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（金黄色葡萄球菌）感染复杂化，因为医院相关金黄色葡萄球菌感染的三分之二和社区获得的金黄色葡萄球菌感染的约50%现在是耐甲氧西林（MRSA）。金黄色葡萄球菌和其他细菌的多药耐药发生率日益增加，这突出表明需要能够对抗这些危险病原体的下一代抗生素。理想情况下，新药不仅能有效地治疗现有的耐药菌株，而且还能延缓新的耐药表型的发展。要做到这一点，新药的作用机制必须与传统的抑制性抗生素不同。人类免疫系统已经进化出了强大的杀菌剂武器库，其中许多杀菌剂攻击的细菌细胞结构，其固有可塑性不如传统抑制药物靶向的蛋白质和核糖核酸。一个例子是人溶菌酶（hlyys），它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。虽然所有致病菌都依赖肽聚糖来保持结构稳定性，但有些菌株能够逃避hys的破坏。hys耐药的一个常见机制是细胞壁肽聚糖的细微结构修饰。特别是，o -乙酰化可以消除hys活性，并且是已知存在于至少39种不同细菌中的一种修饰，包括致病性葡萄球菌和链球菌。重要的是，金黄色葡萄球菌的hys耐药可以仅仅归因于肽聚糖的o -乙酰化。在这一建议中，组合蛋白工程将用于开发全新的hys变体，能够有效地降解金黄色葡萄球菌的o -乙酰化肽聚糖，从而杀死病原体。特异性目标1将侧重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与细菌靶点共包被在50米的水凝胶微滴（GMDs）中。分泌蛋白杀死细菌目标的gmd用活/死染色进行荧光标记，随后使用高速荧光激活细胞分选（FACS）进行分离。GMD-FACS检测已在概念验证实验中得到验证，并将通过从大量过量的阴性对照中人工富集阳性对照细胞来充分优化。特异性目的2将验证hLYS可以有效水解o -乙酰化肽聚糖和裂解金黄色葡萄球菌的假设。利用来自aim 1的GMD-FACS分析，将对突变hys酶的大型组合文库进行抗s筛选。金黄色裂解活性。分离的酶变体将在抗生素效力方面进行定量表征。成功实现项目目标将产生高通量筛选，在开发抗菌蛋白和肽方面具有广泛的用途，并且还将产生全新的、治疗性的、能够有效杀死耐药金黄色葡萄球菌病原体的人类酶。

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.