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

摘要

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 Ba?l?k: Konak k?kenli Doku Ko-kültivasyon Sisteminde üretilen Lyme Hastal??? Spiroketi Borrelia burgdorferi'de Adezyon ve OspC Proteini Sentezinin Art??? 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 anla??lamam??t?r ve infeksiyon mekanizmas? tart??mal?d?r. Ama?: B.burgdorferi'nin infeksiyon mekanizmas?n? analize etmek amac?yla bu bakterinin patojenik (BSK besiyerinde az pasajl? veya duyarl? rat eklem doku kültüründe ko-kültivasyonlu) veya patojen olmayan (BSK besiyerinde ?ok pasajl?) bir izolat?n?n (FNJ) mikroskop lamelleri üzerinde üretilmi? insan umbilikal ven hücrelerine (HUVEC) adezyon h?z? ve OspA ve OspC proteinlerinin sentezi ara?t?r?ld?. ?al??ma Tasar?m?: Spiroketler BSK besiyerinde veya LEW/N rat tibiyotarsal eklem doku besleyici tabakas? ve ESG besiyeri i?eren ko-kültivasyon sisteminde üretildi ve 3H-adenin ile 48 saat i?aretlendi. Y?ntemler: Patojen olan ve patojen olmayan spiroketlerin adezyonlar?n?n kar??la?t?r?lmas? i?in SDS-PAGE, Western Blotting, Immunogold A i?aretlemesi ve ayr?ca radyoaktif i?aretleme y?ntemleri kullan?ld?. Bulgular: Doku ko-kültüründe üretilen B. burgdorferi , BSK besiyerinde üretilmi? spiroketlerden 10 kat h?zl? adezyon ?zelli?i g?sterdi. Tripsin uygulamas? spiroketlerin HUVEC tabakas?na yap??mas?n? ?nledi ve tripsinize edilmi? spiroketlerin tekrar doku kültüründe üretilmesi bu inhibisyonu ortadan kald?rd?. Ayr?ca, spiroketlerin OspC proteininin üretiminin doku ko-kültivasyonundan sonra olduk?a artt??? SDS-PAGE ve proteinA-immunogold i?aretlemesi ve anti-OspC antikorlar? kullan?larak yap?lan elektron mikroskopu incelemeleriyle saptand?. Ayn? tekniklerle yap?lan analizlerde tüm Borrelia kültürlerinde OspA proteininin e?it miktarlarda sentezlendi?i belirlendi. Sonu?: BSK besiyerinde az pasajl? veya doku ko-kültüründe üretilerek patojenli?i korunmu? Lyme hastal??? spiroketleri HUVEC tabakas?na yüksek BSK pasajl?, patojenli?ini kaybetmi? spiroketlerden daha h?zl? yap??t?lar. Spiroketler konak dokular? ile birlikte üretildiklerinde OspC proteini sentezlediler. Ancak, in vitro konak doku ko-kültürlerinde üretilen spiroketlerde OspA sentezinde azalma g?zlemlenmedi.