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

Yıl 2013, , 215 - 224, 01.02.2013

Ece Şen Leonard H. Sigal

https://doi.org/10.5152/balkanmedj.2013.7059

Öz

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.

Anahtar Kelimeler

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

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