Molecular Engineering of Humanized Anti-Staphlococcal Lytic Enzymes

人源化抗葡萄球菌裂解酶的分子工程

基本信息

批准号：
8093306
负责人：
Karl E Griswold
金额：
$ 19.75万
依托单位：
DARTMOUTH COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-03-01 至 2013-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8093306
关键词：
Acetylation Active Sites Agar Amino Acids Anti-Bacterial Agents Antibiotic Resistance Antibiotics Bacteria Bacterial Infections Binding Biological Assay Cell Wall Cells Cellular Structures Cleaved cell Clinical Communities Community Hospitals Cytolysis Development Drug Industry Drug resistance Engineering Enzymes Experimental Designs Fluorescence Fluorescence-Activated Cell Sorting Future Genes Genetic Genus staphylococcus Goals Gram-Positive Bacteria Hospitals Human Human Engineering Hydrogels Hydrolysis Hydroxyl Radical Immune system Immunocompetence Incidence Individual Infection Label Libraries Life Lung LytA enzyme Lytic Medical Methicillin Resistance Methods Modification Molecular Multi-Drug Resistance Muramidase Mutagenesis Mutate N-acetylmuramic acid Nature Outcome Peptide Synthesis Peptides Peptidoglycan Pharmaceutical Preparations Phenotype Physicians Property Protein Engineering Proteins RNA Recombinant DNA Recombinants Resistance Resistance development Saccharomyces cerevisiae Screening procedure Skin Sorting - Cell Movement Specificity Speed Staining method Stains Staphylococcus aureus Streptococcus Techniques Testing Therapeutic Therapeutic Agents Transferase Variant Virulence Work Yeasts antimicrobial bactericide base combat combinatorial design drug resistant bacteria high throughput screening improved innovation killings knowledge base molecular dynamics mutant next generation novel novel therapeutics pathogen pathogenic bacteria prospective research study resistant strain validation studies

项目摘要

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.

描述（由申请人提供）：抗生素抗性使金黄色葡萄球菌（金黄色葡萄球菌）感染的大多数复杂化，因为全部三分之二的医院与金黄色葡萄球菌感染了，并且在社区中获得的抗生素是甲壳虫蛋白耐药蛋白耐药（MRSA）。金黄色葡萄球菌和其他细菌中多药抗性的发生率的增加强调了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，新药不仅可以有效地治疗当代抗性菌株，而且还将延迟新的抗性表型的发展。为此，新药必须通过与常规抑制性抗生素的机制正交机制作用。人类免疫系统已经进化出了杀菌剂的强大武器库，其中许多具有固有可塑性的攻击细菌细胞结构，而不是传统抑制性药物靶向的蛋白质和核糖核酸。一个例子是人酶溶菌酶（HLYS），它通过细胞壁肽聚糖和随后的细菌裂解而部分杀死革兰氏阳性病原体。尽管所有致病细菌都依赖于肽聚糖来实现结构稳定性，但有些菌株能够逃避Hlys的破坏。 Hlys抗性的一种常见机制是对细胞壁肽聚糖的微妙结构修饰。特别是，O-乙酰化可以消除Hlys活性，并且是一种已知存在于至少39种不同细菌中的修饰，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HLYS抗性仅归因于肽聚糖的O-乙酰化。在此提案中，组合蛋白工程将用于开发完全新颖的Hlys变体，能够有效降解金黄色葡萄球菌的O-乙酰化肽聚糖，从而杀死病原体。特定的目标1将重点放在优化创新的超高吞吐量抗生素屏幕上。重组酵母分泌前瞻性抗菌蛋白与50 5M水凝胶微核（GMDS）中的细菌靶标共同体。分泌蛋白质杀死细菌靶的GMD被荧光标记为活/死污渍，随后使用高速荧光激活的细胞分选（FACS）将其分离出来。 GMD-FACS分析已在概念验证实验中得到证明，并将使用来自大量的阴性对照组中的阳性对照细胞的人工富集进行完全优化。特定的目标2将测试可以设计Hlys以有效水解O-乙酰化肽聚糖和金黄色葡萄球菌的假设。利用AIM 1的GMD-FACS分析，将筛选出突变的Hlys酶的大型组合库以筛选抗S。金黄色的裂解活性。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吞吐量筛选，并在开发抗菌蛋白和肽方面具有广泛的效用，并且还将产生完全新颖的，治疗性的，人类的酶，能够有效地杀死耐药的金黄色葡萄球菌病原体。公共卫生相关性：细菌病原体通常在第一次治疗后不久就开始对传统抗生素产生抗药性。耐药细菌（例如MRSA）变得越来越普遍，它们可以将普通感染转化为威胁生命的疾病。该建议旨在开发强大的，新的抗菌酶，其治疗特性不能轻易破坏。这些下一代治疗剂可以在与耐药细菌感染的斗争中重新治疗医生。