Structure, Dynamics and Activity of 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. This barrier needs to be traversed in a controlled manner to allow the import of nutrients, and removal of waste products, and for communication with the outside world. This is achieved by a wide range of proteins that reside in the various membranes. 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 gain a complete understanding about how proteins are exported-protein secretion. The secretory ('Sec' for short) machinery is essential for life-for every cell in every organism. The project concerns this process, in 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 or envelope.

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 from the cell interior through the Sec machinery. Proteins of the periplasm can readily fold and remain there, while those destined for the cell surface are delivered to another transport machine called the BAM complex, for incorporation into the outer-membrane. The journey from the inner- to the outer-membrane through the envelope, is poorly understood. We are concerned with the question: how do proteins make their way rapidly and efficiently through this very crowded and challenging environment?

We have discovered that the protein transport machineries of the inner- (Sec) and outer-membranes (BAM) as well as various accessory factors concerned with quality control interact to form an assembly that spans the bacterial envelope, which we have called the bacterial secretosome. The existence of a contiguous conduit through the envelope will have far reaching implications for our understanding of outer-membrane biogenesis, including an answer to the question posed above.

The project will harness complementary expertise in biochemistry and new breakthrough technologies for high resolution imaging (electron microscopy) and accurate mass measurements (mass spectrometry). This powerful combination will allow us to examine the activity, structure and dynamics of the assembly in order to understand how it works-how proteins are delivered through the inner-membrane to the periplasm and the outer-membrane. This knowledge is important because it will enlighten our understanding of a fundamental aspect of bacterial physiology, and inter-membrane transport throughout biology. Moreover, new advances in our understanding of the bacterial secretosome will help develop strategies to compromise envelope biogenesis and its regenerative capabilities-essential for survival. This would generate much needed ammunition in our fight against antimicrobial resistance (AMR).

Bacteria secrete proteins for a wide range of activities, including envelope biogenesis, pathogenicity and degradation of antibiotics. The major export route is via the ubiquitous Sec translocon. This proposal concerns the mechanism of this process and subsequent poorly understood downstream transit through the bacterial envelope.

The Gram-negative envelope is composed of an inner- and outer-membrane; sandwiched between them is the periplasm incorporating a peptidoglycan (PG) layer. The mechanism of transport across the inner-membrane is relatively well understood. However, it is unclear how they are then rapidly and specifically sorted into the periplasm, or delivery to the outer-membrane-all done in the absence of energy.

We have identified interactions between the holo-translocon (HTL), periplasmic chaperones and the beta-barrel assembly machinery (BAM), forming a super structure spanning the entirety of the envelope. This assembly-the secretosome-could form a contiguous conduit for efficient and quality controlled passage of proteins to the outer-membrane. Its existence has far reaching implications for our understanding of envelope biogenesis.

Our exploration of this unexpected aspect of bacterial biology will harness complementary expertise in biochemistry and cryo-EM at Bristol, combined with recent technological advances for hydrogen deuterium exchange mass spectrometry (HDX-MS), with cyclic ion mobility for enhanced resolution, in Manchester. This powerful combination will enable an interrogation of the activity, structure and dynamics of the secretosome-leading to greater knowledge of the molecular mechanisms underlying protein transport across the inner-membrane and through the envelope. The project will enlighten our understanding of a fundamental process, essential for survival. Moreover, the results will suggest strategies to subvert the process of envelope biogenesis and maintenance, and thereby help towards the development new antibiotics.