B. anthracis Peptidoglycan Deacetylase as a Drug Target

描述(由申请人提供)：炭疽杆菌营养细胞中的肽聚糖层中的很高比例的乙酰氨基糖残留物是通过一个或多个特定的肽聚糖脱乙酰酶的作用进行脱乙酰化的(S)。这种修饰的效果是使细菌细胞壁抵抗溶菌酶的消化，溶菌酶是人体分泌物、血液和组织中普遍存在的一种酶。在R21的应用中，我们评估了炭疽杆菌的肽聚糖脱乙酰酶作为新药靶点的可行性。这种酶的特定抑制剂可能会使细菌无法保护它们的细胞壁免受宿主溶菌酶的影响，然后它们就会迅速被杀死。在炭疽杆菌基因组中已初步鉴定出12个候选的肽聚糖脱乙酰酶基因。第一个特定的目的是筛选表达的候选脱乙酰酶的酶活性。为了便于开发适用于炭疽杆菌脱乙酰酶的分析方法和生化程序，将首先克隆和表达唯一被证实的肽聚糖脱乙酰酶基因，来自肺炎链球菌的pgdA基因。将确定酶的重要性质，包括它们的最适pH、金属离子需求(如果有的话)以及还原剂对酶活性的影响。此外，对酶活性至关重要的氨基酸残基将通过酶的高度保守区域的定点突变来鉴定。第二个特定目标是突变灭活炭疽杆菌候选脱乙酰酶基因，然后评估突变对肽聚糖脱乙酰化和对溶菌酶的敏感性的影响。在不太可能的情况下，没有一个候选基因编码活性脱乙酰酶，该基因将使用转座子突变程序进行鉴定。第三个具体目标是确定脱乙酰酶缺陷突变体在炭疽杆菌感染的小鼠模型中是否不再具有毒力。用对接种Sterne孢子不同敏感的A/J和BALB/c小鼠比较炭疽斯特恩菌株野生型和突变(即肽聚糖脱乙酰酶缺失)孢子的毒力。小鼠将受到三种不同途径的挑战，即皮下、鼻腔和气管内接种孢子。该项目的成功可能会导致开发另一种有价值的方法来治疗由炭疽杆菌以外的其他革兰氏阳性细菌引起的感染，方法是抑制脱乙酰酶，这种脱乙酰酶专门使细胞壁肽聚糖抵抗溶菌酶的作用。

DESCRIPTION (provided by applicant): A high proportion of the acetamido sugar residues in the peptidoglycan layer of B. anthracis vegetative cells are de-N-acetylated by the action of one or more specific peptidoglycan deacetylase(s). This modification has the effect of making the bacterial cell wall resistant to digestion by lysozyme, a ubiquitous enzyme in human secretions, blood and tissues. The feasibility of using the peptidoglycan deacetylase of B. anthracis as a new drug target is assessed in this R21 application. A specific inhibitor of the enzyme could render the bacteria unable to protect their cell walls from host lysozyme and they would then be rapidly killed. Twelve candidate peptidoglycan deacetylase genes have been tentatively identified in the genome of B. anthracis. The first specific aim is to screen expressed candidate deacetylases for enzyme activity. To facilitate development of suitable analytical methods and biochemical procedures for use with the B. anthracis deacetylase, the only confirmed peptidoglycan deacetylase gene, the pgdA gene from S. pneumoniae, will be cloned first and expressed. Important properties of the enzymes will be determined, including their pH optima, metal ion requirements, if any, and the effect of reducing agents on enzyme activity. In addition, amino acid residues critical for enzymatic activity will be identified by means of site-directed mutagenesis of highly conserved regions of the enzymes. The second specific aim is to mutationally inactivate candidate deacetylase genes in B. anthracis and then assess the effects of the mutations on peptidoglycan deacetylation and sensitivity to lysozyme. In the unlikely event that none of the candidate genes encode the active deacetylase, the gene will be identified using a transposon mutagenesis, procedure. The third specific aim is to determine if deacetylase-deficient mutants are no longer virulent in a mouse model of B. anthracis infection. Virulence of wild type and mutant (i.e., peptidoglycan deacetylase-deficient) spores of the Sterne strain of B. anthracis will be compared using A/J and BALB/c mice, which are differentially sensitive to inoculation of Sterne spores. Mice will be challenged by three different routes, subcutaneous, intranasal, and intratracheal inoculation of spores. Success of this project may lead to the development of another valuable means of treating infections caused by other Gram-positive bacterial pathogens, in addition to B. anthracis, by inhibiting the deacetylases which specifically render their cell wall peptidoglycans resistant to the action of lysozyme.