Characterization of Zif, a unique FemABX-like protein

描述（由申请人提供）：我们的长期目标是确定允许某些细菌修改其细胞壁肽聚糖（PG）以对PG水解酶，细菌素和细胞壁靶向抗微生物药物产生抗性的机制。我们对femabx样免疫蛋白特别感兴趣，它修饰PG交叉桥，使它们能够抵抗溶菌内肽酶的水解。在这个项目中，我们将定义zoocin a免疫因子（Zif）的活性，Zif是该家族的一员，它的独特之处在于它可以修饰PG而不会明显改变交叉桥的组成。我们将通过以下具体目标来实现这一目标：1)确定zoocin a的作用位点。确定该酶的作用位点将有助于确定Zif最有可能的靶点。首先，我们将鉴定由酶消化合成肽底物的产物。接下来，我们将使用高效液相色谱/质谱分析马链球菌（Streptococcus equi subsp） zoocin a -阴性、zoocin a敏感敲除突变体纯化PG后产生的片段。zooepidemicus 4881， zoocin a的产生生物，以确认zoocin a水解的一个或多个键。2)确定Zif引起的PG结构变化。根据初步数据，我们假设Zif在PG交叉桥中插入d -丙氨酸代替l -丙氨酸。首先，我们将测定zif+和zif-菌株pg中d -丙氨酸与l -丙氨酸的比例。如果我们的假设是不正确的，将使用HPLC/MS分析比较来自等基因zif+和zif-菌株的各种pg的片段，以确定zif引起的变化。我们将寻找Zif与MurM和MurN之间的相互作用，它们在PG合成过程中将氨基酸插入链球菌的交叉桥中，并通过拉下与其他蛋白质相互作用。细菌双杂交系统也将用于寻找相互作用。此外，Zif似乎对金黄色葡萄球菌有毒性。因此，我们将确定Zif是否与FemA、FemB或FemX形成复合物（在PG合成过程中将氨基酸插入葡萄球菌的交叉桥中），或与另一种类似femabx的免疫蛋白Epr（也称为liff）通过下拉和细菌双杂交系统形成复合物。参与细胞壁合成的因子是许多抗生素（如青霉素和头孢菌素）的靶标，而其他细胞壁合成途径与对这些抗菌剂的耐药性有关。该项目将提供关于细胞壁合成和降解的额外信息，可能为化疗干预提供新的靶点。

DESCRIPTION (provided by applicant): Our long-term objective is to define the mechanisms that allow some bacteria to modify their cell wall peptidoglycans (PGs) to become resistant to PG hydrolases, bacteriocins, and cell wall-targeted anti-microbials. In particular we are interested in FemABX-like immunity proteins, which modify PG cross bridges so that they become resistant to hydrolysis by bacteriolytic endopeptidases. In this project we will define the activity of the zoocin a immunity factor (Zif), a member of this family that is unique because it modifies PG without any apparent change in cross bridge composition. We will accomplish this through the following specific aims: 1) Define the site of action of zoocin A. Identification of the site of action of the enzyme will help identify the most likely target for Zif. First we will identify the products from the digestion of synthetic peptide substrates by the enzyme. Next, we will use HPLC/MS analysis of fragments generated from a zoocin A, mutanolysin digestion of purified PG of a zoocin A-negative, zoocin A-sensitive knockout mutant of Streptococcus equi subsp. zooepidemicus 4881, the zoocin A-producing organism, to confirm the bond or bonds hydrolyzed by zoocin A. 2) Determine the change in PG structure caused by Zif. Based on preliminary data, we hypothesize that Zif inserts D-alanine for L-alanine in PG cross bridges. First we will determine the ratio of D-alanine to L-alanine in PGs from zif+ and zif- strains. If our hypothesis is incorrect, comparison of HPLC/MS analyses of fragments from various digests of PGs from isogenic zif+ and zif- strains will be used to identify the change caused by Zif. 3) Determine if Zif forms complexes with other proteins. We will look for interactions between Zif and both MurM and MurN, which insert amino acids into streptococcal cross bridges during PG synthesis, and with other proteins using pulldowns. A bacterial two-hybrid system will also be used to look for interactions. Furthermore, Zif appears to be toxic in Staphylococcus aureus. Therefore we will determine if Zif forms complexes with FemA, FemB, or FemX, which insert amino acids into the staphylococcal cross bridges during PG synthesis, or with another FemABX-like immunity protein, Epr (also known as Lif), by pulldowns and with a bacterial two-hybrid system. The factors that are involved in cell wall synthesis are targets for a number of antibiotics, such as penicillins and cephalosporins, and alternative cell wall synthesis pathways are involved in resistance to these antimicrobial agents. This project will provide additional information regarding cell wall synthesis and degradation that may offer new targets for chemotherapeutic intervention.