Molecular Engineering of Humanized Anti-Staphlococcal Lytic Enzymes

描述（由申请人提供）：抗生素耐药性使大多数金黄色葡萄球菌（S。金黄色葡萄球菌）感染，作为整整三分之二的医院相关的S.金黄色葡萄球菌感染和~50%的那些在社区获得的现在是耐甲氧西林（MRSA）。耐多药发生率的增加是导致耐药的主要原因。金黄色葡萄球菌和其他细菌的感染强调了对能够对抗这些危险病原体的下一代抗生素的需求。理想情况下，新药不仅能有效治疗当前的耐药菌株，而且还能延缓新的耐药表型的发展。要做到这一点，新药必须通过与传统的抑制性抗生素正交的机制发挥作用。人类免疫系统已经进化出了强大的杀菌剂武器库，其中许多攻击细菌细胞结构的固有可塑性比常规抑制药物靶向的蛋白质和核糖核酸要小。一个例子是人溶菌酶（hLYS），其部分地通过水解细胞壁肽聚糖和随后的细菌裂解来杀死革兰氏阳性病原体。虽然所有病原性细菌都依赖于肽聚糖的结构稳定性，但一些菌株能够逃避hLYS的破坏。hLYS抗性的一种常见机制是对细胞壁肽聚糖的细微结构修饰。特别地，O-乙酰化可以消除hLYS活性，并且是已知存在于至少39种不同细菌（包括致病性葡萄球菌和链球菌）中的修饰。重要的是，S.金黄色葡萄球菌的感染可以仅仅归因于肽聚糖的O-乙酰化。在这个提议中，组合蛋白质工程将用于开发能够有效降解S的O-乙酰化肽聚糖的全新hLYS变体。金黄色葡萄球菌，从而杀死病原体。Specific Aim 1将专注于优化创新的超高通量抗生素筛选。将分泌抗菌蛋白的重组酵母菌与细菌靶标共包封在50 μ m水凝胶微滴（GMD）中。其中分泌的蛋白质杀死细菌靶标的GMD用活/死染色剂荧光标记，并且随后使用高速荧光激活细胞分选（FACS）分离。GMD-FACS测定已在概念验证实验中得到证实，并将使用来自大量过量阴性对照的阳性对照细胞的人工富集进行充分优化。具体目标2将检验hLYS可经工程改造以有效水解O-乙酰化肽聚糖并裂解S的假设。金黄色。利用来自目的1的GMD-FACS测定，将针对抗S筛选突变的hLYS酶的大型组合文库。金细胞溶解活性。将对分离的酶变体的抗生素效价进行定量表征。成功实现项目目标将产生一个高通量筛选，在开发抗菌蛋白和肽方面具有广泛的实用性，并且还将产生能够有效杀死耐药S.金黄色葡萄球菌 公共卫生相关性：细菌病原体通常在常规抗生素首次治疗应用后不久就开始开始对常规抗生素产生耐药性。耐药性细菌，如MRSA，正变得越来越普遍，它们可以将普通感染转化为危及生命的疾病。该提案旨在开发强大的新型抗菌酶，其治疗特性不易被破坏。这些下一代治疗药物可以重新武装医生，对抗耐药细菌感染。

DESCRIPTION (provided by applicant): Antibiotic-resistance complicates the majority of Staphylococcus aureus (S. aureus) infections, as a full two thirds of hospital associated S. aureus infections and ~50% of those acquired in the community are now methicillin-resistant (MRSA). The increasing incidence of multidrug-resistance in S. aureus and other bacteria underscores the need for next generation antibiotics capable of combating these dangerous pathogens. Ideally, new drugs will not only efficaciously treat contemporary resistant strains, but they will also delay the development of new resistance phenotypes. To do so, new drugs will have to function by mechanisms orthogonal to that of conventional, inhibitory antibiotics. The human immune system has evolved a formidable arsenal of bactericidal agents, and many of these attack bacterial cell structures with less inherent plasticity than the proteins and ribonucleic acids targeted by conventional inhibitory drugs. One example is the human enzyme lysozyme (hLYS), which kills Gram-positive pathogens in part by hydrolysis of cell wall peptidoglycan and subsequent bacterial lysis. While all pathogenic bacteria rely on peptidoglycan for structural stability, some strains are able to evade destruction by hLYS. One common mechanism of hLYS-resistance is subtle structural modifications to cell wall peptidoglycan. In particular, O-acetylation can abrogate hLYS activity, and is a modification known to exist in at least 39 different bacteria including pathogenic Staphylococci and Streptococci. Importantly, the hLYS-resistance of S. aureus can be attributed solely to O-acetylation of peptidoglycan. In this proposal, combinatorial protein engineering will be used to develop entirely novel hLYS variants capable of efficiently degrading the O-acetylated peptidoglycan of S. aureus, thus killing the pathogen. Specific Aim 1 will focus on optimizing an innovative ultra-high throughput antibiotic screen. Recombinant yeast secreting prospective antibacterial proteins are coencapsulated with bacterial targets in 50 5m hydrogel microdroplets (GMDs). GMDs in which the secreted protein kills the bacterial target are fluorescently tagged with a live/dead stain, and are subsequently isolated using high speed fluorescence activated cell sorting (FACS). The GMD-FACS assay has been demonstrated in proof-of-concept experiments, and will be fully optimized using artificial enrichments of positive control cells from large excesses of negative controls. Specific Aim 2 will test the hypothesis that hLYS can be engineered to efficiently hydrolyze O-acetylated peptidoglycan and lyse S. aureus. Leveraging the GMD-FACS assay from aim 1, large combinatorial libraries of mutated hLYS enzymes will be screened for anti-S. Aureus lytic activity. Isolated enzyme variants will be quantitatively characterized with respect to antibiotic potency. Successfully achieving the project objectives will yield a high throughput screen with broad utility in developing antibacterial proteins and peptides, and will also produce entirely novel, therapeutic, human enzymes capable of efficiently killing drug-resistant S. aureus pathogens. PUBLIC HEALTH RELEVANCE: Bacterial pathogens typically begin to develop resistance towards conventional antibiotics shortly after their first therapeutic application. Drug-resistant bacteria, such as MRSA, are becoming increasingly common, and they can transform a common infection into a life threatening illness. This proposal seeks to develop powerful, new, antibacterial enzymes whose therapeutic properties cannot be undermined easily. These next generation therapeutic agents could rearm physicians in the battle against drug-resistant bacterial infections.