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产生抗药性的机制。特别是，我们对FemABX样免疫蛋白感兴趣，它可以修饰PG交叉桥，使它们对溶菌内肽酶的水解产生抵抗力。在这个项目中，我们将定义Zoocin a免疫因子(ZIF)的活性，ZIF是这个家族中独一无二的成员，因为它在交叉桥组成没有任何明显变化的情况下修饰PG。我们将通过以下具体目标来实现这一点：1)确定Zoocin A的作用部位。确定该酶的作用部位将有助于确定ZIF最有可能的靶点。首先，我们将鉴定合成肽底物被酶消化的产物。接下来，我们将使用高效液相色谱/质谱仪对马链球菌的人凝集素A阴性、人凝集素A敏感的基因敲除突变株的Pg进行裂解，分析产生的片段。2)确定ZIF引起的PG结构的变化。根据初步数据，我们假设ZIF在PG交叉桥中插入D-丙氨酸取代L-丙氨酸。首先，我们将测定ZIF+和ZIF-菌株的PG中D-丙氨酸和L-丙氨酸的比例。如果我们的假设是错误的，我们将通过对来自同基因ZIF+和ZIF-菌株的PGs不同酶切片段的比较来确定ZIF引起的变化。3)确定ZIF是否与其他蛋白质形成复合体。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，并使用下拉菜单与其他蛋白质相互作用。细菌双杂交系统也将被用来寻找相互作用。此外，ZIF似乎对金黄色葡萄球菌有毒性。因此，我们将通过下拉和细菌双杂交系统确定ZIF是与FEMA、FemB或FemX形成复合体，这些复合体在PG合成过程中将氨基酸插入葡萄球菌的交叉桥中，还是与另一种FemABX样免疫蛋白EPR(也称为Lif)形成复合体。参与细胞壁合成的因子是许多抗生素的靶标，如青霉素和头孢菌素，而替代的细胞壁合成途径参与了对这些抗菌剂的耐药性。该项目将提供有关细胞壁合成和降解的更多信息，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.