Resistance of Bacillus anthracis to lysozyme

描述（由申请人提供）：溶菌酶通过抑制细胞外细菌活力在先天免疫中起核心作用，特别是在粘膜表面，如肺。然而，许多革兰氏阳性病原体，包括炭疽芽孢杆菌，在体外对脊椎动物c型溶菌酶具有耐药性，这一特征可能与发病机制有关。在金黄色葡萄球菌中，细胞壁肽聚糖的o -乙酰化已被确定为对溶菌酶抗性负责的修饰。目前尚不清楚在炭疽芽孢杆菌中是否存在类似的修饰。我们假设炭疽芽孢杆菌的溶菌酶抗性是由于o -乙酰化，我们将使用既定的方法来测试炭疽芽孢杆菌肽聚糖的o -乙酰化，并表征这种修饰的生长阶段依赖性。我们将鉴定和表征炭疽芽孢杆菌o -乙酰转移酶的活性，并确定其膜拓扑结构和亚细胞定位。我们将评估o -乙酰化在保护炭疽芽孢杆菌细胞免受培养的哺乳动物细胞分泌的溶菌酶等抗菌因子的作用以及它们在宿主感染期间的存活方面是否重要。与所有动物表达的c型溶菌酶不同，在一些细菌和真菌中发现的ch型溶菌酶可以水解o -乙酰化的细胞壁。因此，重组ch型溶菌酶可能作为一种有效的治疗药物，以补充宿主防御，降低炭疽芽胞杆菌的存活率。我们已经在线虫C. elegans及其近亲C. briggsae的基因组中发现了编码假定的ch型溶菌酶的基因，但在任何其他已测序的后生动物基因组中都没有发现。我们假设这些蛋白能够水解O-乙酰化的细胞壁。我们将通过克隆和纯化这些酶来验证这一假设，并评估它们消化炭疽杆菌细胞壁和裂解炭疽杆菌细胞的能力。我们将进一步表征这些酶，以评估其预测的活性位点残基和底物特异性。与细菌和真菌的同源物不同，这些酶已经进化到在多细胞真核生物的生理环境中起作用，使它们成为克服炭疽芽孢杆菌溶菌酶耐药性的优秀候选治疗剂。我们将研究细菌构建细胞壁的过程，细胞壁是决定和维持细菌形状的结构。尽管这一过程是包括青霉素在内的许多目前使用的抗生素的目标，但细菌正在迅速对这些药物产生耐药性，因此我们期望对这一过程的新理解将导致新抗生素的开发。

DESCRIPTION (provided by applicant): Lysozyme plays a central role in innate immunity by inhibiting extracellular bacterial viability, particularly on mucosal surfaces such as those of the lung. However, many gram-positive pathogens, including Bacillus anthracis, are resistant in vitro to vertebrate c-type lysozyme, a feature likely relevant for pathogenesis. In Staphylococcus aureus, O-acetylation of the cell wall peptidoglycan has been identified as the modification that is responsible for lysozyme resistance. The presence of a similar modification in B. anthracis is unknown. We hypothesize that the lysozyme resistance of B. anthracis is due to O-acetylation and we will test this hypothesis using established methodology to assay the O-acetylation of B. anthracis peptidoglycan and to characterize the growth phase dependence of this modification. We will identify and characterize the activity of the B. anthracis O-acetyl transferase and determine its membrane topology and subcellular localization. We will assess whether O-acetylation is important in protecting B. anthracis cells from the action of antimicrobial factors including lysozyme secreted by cultured mammalian cells and in their survival during host infection. Unlike the c-type lysozyme expressed by all animals, CH-type lysozyme found in some bacteria and fungi can hydrolyze O-acetylated cell wall. Thus, recombinant CH-type lysozyme may serve as a useful therapeutic agent to supplement host defenses in reducing B. anthracis survival. We have identified genes encoding putative CH-type lysozymes in the genomes of the nematode C. elegans and its close relative C. briggsae, but not in any other sequenced metazoan genome. We hypothesize that these proteins will be capable of hydrolyzing O- acetylated cell walls. We will test this hypothesis by cloning and purifying these enzymes and assessing their ability to digest B. anthracis cell wall and to lyse B. anthracis cells. We will further characterize these enzymes to evaluate their predicted active site residues and substrate specificity. Unlike their bacterial and fungal homologs, these enzymes have evolved to work within the physiological context of a multi-cellular eukaryotic organism, making them excellent candidates as therapeutic agents to overcome B. anthracis lysozyme resistance. PUBLIC HEALTH RELEVANCE We will study the process by which bacteria construct their cell wall, the structure that determines and maintains their shape. Although this process is the target of many presently used antibiotics including penicillin, bacteria are quickly becoming resistant to these drugs, so we expect that new understanding of this process will lead to the development of new antibiotics.