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Multi-scale model of bacterial cell-cell interactions

Periodic Reporting for period 4 - T6S (Multi-scale model of bacterial cell-cell interactions)

Reporting period: 2021-07-01 to 2021-12-31

Bacteria are only rarely found as individual cells in the environment and often interact with other organisms. These interactions are usually mediated by macromolecular machines. The goal of this project was to establish multiscale models of these macromolecular machines, i.e. datasets that integrate information from different scales of resolution (molecules to cells to multicellular assemblies). In order to achieve the biological insights, the project had a strong component of development of enabling imaging methods. The biological focus was on contractile injection systems, in particular the Type 6 Secretion System. Besides this system, the new methods were also employed to understand other types of cell-cell interactions.

The project generated insights on the structure, mechanism, function and evolution of the particular macromolecular machines mediating cell-cell interactions.
The project also delivered new methods that can be employed to other problems in the future.

Understanding bacterial cell-cell interactions is important for the fields of infection biology, evolutionary biology, environmental biology, biotechnology and medical sciences.
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.
The following publications are considered most significant with respect to this project, as they represent major advances:

1. Böck et al., Science, 2017
11. Weiss et al., Cell, 2019
12. Zachs et al., Elife, 2020
16. Weiss et al., Science, 2020
XX. Xu et al., Nature Microbiology, in press
XX. Weiss et al., Nature Microbiology, in press

There were also several unexpected findings and developments. First, the diversity of contractile injection systems was much higher than expected. This led to the discovery of a novel mode of action in cyanobacteria (Weiss et al, in press, Nature Microbiology). In combination with the study on cell-cell junctions in the same organism, this led to fundamentally novel aspects of cell biological mechanisms. Second, the bioinformatic analyses revealed a much higher abundance of contractile injection systems than previously thought. This led to the discovery of novel systems that may be useful for biomedical applications in the future. Third, the technology of cryo-electron tomography advanced at an unprecedented pace. This allowed us to tackle projects that were not thought to be feasible at the beginning of the project.

Besides insights into the biology of bacterial cell-cell interactions, we made advances in developing methods for cryo-electron tomography, cryo-light microscopy and cryo-focused ion beam milling (detailed above). These new methods were significant for the success of the project but also allowed the exploration of other cell-cell interactions. The automation of cryo-focused ion beam milling is now implemented on many other instruments world-wide.
T6SS membrane complex
T6SS multibarrel guns from amoebophilus
cyanobacterial contractile injection system