Enhanced Adhesion and OspC Protein Synthesis of the Lyme Disease Spirochete Borrelia Burgdorferi Cultivated in a Host-Derived Tissue Co-Culture System

Abstract

Background: The adhesion process of Borrelia burgdorferi to susceptible host cell has not yet been completely understood regarding the function of OspA, OspB and OspC proteins and a conflict exists in the infection process. Aims: The adhesion rates of pathogenic (low BSK medium passaged or susceptible rat joint tissue co-cultivated ) or non-pathogenic Borrelia burgdorferi (high BSK medium passaged) isolate (FNJ) to human umbilical vein endothelial cells (HUVEC) cultured on coverslips and the synthesis of OspA and OspC proteins were investigated to analyze the infection process of this bacterium. Study Design: In-vitro study. Methods: Spirochetes were cultured in BSK medium or in a LEW/N rat tibiotarsal joint tissue feeder layer supported co-culture system using ESG co-culture medium and labelled with 3H-adenine for 48 hours. SDS-PAGE, Western Blotting, Immunogold A labeling as well as radiolabeling experiments were used to compare pathogenic or non pathogenic spirochetes during the adhesion process. Results: Tissue co-cultured B. burgdorferi adhered about ten times faster than BSK-grown spirochetes. Trypsin inhibited attachment to HUVEC and co-culture of trypsinized spirochetes with tissues reversed the inhibition. Also, the synthesis of OspC protein by spirochetes was increased in abundance after tissue co-cultures, as determined by SDS-PAGE and by electron microscopy analysis of protein A-immunogold staining by anti-OspC antibodies. OspA protein was synthesized in similar quantities in all Borrelia cultures analyzed by the same techniques. Conclusion: Low BSK passaged or tissue co-cultured pathogenic Lyme disease spirochetes adhere to HUVEC faster than non-pathogenic high BSK passaged forms of this bacterium. Spirochetes synthesized OspC protein during host tissue-associated growth. However, we did not observe a reduction of OspA synthesis during host tissue co-cultivation in vitro. Turkish Başlık: Konak kökenli Doku Ko-kültivasyon Sisteminde Üretilen Lyme Hastalığı Spiroketi Borrelia burgdorferi'de Adezyon ve OspC Proteini Sentezinin Artışı Anahtar Kelimeler: Adezyon, Borrelia, OspC, patogenez, hücre kültürü, HUVEC Arka Plan: OspA, OspB ve OspC proteinlerinin B.burgdorferi'nin konak hücrelerine adezyonundaki rolü henüz tamamen anlaşılamamıştır ve infeksiyon mekanizması tartışmalıdır. Amaç: B.burgdorferi'nin infeksiyon mekanizmasını analize etmek amacıyla bu bakterinin patojenik (BSK besiyerinde az pasajlı veya duyarlı rat eklem doku kültüründe ko-kültivasyonlu) veya patojen olmayan (BSK besiyerinde çok pasajlı) bir izolatının (FNJ) mikroskop lamelleri üzerinde üretilmiş insan umbilikal ven hücrelerine (HUVEC) adezyon hızı ve OspA ve OspC proteinlerinin sentezi araştırıldı. Çalışma Tasarımı: Spiroketler BSK besiyerinde veya LEW/N rat tibiyotarsal eklem doku besleyici tabakası ve ESG besiyeri içeren ko-kültivasyon sisteminde üretildi ve 3H-adenin ile 48 saat işaretlendi. Yöntemler: Patojen olan ve patojen olmayan spiroketlerin adezyonlarının karşılaştırılması için SDS-PAGE, Western Blotting, Immunogold A işaretlemesi ve ayrıca radyoaktif işaretleme yöntemleri kullanıldı. Bulgular: Doku ko-kültüründe üretilen B. burgdorferi , BSK besiyerinde üretilmiş spiroketlerden 10 kat hızlı adezyon özelliği gösterdi. Tripsin uygulaması spiroketlerin HUVEC tabakasına yapışmasını önledi ve tripsinize edilmiş spiroketlerin tekrar doku kültüründe üretilmesi bu inhibisyonu ortadan kaldırdı. Ayrıca, spiroketlerin OspC proteininin üretiminin doku ko-kültivasyonundan sonra oldukça arttığı SDS-PAGE ve proteinA-immunogold işaretlemesi ve anti-OspC antikorları kullanılarak yapılan elektron mikroskopu incelemeleriyle saptandı. Aynı tekniklerle yapılan analizlerde tüm Borrelia kültürlerinde OspA proteininin eşit miktarlarda sentezlendiği belirlendi. Sonuç: BSK besiyerinde az pasajlı veya doku ko-kültüründe üretilerek patojenliği korunmuş Lyme hastalığı spiroketleri HUVEC tabakasına yüksek BSK pasajlı, patojenliğini kaybetmiş spiroketlerden daha hızlı yapıştılar. Spiroketler konak dokuları ile birlikte üretildiklerinde OspC proteini sentezlediler. Ancak, in vitro konak doku ko-kültürlerinde üretilen spiroketlerde OspA sentezinde azalma gözlemlenmedi.

