Engineering recombinant lubricin to combat orthopedic infection

项目总结/摘要 该申请寻求资金支持NIAMS K 08收件人在她过渡到研究独立。 广泛的目标是研究润滑素如何减弱骨科生物膜，并确定是否有不同的生物膜。 润滑素O-聚糖介导这些抗生物膜性质。许多骨科植入物相关的能力 细菌形成生物膜使得这些生物体对抗微生物治疗具有抗性。一旦建立，植入- 相关的生物膜通常需要清创术、植入物交换或植入物移除，并可导致严重的 功能恶化需要关节融合术甚至截肢。因此，预防和治疗 骨科相关的生物膜已被确定为肌肉骨骼感染的关键研究重点。 最近的工作已经确定粘蛋白生物聚合物作为生物膜组装和毒力基因的有效拮抗剂 表达，提高了重组润滑素治疗可能是一种有效的策略， 临床相关生物膜。我们的初步数据表明，重组润滑素的发展，作为一部分， 申请人的K 08研究不仅抑制生物膜形成，而且还消除了已建立的铜绿假单胞菌 体外生物膜。在目前的建议中，我们将调查的假设，表面的双重性质， 粘附（N-和C-末端结构域）和抗粘附（粘蛋白O-聚糖结构域）使润滑素成为理想的抗粘附剂。 生物被膜剂在目标1中，我们将研究润滑素抑制生物膜形成的机制， 诱导已形成的生物膜在生长介质、滑液和钛合金（Ti6 Al 4V）中分散。在 目的2，我们将确定不同的O-聚糖是否介导这些抗生物膜功能，以及润滑素 可以通过糖工程优化功能。作为目标2的一部分，我们将操作表达式 的关键糖基转移酶，以产生具有改变的核心-1，核心-2和核心-3组成的重组润滑素。 2和唾液酸化的O-聚糖结构。 该提议建立在申请人的K 08研究的基础上，在该研究中，润滑素已经被主要研究为一种抗氧化剂。 用于治疗创伤后骨关节炎的摩擦剂。通过研究一种新的抗生物膜应用， 对于润滑素治疗，该奖项将使Reesink博士能够分支到骨科研究的一个新的子学科 具有与申请人在小型和大型动物模型中的临床兽医专业知识相结合的潜力 骨科感染Reesink博士的建议得到了一个特殊团队的支持，其中包括 糖工程，细菌-表面相互作用的分子表征，以及 假体周围关节感染和临床平移。

PROJECT SUMMARY / ABSTRACT This application seeks funding to support a NIAMS K08 recipient during her transition to research independence. The broad objective is to investigate how lubricin attenuates orthopedic biofilms and to determine whether distinct lubricin O-glycans mediate these anti-biofilm properties. The ability of many orthopedic implant-associated bacteria to form biofilms renders these organisms resistant to antimicrobial therapy. Once established, implant- associated biofilms typically require debridement, implant exchange or implant removal and can lead to severe functional deterioration necessitating arthrodesis or even amputation. Therefore, prevention and treatment of orthopedic-associated biofilms has been identified as a critical research priority in musculoskeletal infection. Recent work has identified mucin biopolymers as potent antagonists of biofilm assembly and virulence gene expression, raising the possibility that recombinant lubricin therapeutics could be an effective strategy for clinically relevant biofilms. Our preliminary data demonstrate that the recombinant lubricin developed as part of the applicant’s K08 research not only inhibits biofilm formation but also dissipates established P. aeruginosa biofilms in vitro. In the current proposal, we will investigate the hypothesis that the dual properties of surface adhesion (N- and C-terminal domains) and anti-adhesion (mucin O-glycan domains) make lubricin an ideal anti- biofilm agent. In Aim 1, we will investigate the mechanisms by which lubricin inhibits biofilm formation and induces dispersion of established biofilms in growth media, in synovial fluid, and on titanium alloy (Ti6Al4V). In Aim 2, we will determine whether distinct O-glycans mediate these anti-biofilm functions and whether lubricin functionality could be optimized through glycoengineering. As part of Aim 2, we will manipulate the expression of key glycosyltransferase enzymes to produce recombinant lubricin with altered compositions of Core-1, Core- 2 and sialylated O-glycan structures. This proposal builds upon the applicant’s K08 research, in which lubricin has been primarily investigated as a tribological agent for the treatment of post-traumatic osteoarthritis. By investigating a new, anti-biofilm application for lubricin therapy, this award will enable Dr. Reesink to branch into a new sub-discipline of orthopedic research with the potential to integrate with the applicant’s clinical veterinary expertise in small and large animal models of orthopedic infection. Dr. Reesink’s proposal is supported by an exceptional team, including experts in glycoengineering, molecular characterization of bacteria-surface interactions, and in vivo models of periprosthetic joint infection and clinical translation.