Localization matters : new insights into spatio-temporal regulation of type VI secretion systems

Subcellular organization is important for bacterial cell physiology. Especially, bacterial secretion systems are tightly regulated in a temporal and spatial manner to efficiently fulfill their function. Among them, the contact-dependent Type VI secretion (T6SS) has an important role in inter-bacterial competitions and pathogenicity of Gram-negative bacteria.
T6SS translocates effector proteins into target cells using the contraction of a long cytosolic sheath, which pushes an inner tube together with a sharp tip and associated effectors across target cell membranes. This mode of action allows bacteria to use T6SS against a broad range of prokaryotic and eukaryotic organisms. However, the contact-dependency limits the target range and the efficiency of effector translocation is low because only a small number of effectors can be delivered per one round of T6SS assembly.
Recent advances in live-cell fluorescence and super resolution microscopy led to the revelation that T6SS activation patterns and dynamics are surprisingly diverse in different bacteria. These differences in T6SS assembly dynamics likely reflect different strategies to overcome the disadvantages of T6SS mode of action. However, the spatio-temporal regulation behind these different T6SS firing patterns are not well understood.
My PhD thesis provides new insights into how different subcellular localizations of T6SS assembly are achieved.
The Threonine phosphorylation pathway (TPP) is a unique posttranslational regulation mechanism, which allows Pseudomonas aeruginosa to activate its T6SS apparatus in response to membrane damage inflicted by an attack from neighboring bacteria and to localize it to the site of attack. While the involved components are identified, it is not clear how the periplasmic sensor module integrates spatial and temporal information for precise and fast T6SS assembly initiation. To test if relocation of TPP components from outer to inner membrane (IM) is important for T6SS activation, I changed their subcellular localization by mutating their N-terminal signal sequences. Relocation of one TPP component to IM indeed hyper-activated T6SS assembly, however, the exact mechanism of T6SS localization remains to be elucidated.
In collaboration with Prof. Kevin Foster, University of Oxford, we tested the benefit and cost of TPP-dependent localization of T6SS during bacterial competitions. Our results from in silico and imaging experiments suggested that P. aeruginosa uses TPP to kill competing bacteria by localized and repeated T6SS assemblies and thus inflicting more damage than it encounters from attacking competitors.
In collaboration with Prof. Petr Broz, University of Lausanne, we characterized the unique Francisella pathogenicity island (FPI), which encodes a non-canonical T6SS essential for phagosomal escape. The FPI lacks a specialized unfoldase required for recycling of contracted sheaths and for dynamics of canonical T6SS. Furthermore, the FPI encodes genes with unknown function. By live-cell fluorescence microscopy, we showed that F. novicida T6SS dynamics is comparable to canonical T6SS dynamics. Moreover, we found that general-purpose unfoldase ClpB recycles contracted sheaths and is essential for phagosomal escape in vivo. By analyzing T6SS dynamics and virulence of single deletion mutants in vitro and in vivo, we could group FPI components with unknown function into structural components, which are required for T6SS function, and putative effectors, which are critical for virulence but not for T6SS assembly.
Moreover, we showed that F. novicida T6SS assembles exclusively at bacterial poles. This unique polar localization raised the question of how Francisella T6SS is localized to the poles and whether it is important for T6SS function. I analyzed the dynamics of membrane complex formation, which is the first step of T6SS assembly, by live-cell fluorescence microscopy and structured illumination microscopy. I showed that the membrane complex is stably formed on the poles even in the absence of other FPI components. In addition, the membrane complex formation was insufficient to initiate sheath assembly indicating that additional signals are required to activate T6SS in F. novicida.
To investigate the contribution of FPI components and localization of T6SS to Francisella virulence in more detail, I established Galleria mellonella larvae as infection model. Besides, I constructed two expression plasmids for F. novicida, which are mobilized by conjugation and have tetracyline inducible promoters for tunable gene expression. These new tools will be invaluable in the future research of mechanism required for F. novicida pathogenesis.