Identification of factors and processes controlled by Listeria monocytogenes DivIVA/GpsB proteins

DivIVA/GpsB蛋白是一个高度保守的蛋白质家族，仅在革兰氏阳性菌中发现。它们在细胞生长和细胞分裂中发挥重要和部分必要的功能。DivIVA/GpsB蛋白是寡聚膜结合蛋白，其由寡聚化所需的N-末端脂质结合结构域和C-末端结构域（CTD）组成。DivIVA蛋白可以基于其CTD的长度与GpsB蛋白区分开。这两种蛋白质都聚集在细胞分裂期间发生在收缩隔膜处的弯曲膜区域。在那里，它们作为支架蛋白，通过直接的蛋白质-蛋白质相互作用招募过多的不同物种特异性相互作用伴侣，其在细胞分裂、细胞壁生物合成、染色体分离和蛋白质分泌中具有功能。它们的三维结构是独特的，其系统发育分布仅限于革兰氏阳性菌。因此，DivIVA/GpsB蛋白可以被认为是未来开发新型抗菌药物的有前途的药物靶标。人类病原体单核细胞增生李斯特菌的DivIVA通过MinCDJ和-通过迄今未知的机制-SecA 2依赖性自溶素分泌来控制分裂位点选择。李斯特菌GpsB参与细菌细胞壁的生物合成和修饰，并以未知的方式控制双功能青霉素结合蛋白PBP A1和肽聚糖-N-乙酰葡糖胺脱乙酰酶PgdA的活性。DivIVA和GpsB都是L.单核细胞增多症在此研究方案下，提出了L.应鉴定单核细胞增生GpsB蛋白并表征其功能。最初，应筛选已知的细胞分裂蛋白与GpsB的相互作用，并测试其定位的GpsB依赖性。为了鉴定GpsB的新的相互作用配偶体，将分离在升高的温度下抑制gpsB突变体的生长缺陷的抑制突变体。这些抑制突变体自发地以高频率形成，并且初步实验已经证实抑制突变发生在编码新的GpsB相互作用伴侣的基因中。这些基因的功能尚不清楚。它们的功能和其他仍有待确定的gpsB抑制基因的功能将使用互补的遗传，功能和显微镜实验进行研究。GpsB通过PgdA依赖性脱乙酰化在肽聚糖修饰中的作用将在遗传实验、蛋白质-蛋白质相互作用分析和定位研究中进行研究。此外，L.将单核细胞增多症divIVA突变体进行化学诱变，以鉴定在软琼脂上校正该突变体的群集缺陷的抑制突变。受影响的抑制基因的功能将在随后的实验中更详细地研究。

DivIVA/GpsB proteins are a highly conserved protein family, which is exclusively found in Gram-positive bacteria. They play important and partially essential functions in cell growth and cell division. DivIVA/GpsB proteins are oligomeric membrane binding proteins, which consist of an N-terminal lipid binding domain and a C-terminal domain (CTD) required for oligomerization. DivIVA proteins can be discriminated from GpsB proteins based on the lengths of their CTDs. Both proteins cluster at curved membrane areas that occur during cell division at the constricting septum. There they act as scaffold proteins to recruit a plethora of different species-specific interaction partners with functions in cell division, cell wall biosynthesis, chromosome segregation and protein secretion through direct protein-protein interactions to the division site. Their three-dimensional structure is unique and their phylogenetic distribution is limited to the Gram-positive bacteria. Thus, DivIVA/GpsB proteins can be considered as promising drug targets for the future development of novel antibacterials. DivIVA of the human pathogen Listeria monocytogenes controls division site selection through MinCDJ and - by a so far unknown mechanism - SecA2-dependent autolysin secretion. Listerial GpsB is involved in biosynthesis and modification of the bacterial cell wall and controls the activities of the bi-functional penicillin binding protein PBP A1 and of the peptidoglycan-N-acetylglucosamine deacetylase PgdA in unknown ways. Both, DivIVA and GpsB, are absolutely indispensable for virulence of L. monocytogenes. With this research proposal, novel interaction partners of the L. monocytogenes GpsB protein should be identified and their function should be characterized. Initially, known cell division proteins should be screened for interaction with GpsB and the GpsB-dependency of their localization will be tested. In order to identify novel interaction partners of GpsB, suppressor mutants will be isolated that suppress the growth defect of the gpsB mutant at elevated temperature. These suppressor mutants are formed spontaneously with high frequency and preliminary experiments have confirmed that the suppressor mutations occur in genes that code for novel GpsB interaction partners. The function of these genes is unknown. Their function and the function of other still to be identified gpsB suppressor genes will be studied using complementary genetic, functional and microscopic experiments. The role of GpsB in peptidoglycan modification through PgdA-dependent deacetylation will be studied in genetic experiments, protein-protein interaction analyses and localization studies. Furthermore, the L. monocytogenes divIVA mutant will be subjected to chemical mutagenesis in order to identify suppressor mutations that correct the swarming defect of the this mutant on soft agar. The function of the affected suppressor genes will be studied in more detail in subsequent experiments.