The Bacterial Secretosome

All cells are surrounded by membranes, made up from a double layer of fatty molecules called phospholipids. Cell membranes act as a molecular "skin", keeping the cell's insides in, and separating different biochemical reactions. The barrier needs to be breached in a controlled manner to allow transport of nutrients, waste products and for communication with the outside world; this is achieved by a wide range of membrane-inserted proteins. We understand a great deal about the diverse biological functions that membrane proteins bestow, such as transport, respiration, photosynthesis. However, we know very little about how membranes are formed, or about the necessary transport of proteins across or into membranes during their biogenesis.

Our proposal aims to understand more about how the cell's protein secretion occurs. The secretory ('Sec' for short) machinery is essential for life - for every cell in every organism. The project concerns this process, in very simple bacterial cells. Bacteria secrete proteins for a wide range of membrane and extracellular activities including for: cell adherence, pathogenicity, the degradation of antibiotics, including also the biogenesis of the protective cell wall.

A major class of bacteria known as Gram-negatives, possess a cell wall composed of a periplasm with a peptidoglycan (PG) layer, surrounded by an outer-membrane. The biogenesis of the cell wall is dependent on protein secretion through from the cell interior through the Sec machinery. Proteins of the periplasm can readily fold and remain there. Proteins are also transported across or into the outer membrane by another transport machine called the BAM complex, but it is not clear how they are shuttled there, to ensure the process is rapid and efficient.

We have identified an interaction between the Sec machinery of the inner plasma membrane and the BAM complex, forming a structure that spans the entirety of the cell wall. This giant assembly, which we have called the bacterial 'secretosome', could form a contiguous conduit for very efficient passage of proteins from the cytosol to the outer-membrane. Its existence will have far reaching implications for our understanding of outer-membrane biogenesis.

The project will harness complementary expertise in biochemistry and new breakthrough technologies in imaging by light and high-resolution electron cryo-microscopy. These scientific methods will illuminate the architecture of the secretosome, and how it works. The results of the project will be important because the bacterial cell wall, is vulnerable to attack. The weakening of the cell wall, or a compromise in its biogenesis or regenerative capabilities could be lethal. Therefore, new information towards our understanding of the bacterial secretosome, and its action in the maintenance of the cell wall, could suggest ways in which it could be subverted towards the development of new antibiotics. This would generate much needed ammunition in our fight against antimicrobial resistance (AMR).

Protein secretion is essential for life. Bacteria secrete proteins for a wide range of membrane and extracellular activities, including envelope biogenesis, pathogenicity and degradation of antibiotics. The major route for this process is via the ubiquitous Sec machinery of the bacterial plasma membrane. This proposal concerns the mechanism of this process and subsequent poorly understood downstream transit through the bacterial envelope, and ultimately the biogenesis of the Gram-negative outer-membrane.

Gram-negative bacteria possess a cell wall composed of a periplasm with a peptidoglycan (PG) layer, surrounded by an outer-membrane. So how does the bacterial cell ensure rapid and specific sorting of secreted protein for folding into the periplasm, or delivery to the outer-membrane, all done in the absence of energy? The mechanism of ATP driven transport across the inner-membrane by the Sec machinery is relatively well understood. Quality control systems are in place to ensure folding or, if required, degradation of resident periplasmic proteins. However, the route for outer-membrane proteins to the beta-barrel assembly machinery (BAM) is less clear.

We have identified an interaction between the bacterial holo-translocon (HTL) with a periplasmic chaperone and BAM, forming a structure that spans the entirety of the cell envelope. This giant assembly -the bacterial secretosome- could form a contiguous conduit for efficient passage of proteins from the cytosol to the outer-membrane. Its existence will have far reaching implications for our understanding of outer-membrane biogenesis.

The project will harness complementary expertise in biochemistry and high-resolution electron cryo-microscopy (cryo-EM) to explore the structure and mechanism of this new and unexpected aspect of bacterial biology. Moreover, the description of the mechanism of the secretosome will suggest new strategies to subvert outer-membrane biogenesis towards the development new antibiotics.

The overarching and immediate aim of the proposal is to gain an understanding of an important fundamental aspects of bacterial biology: protein secretion and Gram-negative outer-membrane biogenesis. The immediate impact in terms of the current project will lie in scientific advancement and the generation of new knowledge. The project will also present new hypothetical concepts that if proven to be true will have a major impact in our understanding of the bacterial envelope, and have important implication for the development of effective treatments against bacterial infections.

The main areas of impact are:
1. Application and exploitation. While the proposed project is at a "pre-competitive" stage in terms of commercial exploitation, the knowledge generated will have an immediate benefit to both the National and International bioscience community (academic and commercial) in terms of understanding a fundamental process that spans the breadth of biology. The process is of fundamental importance for bacterial survival and certain complex components are specific to bacteria. The bacterial envelope and its biogenesis are particularly vulnerable to attack; its weakening by, for instance, antibiotics can be lethal. Therefore, the subject of this proposal is a particularly fertile area, with respect to the development of new antibiotics and for strategies against anti-microbial resistance (AMR). Therefore, in the medium term the work could lead to new approaches/ targets for antimicrobial drug development. The knowledge gained could support an ongoing drug discovery programme (collaboration with Dr A. Woodland, Drug Discovery Unit, Dundee) aimed at the identification of small molecule inhibitors of the bacterial secretion. Bristol has mechanisms in place to increase the impact of research and to exploit any commercialisation (see main impact summary).

2. Engagement. The benefits to the bioscience community are summarised above. The standard routes to information dissemination (e.g. pre-print submissions, papers in journals and presentations at conferences) will be used throughout the duration of the project. A more general benefit of our work to the UK stems from our commitment to public engagement. The PI and PDRAs routinely participate in public engagement activities, from school children to politicians, and for the promotion science to women and girls. The group will continue with public engagement activities throughout the course of the project, using work generated from the project to exemplify the importance of research.

3. Staff training. The project will ultimately generate trained staff with desirable expertise in complex biochemical and biophysical analysis of membrane protein complexes that are involved in important bacterial activities. The researchers will be in demand in both the academic and commercial sectors. During the project, further PDRA development will be encouraged through attending courses in areas directly and indirectly related to their role as research scientists (e.g. project management and leadership).