]
    [Google Scholar]
  38. Stie J. , Bruni G. , Fox D. . ( 2009; ). Surface-associated plasminogen binding of Cryptococcus neoformans promotes extracellular matrix invasion. . PLoS ONE 4:, e5780. [CrossRef] [PubMed]
    [Google Scholar]
  39. Turner L. H. . ( 1970; ). Leptospirosis III: maintenance, isolation and demonstration of leptospires. . Trans R Soc Trop Med Hyg 64:, 623–646. [CrossRef] [PubMed]
    [Google Scholar]
  40. Verma A. , Brissette C. A. , Bowman A. A. , Shah S. T. , Zipfel P. F. , Stevenson B. . ( 2010; ). Leptospiral endostatin-like protein A is a bacterial cell surface receptor for human plasminogen. . Infect Immun 78:, 2053–2059. [CrossRef] [PubMed]
    [Google Scholar]
  41. Vieira M. L. , Vasconcellos S. A. , Gonçales A. P. , de Morais Z. M. , Nascimento A. L. . ( 2009; ). Plasminogen acquisition and activation at the surface of leptospira species lead to fibronectin degradation. . Infect Immun 77:, 4092–4101. [CrossRef] [PubMed]
    [Google Scholar]
  42. Vieira M. L. , de Morais Z. M. , Gonçales A. P. , Romero E. C. , Vasconcellos S. A. , Nascimento A. L. . ( 2010a; ). Lsa63, a newly identified surface protein of Leptospira interrogans binds laminin and collagen IV. . J Infect 60:, 52–64. [CrossRef] [PubMed]
    [Google Scholar]
  43. Vieira M. L. , Atzingen M. V. , Oliveira T. R. , Oliveira R. , Andrade D. M. , Vasconcellos S. A. , Nascimento A. L. . ( 2010b; ). In vitro identification of novel plasminogen-binding receptors of the pathogen Leptospira interrogans . . PLoS ONE 5:, e11259. [CrossRef] [PubMed]
    [Google Scholar]
  44. Vieira M. L. , Atzingen M. V. , Oliveira R. , Mendes R. S. , Domingos R. F. , Vasconcellos S. A. , Nascimento A. L. . ( 2012; ). Plasminogen binding proteins and plasmin generation on the surface of Leptospira spp.: the contribution to the bacteria–host interactions. . J Biomed Biotechnol 2012:, 758513. [CrossRef] [PubMed]
    [Google Scholar]
  45. Vieira M. L. , Fernandes L. G. , Domingos R. F. , Oliveira R. , Siqueira G. H. , Souza N. M. , Teixeira A. R. , Atzingen M. V. , Nascimento A. L. . ( 2014; ). Leptospiral extracellular matrix adhesins as mediators of pathogen-host interactions. . FEMS Microbiol Lett 352:, 129–139. doi: 10.1111/1574-6968.12349.[CrossRef]
    [Google Scholar]
  46. Yan W. , Faisal S. M. , McDonough S. P. , Chang C. F. , Pan M. J. , Akey B. , Chang Y. F. . ( 2010; ). Identification and characterization of OmpA-like proteins as novel vaccine candidates for Leptospirosis. . Vaccine 28:, 2277–2283. [CrossRef] [PubMed]
    [Google Scholar]
  47. Zhang Y. , Bao L. , Zhu H. , Huang B. , Zhang H. . ( 2010; ). OmpA-like protein Loa22 from Leptospira interrogans serovar Lai is cytotoxic to cultured rat renal cells and promotes inflammatory responses. . Acta Biochim Biophys Sin (Shanghai) 42:, 70–79. [CrossRef] [PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/jmm.0.078378-0
Loading
/content/journal/jmm/10.1099/jmm.0.078378-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error