Mechanism of type VI protein secretion
Final Report Abstract
Type VI secretion systems (T6SSs) represent widespread specialized secretion systems of Gram-negative bacteria. They play crucial roles in interbacterial competition by killing opponent cells via delivery of toxic effector proteins including cell wall degrading enzymes, lipases and nucleases. T6SSs also contribute to virulence of pathogenic bacteria either directly by toxin delivery into host cells or indirectly by outcompeting commensal bacteria. T6SSs therefore represent attractive drug targets. Understanding the structural organization and mechanism of T6SSs is therefore of high scientific but also medical relevance. The T6SS exoproteins Hcp and VgrG exhibit structural similarities to proteins of the infection machinery of tailed bacteriophages. It was therefore suggested that T6SSs function as contractile injection machineries that deliver effectors upon puncturing the membranes of target cells. In the first funding period we demonstrated that the AAA+ protein ClpV functions as an essential ATPase component of T6SSs by disassembling tubular complexes, composed of the conserved and essential T6SS components VipA and VipB. VipA/VipB complexes have a similar architecture and dimension as contractile viral tail sheath proteins and were suggested to function in an analogous manner, providing the energy for T6S. In the second funding period we analyzed the localization and dynamics of VipA/VipB tubules by time lapse fluorescence microscopy and immunoelectron microscopy. We showed that functional VipA-YFP/VipB tubules are highly dynamic, exhibiting cycles of elongation, contraction and disintegration. These findings qualify VipA/VipB as contractile tail sheath proteins that mediate effector export upon contraction. ClpV has a dual role in T6S by preventing the formation of non-productive VipA/VipB off-pathway assemblies and by recycling contracted tubules, allowing for new rounds of T6S. ClpV activity is restricted to the contracted state of VipA/VipB tubules. We could determine the molecular principle of ClpV-VipA/VipB tubule interaction, by (i) determining the ClpV binding site within VipB using peptide libraries, (ii) providing the structural rationale for ClpV binding specificity by determining the crystal structure of the ClpV N-domain and interacting VipB peptide and (iii) derive models of contracted and elongated VipA/VipB tubules by cryo electron microscopy, indicating that the ClpV binding site becomes only accessible upon VipA/VipB tubule contraction. Together our data highlight the role of VipA/VipB tubules as contractile T6SS elements and unravel the mechanism of autonomous ClpV activity control, by restricting its access to contracted VipA/VipB complexes.
Publications
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(2011) Molecular basis for the unique role of the AAA+ chaperone ClpV in type VI protein secretion. J Biol Chem. 2011 Aug 26;286(34):30010-21
Pietrosiuk A, Lenherr ED, Falk S, Boenemann G, Kopp J, Zentgraf H, Sinning I, Mogk A
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(2013) ClpV recycles VipA/VipB tubules and prevents non-productive tubule formation to ensure efficient type VI protein secretion. Mol Microbiol 87: 1013-1028
Kapitein N, Bonemann G, Pietrosiuk A, Seyffer F, Hausser I, Locker JK, Mogk A
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(2013) Deadly syringes: type VI secretion system activities in pathogenicity and interbacterial competition. Curr Opin Microbiol 16: 52-58
Kapitein N, Mogk A
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(2014) Structure of the VipA/B type VI secretion complex suggests a contraction-state-specific recycling mechanism. Cell Rep 8: 20-30
Kube S, Kapitein N, Zimniak T, Herzog F, Mogk A, Wendler P
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(2014) Type VI secretion system helps find a niche. Cell Host Microbe 16: 5-6
Kapitein N, Mogk A