Keywords

• Adhesion, Borrelia burgdorferi, OspA, OspC, pathogenicity, cell culture, HUVEC

Enhanced Adhesion and OspC Protein Synthesis of the Lyme Disease Spirochete Borrelia Burgdorferi Cultivated in a Host-Derived Tissue Co-Culture System

Öz

Kaynakça

1. Burkot TR, Piesman J, Wirtz RA. Quantitation of the Borrelia burgdorferi outer surface protein A in Ixodes scapularis: fluctuations during the tick life cycle, doubling times, and loss while feeding. J Inf Dis 1994;170:883-9. [CrossRef]
2. Coleman JL, Gebbia JA, Piesman J, Degen JL, Bugge TH, Benach JL. Plasminogen is required for efficient dissemination of B.burgdorferi in ticks and for enhancement of spirochetemia in mice. Cell 1997;89:1111-9. [CrossRef]
3. De Silva AM, Fikrig EGrowth and migration of Borrelia burgdorferi in Ixodes ticks during blood feeding. Am J Trop Med Hyg 1995;53:397-404. de Silva AM, Telford SR 3rd, Brunet LR, Barthold SW, Fikrig E. Borrelia burgdorferi OspA is an arthropod-specific transmission-blocking Lyme disease vaccine. J Exp Med 1996;183:271-5. [CrossRef]
4. Roehrig JT, Piesman J, Hunt AR, Keen MG, Happ CM, Johnson BJ. The hamster immune response to tick-transmitted B.burgdorferi differs from the response to needle-inoculated, cultured organisms. J Immunol 1992;149:3648-53.
5. Masuzawa T, Kurita T, Kawabata H, Yanagihara Y. Relationship between infectivity and OspC expression in Lyme disease Borrelia. FEMS Microbiol Lett 1994;123:319-24. [CrossRef]
6. Grimm D, Tilly K, Byram R, Stewart PE, Krum JG, Bueschel DM, et al. Outer-surface protein C of the Lyme disease spirochete: a protein induced in ticks for infection of mammals. Proc Natl Acad Sci U S A 2004;101:3142-7. [CrossRef]
7. Mulay VB, Caimano MJ, Iyer R, Dunham-Ems S, Liveris D, Petzke MM, et al. Borrelia burgdorferi bba74 is expressed exclusively during tick feeding and is regulated by both arthropod- and mammalian host-specific signals. J Bacteriol 2009;191:2783-94. [CrossRef] Brissette CA, Bykowski T, Cooley AE, Bowman A, Stevenson B. Borrelia burgdorferi RevA antigen binds host fibronectin. Infect Immun 2009;77:2802-12. [CrossRef]
8. Angel TE, Luft BJ, Yang X, Nicora CD, Camp DG 2nd, Jacobs JM, et al. Proteome analysis of Borrelia burgdorferi response to environmental change. PLoS One 2010;5:e13800. [CrossRef]

