Dynamics of bacterial killing by the membrane attack complex

Lead Research Organisation: University College London
Department Name: London Centre for Nanotechnology

Abstract

To avoid the spreading of bacteria in the blood stream, our immune system contains the so-called complement system, a large arsenal of proteins that collectively promote inflammation and target microbes. One of complement's rather spectacular functions is that of piercing the membranes of Gram-negative bacteria such as E. coli, thus killing the bacteria. In some cases, we may wish to enhance this immune function, for example to counter bacterial infections; in other cases, such as sepsis, it may be beneficial to prevent a patient's overactive immune system from causing potentially lethal effects. For the development of any such therapeutic approaches, it helps to have a sound understanding of how complement works.

In this project, we focus on the membrane attack complex, used by complement to form pores in the membranes of bacteria. So far, many structural and functional studies have been carried out on the MAC attacking single-membrane model systems such as lipid vesicles and red blood cells. However, the envelope of Gram-negative bacteria consists of a double membrane with a mesh-like peptidoglycan layer in between. At present, it is not clear how the MAC overcomes this triple barrier. To address this question, we will use a special type of microscopy, atomic force microscopy, in which a sharp needle can trace the surface of a bacterium as it is being attacked by the MAC. Briefly, by thus visualising the formation of MAC on the bacterial surface and the subsequent effects on the bacterial envelope, we will determine the mechanisms by which the MAC kills bacteria.

Technical Summary

Complement is a key part of the innate immune system. Its activation can lead to the formation of pore forming membrane attack complexes (MACs) to lyse bacteria. Much of our current understanding of the MAC results from studies of the MAC perforating single membrane model systems such as synthetic lipid bilayers and red blood cells. However, in physiological context, the MAC targets the much more complex and composite bacterial envelope, and - while of great medical relevance - its mechanisms of (anti)bacterial attack remain unclear. With this project we aim to understand the sequence of events from MAC formation on the bacterial envelope to bacterial killing, presumably by lysis. To this end, we will carry out atomic force microscopy experiments, resolving the MAC as it is assembling and the bacterial envelope as it is degraded, and identify the different steps in membrane pore formation and bacterial killing by the MAC.

Planned Impact

Infections by Gram-negative bacteria such as E. coli are on the rise, and in a broader context antimicrobial resistance poses a significant threat to human health. These developments make it imperative to research new therapeutic avenues that prevent or target bacterial infections. One route is to optimise bacterial killing by the immune system, e.g., by labelling bacteria for lysis by the membrane attack complex. Such approaches are highly dependent on molecular-scale insight into the immune system and in particular into its ways of killing bacteria.

On the hand, at least 30 different diseases can be traced to unwanted complement activation and there is an urgent medical need for improved treatments. In sepsis, excessive production of the complement component C5a is thought to trigger a series of events leading to septic shock, multi-organ failure, and lethality. It can therefore be an effective target for antibody therapy, but - since many C5a-targetting antibodies also target C5 - implies the risk of disabling the formation of the membrane attack complex and its function of lysing Gram-negative bacteria. Again, it is desirable to better understand how the membrane attack complex kills bacteria, here to better identify which parts of the complement activation pathway can and cannot be targeted without prohibitive side effects.

In summary, we expect our research to be able to guide new therapeutic developments against bacterial infections and against unwanted or excessive complement activation such as in sepsis. Such developments can have a major impact on health care and promote activities in biotechnology and pharmaceutical industry.

Our microscopy-based approach has the additional advantage of yielding powerful images and movies of biomolecular machines at work. E.g., atomic force microscopy has provided real-time images of the molecular motor myosin V walking along an actin filament; published in 2010 (by Ando's group in Kanazawa), these images have already become textbook material, changing students' view on the molecular machinery that underpins health and disease. Moreover, such images have a great appeal to a broad audience, conveying the importance and beauty of science. By filming the immune system at work while killing bacteria, we anticipate to generate molecular-scale movies of similar appeal, thus promoting science to (potential) students, medics, and lay audiences.

Publications

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Title Correlative microscopy method 
Description We have developed and applied a method to analyse protein binding to supported lipid bilayers across microscopy platforms 
Type Of Material Biological samples 
Year Produced 2018 
Provided To Others? No  
Impact We used this method to determine the rate-limiting step in the assembly of the membrane attack complex, one of the molecular machines of the immune system to kill Gram-negative bacteria. We have also determined the pharmacokinetic parameters for drug binding to its cell surface receptor, in collaboration with MedImmune Ltd. Results are expect to be published over the coming year. 
 
Title High-resolution microscopy of bacteria 
Description We have developed microscopy protocols to image the bacterial outer membrane at ~1 nm resolution. 
Type Of Material Technology assay or reagent 
Year Produced 2019 
Provided To Others? Yes  
Impact Not yet 
 
Description Collaboration with Oxford University (Colin Kleanthous lab) 
Organisation University of Oxford
Country United Kingdom 
Sector Academic/University 
PI Contribution Our investigations have led to higher-resolution views of the bacterial outer membrane and contribute to understanding its organisation.
Collaborator Contribution Our partners have provided us with scientific advice and labels to chemically identify structures we observed on the outer membrane.
Impact Not yet.
Start Year 2019
 
Description Partnership with Princeton (Tom Silhavy) 
Organisation Princeton University
Country United States 
Sector Academic/University 
PI Contribution Our microscopy data have revealed details on the molecular organisation of the outer membrane.
Collaborator Contribution Our partners have provided different bacterial strains and scientific advice to help us interpret our results.
Impact Not yet
Start Year 2019