Mechanism of type VI protein secretion

• Applicant: Privatdozent Dr. Axel Mogk
• Subject Area: Biochemistry
• Term: from 2011 to 2014
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 194363196

Type VI secretion systems (T6SSs) are found in many pathogenic Gram-negative bacteria and contribute to virulence in various species by delivering effectors into target cells. Exoproteins of T6SSs (Hcp, VgrG) exhibit structural similarities to components of the cell puncturing device of tailed bacteriophages. It is therefore suggested that T6SSs represent contractile injection machineries, thereby using a unique mechanism to target toxins into host cells. We could demonstrate that the AAA+ chaperone ClpV acts as an essential ATPase of T6SSs by severing VipA/VipB tubules, which are conserved and essential components of T6SSs. The architecture of VipA/VipB tubules suggests that they might function in analogy to tail sheath proteins of bacteriophages, driving the ejection of the T6SS exoproteins through contraction. We propose that such conformational change of VipA/VipB tubules is triggered by ClpV. Here, we plan to unravel the basic working principle of the V. cholerae T6SS. We aim at providing experimental evidence for the function of VipA/VipB as contractile proteins and to identify novel interaction partners of ClpV and VipA/VipB, which might control tubule severing. Based on our recent findings showing a distinct cellular localization of T6SSs, we will employ immunofluorescence and immunoelectron microscopy to determine the subcellular organization of T6SSs and to position VipA/VipB tubules, ClpV and the exoprotein Hcp in a T6S assembly. Together these approaches will lead to an advanced mechanistic understanding of T6SSs. Our second main goal is to characterize the interaction of ClpV and VipA/VipB at the molecular level and to identify small compounds that inhibit T6SSs by affecting the interaction between ClpV and VipA/VipB tubules. We recently determined the crystal structure of the ClpV N-domain and identified its interaction site in the VipA/VipB tubule within the N-terminal region of VipB. We are aiming at determining the precise binding mode by co-crystallization and validating the structure by subsequent mutagenesis. A meanwhile established peptide binding assay sets the basis for establishing a medium-throughput ALPHA screen to identify small compounds that prevent the ClpV-VipA/VipB interaction. The potential of such molecules as antimicrobial agents will then be tested in vitro and in vivo.

• DFG Programme: Research Grants