9. Güner ES. Complement evasion by the Lyme disease spirochete Borrelia burgdorferi grown in host-derived tissue co-cultures: role of fibronectin in complement-resistance. Experientia 1996;52:364-72. [CrossRef]
10. Comstock LE, Thomas DD. Penetration of endothelial cell monolayers by Borrelia burgdorferi. Infect Immun 1989;57:1626-8.
11. Thomas DD, Comstock LE. Interaction of Lyme disease spirochetes with cultured eucaryotic cells. Infect Immun 1989;57:1324-26.
12. Steiner BM, Sell S. Characterization of the interaction between fibronectin and T pallidum. Curr Microbiol 1985;12:157-62. [CrossRef]
13. Comstock LE, Thomas DD. Characterization of B.burgdorferi invasion of cultured endothelial cells. Microb Patho 1991;10:137-48. [CrossRef] Ma Y, Sturrock A, Weis JJ. Intracellular localization of Borrelia burgdorferi within human endothelial cells. Infect Immun 1991;59:671-8.
14. Montgomery RR, Malawista SE, Feen KJ, Bockenstedt LK. Direct demonstration of antigenic substitution of Borrelia burgdorferi ex vivo: exploration of the paradox of the early immune response to outer surface proteins A and C in Lyme disease. J Exp Med 1996;183:261-9. [CrossRef]
15. Güner ES. Retention of B.burgdorferi pathogenicity and infectivity after multiple passages in a co-culture system. Experientia 1994;50:54-9. [CrossRef]
16. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248-54. [CrossRef]
17. Beachey EH. Bacterial adherence: Adhesin-receptor interactions mediating the attachment of bacteria to mucosal surfaces. J Infect Dis 1981;143:325-45. [CrossRef]
18. Johnson RC, Marek N, Kodner C. Infection of Syrian hamsters with Lyme disease spirochetes. J Clin Microbiol 1984;20:1099-101.
19. Schwan TG, Burgdorfer W, Garon CF. Changes in infectivity and plasmid profile of the Lyme disease spirochete, Borrelia burgdorferi, as a result of in vitro cultivation. Infect Immun 1988;56:1831-6. Alderete JF, Baseman JB. Surface-associated host proteins on virulent Treponema pallidum. Infect Immun 1979;26:1048-56.
20. Peterson KM, Baseman JB, Alderete JF. Treponema pallidum receptor binding proteins interact with fibronectin. J Exp Med 1983;157:1958-70. [CrossRef]
21. Thomas DD, Baseman JB, Alderete JF. Fibronectin mediates Treponema pallidum cytadherence through recognition of fibronectin cell-binding domain. J Exp Med 1985;161:514-25. [CrossRef]
22. Thomas DD, Baseman JB, Alderete JF. Enhanced levels of attachment of fibronectin-primed Treponema pallidum to extracellular matrix. Infect Immun 1986;52:736-41.
23. Moroni A, Sambri V, Massaria F, La Placa M Jr, Brocchi E, De Simone F, et al. Differential cleavage of surface proteins of B.burgdorferi by proteases. Microbiologica 1992;15:99-106.
24. Kurtti TJ, Munderloh UG, Krueger DE, Johnson RC, Schwan TG. Adhesion to and invasion of cultured tick (Acarina: Ixodidae) cells by Borrelia burgdorferi (Spirochaetales: Spirochaetaceae) and maintenance of infectivity. J Med Entomol 1993;30:586-96.
25. Gilmore RD Jr, Piesman J. Inhibition of Borrelia burgdorferi migration from the midgut to the salivary glands following feeding by ticks on OspC-immunized mice. Infect Immun 2000;68:411-4. [CrossRef]
26. Fingerle V, Liegl G, Munderloh U, Wilske B. Expression of outer surface proteins A and C of B.burgdorferi in I.ricinus ticks removed from humans. Med Microbiol Immunol 1998;187:121-6. [CrossRef]
27. Fingerle V, Laux H, Munderloh UG, Schulte-Spechtel U, Wilske B. Differential expression of outer surface proteins A and C by individual Borrelia burgdorferi in different genospecies. Med Microbiol Immunol 2000;189:59-66. [CrossRef]
28. Norman MU, Moriarty TJ, Dresser AR, Millen B, Kubes P, Chaconas G. Molecular mechanisms involved in vascular interactions of the Lyme disease pathogen in a living host. PLoS Pathog 2008;4:169-74. [CrossRef]
29. Terekhova D, Iyer R, Wormser GP, Schwartz I. Comparative genome hybridization reveals substantial variation among clinical isolates of Borrelia burgdorferi sensu stricto with different pathogenic properties. J Bacteriol 2006;188:6124-34. [CrossRef]
30. Schwan TG, Piesman J. Temporal changes in outer surface proteins A and C of the lyme disease-associated spirochete, Borrelia burgdorferi, during the chain of infection in ticks and mice. J Clin Microbiol 2000;38:382-8.
31. Crother TR, Champion CI, Whitelegge JP, Aguilera R, Wu XY, Blanco DR, et al. Temporal analysis of the antigenic composition of Borrelia burgdorferi during infection in rabbit skin. Infect Immun 2004;72:5063-72. [CrossRef]
