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

• Applicant: Privatdozent Dr. Sven Halbedel
• Subject Area: Parasitology and Biology of Tropical Infectious Disease Pathogens ; Metabolism, Biochemistry and Genetics of Microorganisms
• Term: from 2012 to 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 223759939

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.

• DFG Programme: Research Grants