Resistance of Bacillus anthracis to lysozyme

描述(申请人提供)：溶菌酶通过抑制细胞外细菌的存活，特别是在诸如肺的粘膜表面，在先天免疫中发挥核心作用。然而，许多革兰氏阳性病原体，包括炭疽杆菌，在体外对脊椎动物的c型溶菌酶具有耐药性，这一特征可能与发病相关。在金黄色葡萄球菌中，细胞壁肽聚糖的O-乙酰化是导致溶菌酶抗性的修饰。炭疽杆菌中存在类似的修饰尚不清楚。我们假设炭疽杆菌对溶菌酶的抗性是由于O-乙酰化，我们将使用已建立的方法学来验证这一假设，以检测炭疽杆菌肽聚糖的O-乙酰化，并表征这种修饰的生长阶段依赖性。我们将鉴定和鉴定炭疽杆菌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-乙酰化在保护炭疽杆菌细胞免受包括培养的哺乳动物细胞分泌的溶菌酶在内的抗微生物因子的作用以及在宿主感染期间存活方面是否重要。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-乙酰化细胞壁。因此，重组CH型溶菌酶可以作为一种有效的治疗剂来补充宿主防御，降低炭疽杆菌的存活率。我们已经在线虫及其近缘线虫基因组中发现了可能编码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没有发现。我们假设这些蛋白质将能够水解O-乙酰化细胞壁。我们将通过克隆和纯化这些酶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来检验这一假设。我们将进一步对这些酶进行表征，以评估其预测的活性部位残基和底物专一性。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有机体的生理环境中工作，使它们成为克服炭疽杆菌溶菌酶耐药性的优秀治疗剂。公共卫生相关性我们将研究细菌构建细胞壁的过程，这种结构决定并保持它们的形状。虽然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抗药性，因此我们预计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.