Project Details
Projekt
Functional characterization of the membrane-depolarizing toxin TisB in Escherichia coli
Applicant
Professor Dr. Bork Berghoff
Subject Area
Metabolism, Biochemistry and Genetics of Microorganisms
Term
since 2017
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 379070278
In prokaryotes, chromosomal toxin-antitoxin (TA) systems are involved in many processes, including stress adaptation and persister cell formation. Persister cells are phenotypic variants that exhibit an extremely high tolerance towards antibiotics, and gain increasing attention in clinical settings. Persister formation is favored by growth inhibition through toxins from TA systems. In type I TA systems, toxins are typically small proteins (< 50 amino acids) that target the inner membrane and perturb membrane functioning. This also applies to toxin TisB from the type I TA system TisB/IstR-1 in Escherichia coli. TisB (29 amino acids) is produced upon DNA damage and breaks down the proton motive force by a yet unknown mechanism, leading to membrane depolarization. The subsequent ATP depletion is suspected to trigger persister formation. Current models suggest the formation of water-filled TisB pores, through which protons traverse the inner membrane. In the first funding period, we could demonstrate that membrane targeting by TisB also inflicts stress on cells, and that superoxide detoxification plays an important role for survival of TisB-dependent persister cells. Furthermore, we observed that several other genes with a role in stress responses are upregulated. This applies, for example, to genes from the inner membrane stress response (CxpR regulon) and to genes encoding poorly characterized DUF1471 domain-containing stress proteins. We will unravel the functional relationship between TisB and selected stress proteins with regard to persister formation and post-antibiotic recovery using genetic approaches and physiological assays. TisB forms an α-helix with a transmembrane (TM) domain, and contains the five charged amino acid residues Asp5, Lys12, Asp22, Lys26, and Lys29. We could already show that the charged residues within the TM domain (Lys12 and Asp22) are crucial for TisB functionality. The charged residues might also determine the orientation of TisB in the inner membrane. We will investigate TisB with regard to the functional importance of charged residues and their impact on TisB topology using physiological assays and biochemical methods. In addition, the potential interaction of TisB with other proteins will be investigated by co-purification experiments. We also initiated a project on archaeal type I toxins and their effects on growth in E. coli. We will study these toxins in more detail and compare them to TisB. The proposed project will contribute to our understanding of type I toxins as factors for survival in unpredictable environments.
DFG Programme
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