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.
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.
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
Benn G
(2019)
Imaging live bacteria at the nanoscale: comparison of immobilisation strategies.
in The Analyst
Pang SS
(2019)
The cryo-EM structure of the acid activatable pore-forming immune effector Macrophage-expressed gene 1.
in Nature communications
Parsons ES
(2019)
Single-molecule kinetics of pore assembly by the membrane attack complex.
in Nature communications
Heesterbeek DA
(2019)
Bacterial killing by complement requires membrane attack complex formation via surface-bound C5 convertases.
in The EMBO journal
Suthar J
(2020)
Acoustic Immunosensing of Exosomes Using a Quartz Crystal Microbalance with Dissipation Monitoring.
in Analytical chemistry
Shah NR
(2020)
Structural basis for tuning activity and membrane specificity of bacterial cytolysins.
in Nature communications
Description | Disruption And Resistance In Bacterial Cell Envelopes Challenged By Polymyxins |
Amount | £315,400 (GBP) |
Funding ID | BB/X001547/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 03/2023 |
End | 03/2026 |
Description | Turnkey video-rate atomic force microscopy for nanometre resolution imaging of functional biomolecules and cellular surfaces |
Amount | £412,467 (GBP) |
Funding ID | BB/W019345/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 07/2022 |
End | 07/2023 |
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 |
Title | Molecular-resolution imaging of living bacterial cells |
Description | A method to resolve molecules and molecular-scale changes at the surface of living bacteria |
Type Of Material | Technology assay or reagent |
Year Produced | 2021 |
Provided To Others? | Yes |
Impact | A review of the textbook image of bacterial outer membranes, next having initiated research on how these are affected by antibiotics |
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 | Contract research for Procter & Gamble |
Organisation | Procter & Gamble |
Department | Procter & Gamble (United Kingdom) |
Country | United Kingdom |
Sector | Private |
PI Contribution | Providing microscopy data on how P&G materials affect bacteria |
Collaborator Contribution | Technical advise and materials |
Impact | Characterisation data on commercial reagents |
Start Year | 2021 |
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 |
Description | Utrecht collaboration |
Organisation | University Medical Center Utrecht (UMC) |
Country | Netherlands |
Sector | Academic/University |
PI Contribution | We carry out nanoscale characterisation of life bacteria as they are attacked by immune proteins (complement) in serum, with the aim to determine which are the mechanism by which the immune system clears our body from harmful bacteria. |
Collaborator Contribution | Scientific expertise, different strains of bacteria, purified proteins. |
Impact | We have identified a new role of certain immune enzymes in killing bacteria. This can now be used to guide immune therapies to bacterial infections. |
Start Year | 2016 |
Description | School talk |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Schools |
Results and Impact | Enthusing school children for microscopy research in the framework of science week |
Year(s) Of Engagement Activity | 2024 |