Engineering recombinant lubricin to combat orthopedic infection

项目摘要/摘要 这项申请寻求资金，以支持一位NIAMS K08获奖者在过渡到研究独立的过程中。 广泛的目标是调查润滑素是如何减弱骨科生物膜的，并确定不同的 润滑素O-葡聚糖介导了这些抗生物被膜特性。许多骨科植入物相关能力 细菌形成生物膜，使这些微生物对抗菌治疗产生抗药性。一旦确定，植入- 相关的生物膜通常需要清创、植入物交换或植入物移除，并可能导致严重的 功能恶化需要进行关节融合术或甚至截肢。因此，预防和治疗该病 骨科相关生物膜已被确定为肌肉骨骼感染的关键研究重点。 最近的工作发现，粘蛋白生物聚合物是生物膜组装和毒力基因的有效拮抗剂。 表达，增加了重组润滑素疗法可能是一种有效的策略 临床上相关的生物膜。我们的初步数据表明，重组润滑剂是作为 申请人的K08研究不仅抑制了生物膜的形成，而且还驱散了已建立的铜绿假单胞菌 体外生物膜。在目前的方案中，我们将研究曲面的对偶性质的假设 粘附性(N-和C-端域)和抗粘附性(粘蛋白O-糖域)使润滑剂成为理想的抗粘附剂 生物膜剂。在目标1中，我们将研究润滑素抑制生物膜形成和 导致已建立的生物膜在生长介质、滑液和钛合金(Ti6Al4V)上分散。在……里面 目的2，我们将确定不同的O-糖链是否介导这些抗生物膜功能，以及润滑剂是否 通过糖工程可以优化功能。作为目标2的一部分，我们将操作该表达式 产生重组润滑油的关键糖基转移酶，其中Core-1、Core-1、Core-1 2和唾液酸化的O-糖链结构。 这项建议建立在申请人的K08研究基础上，在该研究中，润滑剂主要是作为一种 用于治疗创伤后骨关节炎的摩擦学试剂。通过研究一种新的抗生物被膜应用 在润滑剂治疗方面，这一奖项将使里辛克博士能够进入整形外科研究的一个新的分支学科 有可能与申请人在小动物和大动物模型方面的临床兽医专业知识相结合 整形外科感染。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，以及活体模型 假体周围关节感染与临床转位。

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.