32. Liang FT, Yan J, Mbow ML, Sviat SL, Gilmore RD, Mamula M, et al. Borrelia burgdorferi changes its surface antigenic expression in response to host immune responses. Infect Immun 2004;72:5759-67. [CrossRef]
33. Tilly K, Krum JG, Bestor A, Jewett MW, Grimm D, Bueschel D, et al. Borrelia burgdorferi OspC protein required exclusively in a crucial early stage of mammalian infection. Infect Immun 2006;74:3554-64. [CrossRef]
34. Seinost G, Dykhuizen DE, Dattwyler RJ, Golde WT, Dunn JJ, Wang IN, et al. Four clones of B. burgdorferi sensu stricto cause invasive infection in humans. Infect Immun 1999;67:3518-24.
35. Lagal V, Portnoï D, Faure G, Postic D, Baranton G. Borrelia burgdorferi sensu stricto invasiveness is correlated with OspC-plasminogen affinity. Microbes Infect 2006;8:645-52. [CrossRef]
36. Alghaferi MY, Anderson JM, Park J, Auwaerter PG, Aucott JN, Norris DE, et al. Borrelia burgdorferi ospC heterogeneity among human and murine isolates from a defined region of northern Maryland and southern Pennsylvania: lack of correlation with invasive and noninvasive genotypes. J Clin Microbiol 2005;43:1879-84. [CrossRef]
37. Earnhart CG, Buckles EL, Dumler JS, Marconi RT. Demonstration of OspC type diversity in invasive human Lyme disease isolates and identification of previously uncharacterized epitopes that define the specificity of the OspC murine antibody response. Infect Immun 2005;73:7869-77. [CrossRef]
38. Fuchs H, Wallich R, Simon MM, Kramer MD. The outer surface protein A of the spirochete B. burgdorferi is a plasmin(ogen) receptor. Proc Natl Acad Sci USA 1994;91:12594-8. [CrossRef]
39. Hu LT, Perides G, Noring R, Klempner MS. Binding of human plasminogen to Borrelia burgdorferi. Infect Immun 1995;63:3491-6.
40. Batsford S, Rust C, Neubert U. Analysis of antibody response to the outer surface family in Lyme borreliosis patients. J Infect Dis 1998;178:1676-83. [CrossRef]
41. Bockenstedt LK, Hodzic E, Feng S, Bourrel KW, de Silva A, Montgomery RR, et al. Borrelia burgdorferi strain-specific Osp C-mediated immunity in mice. Infect Immun 1997;65:4661-71.
42. Garcia-Monco JC, Fernandez-Villar B, Benach JL. Adherence of the Lyme disease spirochete to glial cells and cells of glial origin. J Infect Dis 1989;160:497-506. [CrossRef]
43. Hindersson P, Thomas D, Stamm L, Penn C, Norris S, Joens LA. Interaction of spirochetes with the host. Res Microbiol 1992;143:629-39. [CrossRef]
44. Ohnishi J, Piesman J, de Silva AM. Antigenic and genetic heterogeneity of B.burgdorferi populations transmitted by ticks. Proc Nat Acad Sci 2001;98:670-5. [CrossRef]
45. Rosa PE, Schwan T, Hogan D. Recombination between genes encoding major outer surface proteins A and B of Borrelia burgdorferi. Mol Microbiol 1992;6:3031-40. [CrossRef]
46. Schutzer SE, Coyle PK, Krupp LB, Deng Z, Belman AL, Dattwyler R, et al. Simultaneous expression of Borrelia OspA and OspC and IgM response in cerebrospinal fluid in early neurologic Lyme disease. J Clin Invest 1997;100:763-7. [CrossRef] 5 Seemanapalli SV, Xu Q, McShan K, Liang FT. Outer Surface Protein C Is a Dissemination-Facilitating Factor of B. burgdorferi during Mammalian Infection . PLoS One 2010;5:e15830. [CrossRef] 5 Schmit VL, Patton TG, Gilmore RD Jr. Analysis of Borrelia burgdorferi Surface Proteins as Determinants in Establishing Host Cell Interactions. Front Microbiol 2011;2:141. [CrossRef] 5 Comstock LE, Fikrig E, Shoberg RJ, Flavell RA, Thomas DD. A monoclonal antibody to OspA inhibits association of B. burgdorferi with human endothelial cells. Infect Immun 1993;61:423-31. 5 Schwan TG, Piesman J. Temporal changes in outer surface proteins A and C of the lyme disease-associated spirochete, Borrelia burgdorferi, during the chain of infection in ticks and mice. J Clin Microbiol 2000;38:382-8. 5 Ouyang Z, Narasimhan S, Neelakanta G, Kumar M, Pal U, Fikrig E, et al. Activation of the RpoN-RpoS regulatory pathway during the enzootic life cycle of Borrelia burgdorferi. BMC Microbiol 2012;12:44. [CrossRef] 5 Tilly K, Krum JG, Bestor A, Jewett MW, Grimm D, Bueschel D, et al. Borrelia burgdorferi OspC protein required exclusively in a crucial early stage of mammalian infection. Infect Immun 2006;74:3554-64. [CrossRef] 5 Bockenstedt LK, Hodzic E, Feng S, Bourrel KW, de Silva A, Montgomery RR, et al. Borrelia burgdorferi strain-specific Osp C-mediated immunity in mice. Infect Immun 1997;65:4661-7.