B. anthracis Peptidoglycan Deacetylase as a Drug Target

描述（由申请人提供）：炭疽芽孢杆菌营养细胞的肽聚糖层中的大部分乙酰氨基糖残基通过一种或多种特定的肽聚糖去乙酰化酶的作用被去n -乙酰化。这种修饰的作用是使细菌细胞壁抵抗溶菌酶的消化，溶菌酶是人类分泌物、血液和组织中普遍存在的酶。本R21应用评估了炭疽芽孢杆菌肽聚糖脱乙酰酶作为新药物靶点的可行性。一种特定的酶抑制剂可以使细菌无法保护其细胞壁免受宿主溶菌酶的侵害，然后它们就会被迅速杀死。在炭疽芽孢杆菌基因组中初步鉴定了12个候选肽聚糖脱乙酰酶基因。第一个具体目标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，将首先克隆并表达唯一确认的肽聚糖脱乙酰酶基因，即来自肺炎链球菌的pgdA基因。酶的重要性质将被确定，包括它们的最佳pH值，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诱变来鉴定。第二个具体目标是突变失活炭疽杆菌候选去乙酰化酶基因，然后评估突变对肽聚糖去乙酰化和对溶菌酶敏感性的影响。在不太可能发生的情况下，没有候选基因编码活性去乙酰化酶，该基因将使用转座子诱变程序进行鉴定。第三个具体目的是确定脱乙酰酶缺陷突变体在炭疽杆菌感染小鼠模型中是否不再具有毒性。利用A/J和BALB/c小鼠对接种Sterne孢子的不同敏感性，比较了野生型和突变型（即肽聚糖去乙酰酶缺陷）炭疽芽孢杆菌Sterne菌株孢子的毒力。小鼠将通过三种不同的途径，皮下、鼻内和气管内接种孢子。这个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其他革兰氏阳性细菌病原体引起的感染，除了炭疽芽孢杆菌，通过抑制脱乙酰酶，特异性地使其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.