Shining light on T6SS mode of action and function within single cells and bacterial communities

Bacteria are ubiquitously found in the environment and form the basis for all known ecosystems on our planet. Most bacterial cells reside in complex multi-species bacterial communities, which are often associated with a host, such as the human microbiota. These bacterial communities are shaped by cooperative and competitive interactions amongst their members. Like higher animals, bacteria also compete with their conspecifics for nutrients and space. This evolutionary arms race resulted in a diverse set of strategies for microbial competition. In particular, bacteria residing on solid surfaces can compete with their neighbors through the use of specialized nanomachines, called secretion systems, enabling the direct delivery of toxic effector molecules into by-standing target cells. The most commonly used weapon for contact-dependent antagonism is the bacterial Type VI secretion system (T6SS). The T6SS belongs to the family of contractile injection systems (CISs). All CISs are structurally and functionally related to contractile bacteriophages (e.g. phage T4) and translocate proteins into target cells by means of physical force, which is generated by rapid sheath contraction. This results in the ejection of the inner tube associated with a sharp tip and effector proteins at its end. Effector translocation leads ultimately to target cell death. Importantly, the T6SS is capable translocating effectors across broad ranges of biological membranes making it a powerful weapon in microbial warfare as well as potent virulence mechanism towards eukaryotic host cells. Our current understanding of T6SS mode of action is primarily based on the combination of structural biology and fluorescence live-cell microscopy studies. While in particular cryo-electron microscopy (cryo-EM) revealed the detailed architecture of the T6SS in situ and of isolated subassemblies, fluorescence live-cell microscopy uncovered the remarkable dynamics of T6SS biogenesis. However, a complete understanding of T6SS dynamics is hampered in standard fluorescent microscopy due to: (i) the spatial and temporal resolution limit, (ii) the inability to efficiently label secreted components of the machinery, (iii) the weak signals due to low protein abundance and rapid photobleaching, (iv) the difficulty to perform long-term co-incubation experiments as well as (v) the inability to precisely control spatial and chemical environment. My doctoral thesis aimed to overcome these limitations to allow novel insights into dynamics of the T6SSs of Vibrio cholerae, Pseudomonas aeruginosa and Acinetobacter baylyi. Specifically sheath assembly, initiation of sheath contraction, T6SS mediated protein translocation in to sister cells as well as strategies for prey cell inhibition were studied in this thesis. First, I studied sheath assembly in ampicillin induced V. cholerae spheroplasts. These enlarged cells assemble T6SS sheaths which are up to 10x longer as compared to rod shaped cells. This allowed us to photobleach an assembling sheath structure and demonstrate that new sheath subunits are added to the growing sheath polymer at the distal end opposite the baseplate. Importantly, this was the first direct observation made for any contractile machines studied to date. Moreover, I showed that unlike for all other CISs, T6SS sheath length is not regulated and correlates with cell size. In order to monitor protein translocation into target cells, we developed a T6SS dependent interbacterial protein complementation assay, enabling the indirect detection of translocated T6SS components into the cytosol of recipient cells. This allowed us to demonstrate that secreted T6SS components are exchanged among by-standing sister cells within minutes upon initial cell contact. Importantly, these results were the first experimental indication that T6SS is capable of translocating its components into the cytosol of Gram-negative target cells. Furthermore, we showed that the amount and the composition of the secreted tip influences the number of T6SS assemblies per cell, whereas different concentration of the tube protein influenced sheath length. We also provided evidence that precise aiming of T6SS assemblies through posttranslational regulation in P. aeruginosa increases the efficiency of substrate delivery. In addition, together with two Nanoscience master students we have also been implementing microfluidics in the Basler laboratory. This powerful technology enabled us to control the spatial arrangements of aggressor and prey populations and to follow these populations at single-cell level over time scales of several hours. In collaboration with Prof. Kevin Forster, University of Oxford, we demonstrated that the rate of target cell lysis heavily influences the outcome of contact-dependent T6SS killing and thus drives evolution of lytic effectors. Moreover, microfluidics allows for the dynamic change of the chemical microenvironment during imaging experiments. By following the T6SS dynamics in response to hyperosmotic shocks resulting in a rapid cell volume reduction, we found that physical pressure from the collapsing cell envelope could trigger sheath contraction. This led us to propose a model for sheath contraction under steady-state conditions where continued sheath polymerization against membrane contact site leads to a gradual increase in pressure applied to the assembled sheath. We propose that this could be potentially sensed by the baseplate, which in turn would trigger sheath contraction