B. anthracis Peptidoglycan Deacetylase as a Drug Target

性状（由申请方提供）：B的肽聚糖层中有高比例的乙酰氨基糖残基。炭疽营养细胞通过一种或多种特异性肽聚糖脱乙酰酶的作用而脱N-乙酰基。这种修饰具有使细菌细胞壁抵抗溶菌酶消化的作用，溶菌酶是人体分泌物、血液和组织中普遍存在的酶。利用B的肽聚糖脱乙酰酶的可行性。炭疽作为一种新的药物靶标在R21申请中进行了评估。这种酶的一种特异性抑制剂可以使细菌无法保护它们的细胞壁免受宿主溶菌酶的侵害，然后它们会被迅速杀死。在B的基因组中初步鉴定出12个候选肽聚糖脱乙酰酶基因。炭疽病第一个具体目标是筛选表达的候选脱乙酰酶的酶活性。促进开发适用于B的分析方法和生化程序。炭疽菌的肽聚糖脱乙酰酶基因、炭疽菌的pgdA基因和炭疽菌的pgdA基因。pneumoniae，将首先克隆并表达。将确定酶的重要性质，包括它们的最适pH、金属离子要求（如果有的话）以及还原剂对酶活性的影响。此外，将通过酶的高度保守区域的定点诱变来鉴定对酶活性至关重要的氨基酸残基。第二个具体目的是在B中突变地修饰候选脱乙酰酶基因。炭疽杆菌，然后评估突变对肽聚糖脱乙酰化和对溶菌酶敏感性的影响。在不太可能的情况下，没有候选基因编码活性脱乙酰酶，该基因将使用转座子诱变程序进行鉴定。第三个具体目标是确定脱乙酰酶缺陷突变体在B小鼠模型中是否不再具有毒性。炭疽感染。野生型和突变体的毒力（即，肽聚糖脱乙酰酶缺陷型）B的Sterne菌株的孢子。将使用A/J和BALB/c小鼠比较炭疽菌，这两种小鼠对Sterne孢子接种的敏感性不同。小鼠将通过三种不同途径进行攻毒，即皮下、鼻内和阴道内接种孢子。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除B以外的其他革兰氏阳性细菌病原体引起的感染。炭疽，通过抑制脱乙酰酶，使其细胞壁肽聚糖特异性地抵抗溶菌酶的作用。

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.