Aim 1: Molecular scale – Build an atomic model of the Type 6 Secretion System (T6SS) membrane complex in extended and fired conformational states
1A: We intensely screened a number of different possible model organisms and growth conditions and also engineered skinny E. coli and P. aeruginosa cells. We also identified Amoebophilus and Aureispira as promising divergent systems.
1B: We established a pipeline for state-of-the-art cryo-electron tomography and subtomogram averaging and also published the methods [4/9]. The pipeline allowed us to determine the structures of the trans-envelope complex in Amoebophilus in extended and contracted confirmations [1]. In a collaborative study, we solved atomic resolution structures as well as in situ structures of TssJLM in E. coli [5]. The T6SS in Aureispira revealed as yet unknown structural diversity [Lien et al, manuscript in preparation]. Strikingly, we identified a new mode of action of a contractile injection system in cyanobacteria and revealed its anchoring to thylakoid membranes at molecular detail [Weiss et al, in press, Nature Microbiology].
1C: The identification of subcomplexes was achieved by single particle cryoEM of purified complexes [5; Weiss et al, in press, Nature Microbiology; Xu et al, in press, Nature Microbiology]. These structures revealed mechanistic insights and evolutionary hotspots.
Finally, on the molecular scale we also determined the mechanism of effector loading into a contractile injection system [7].
Aim 2: Cellular scale – Understand clustering and coordination of multiple T6SS machines
2A: We did not make progress on understanding the clustering of T6SSs in Pseudomonas. Instead we discovered the presence of bidirectional firing in E. coli T6SSs [6].
2B: Our landmark study of clustered T6SSs in Amoebophilus revealed that neighboring T6SSs interact laterally via their baseplates and are therefore arranged in hexagonal patters [1].
Aim 3: Intercellular scale – Image T6SS engaged in cell-cell interactions
3A: We successfully developed the methodologies to image cell-cell interactions by cryo-electron tomography. First, we developed a highly-successful cryo-sample thinning technique by cryo-focused ion beam milling [1/2/4/12]. Second, we developed cryo-light microscopy workflows in order to find rare events of interest [6]. Further improvements are currently being explored and will be published in 2022. Third, we advanced tomography data collection procedures [9].
3B: We employed the new methods to study T6SS interactions between bacteria and were able to capture the killing event by tomography [Lien et al, manuscript in preparation].
3C: We also employed to methods to study T6SS-mediated interactions with eukaryotic cells, i.e. Amoebophilus-amoeba interactions [1] and Algoriphagus-choanoflagellate interactions [Xu et al, in press, Nature Microbiology]. Besides these significant advances, we employed the methods in seminal studies to understand other types of cell-cell interactions in cyanobacteria [11], uropathogens [16/17], Legionella [21], Chloroflexi [15], and Salmonella [13/14].
In conclusion, we reached our goal of generating true multiscale models of different macromolecular complexes that mediate cell-cell interactions. These models delivered insights on structure, mechanism, function and evolution.
