Depicting the proposed mechanism by which MPEG1 acts to kill bacteria within the macrophage phagolysosome. MPEG1 orientation prevents damaging the macrophage membrane.

PAPER: The Cryo-EM structure of the acid activatable pore-forming immune effector Macrophage-expressed gene 1

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Scientists make inroads in understanding how immune cells kill bacteria

Gaining knowledge on how our bodies manage to kill bacteria helps scientists learn why one person’s immune system may be working better, worse or differently than others. This is key to ensuring we understand how to fight infections and unlocking the mysteries of our immune systems. The work is published in the journal Nature Communications.

A macrophage is a type of immune cell that is responsible for detecting, engulfing and destroying pathogens, particularly bacteria. By engulfing the bacteria, a macrophage can safely kill the bacteria inside a small sac it creates, known as a phagolysosome. Once this process is complete it then passes debris and information onto other immune cells, kick starting an immune process that allows our bodies to clear the infection.

Scientists know from previous studies that proteins inside the phagolysosome can create small holes in the membrane, known as a pore, to help destroy the engulfed bacteria. One such protein, MPEG1, which this study focuses on, is in a super family of proteins that are known to attack membranes by making pores.

“We knew MPEG1 was found in macrophages, and we also knew that it was related to the membrane attack complex, which targets bacteria. But we didn’t know how the MPEG1 targeted the bacteria inside the macrophage, and we didn’t know how it didn’t damage itself while destroying the bacteria.” said A/Professor Michelle Dunstone, Associate Investigator from the ARC Imaging Centre of Excellence.

Now, in a world-first, the scientists from the ARC Centre of Excellence in Advanced Molecular Imaging have used cryo-Electron Microscopy (cryo-EM) to discern the first three-dimensional views of MPEG1. Their work demonstrates that MPEG1 is in a state primed and ready to make a pore. Importantly, this exists in macrophages but is inactive until the phagolysosome produces acid (which occurs during the maturation of a phagolysosome). The change in pH activates the pre-pore structure, causing the MPEG1 protein to rearrange themselves into the final pore structure. This impressive change in shape enables pore forming proteins, like MPEG1, to punch holes into bacterial cell membrane surface leading to their death.

Additionally, the study also revealed that MPEG1 seems to tether itself to the macrophage membrane and points the pore forming machinery away from the macrophage cell. This way it can stay attached to the membrane of the phagolysosome, while damaging nearby engulfed bacteria. This appears to be a unique property of this protein and might be another way through which the sac safeguards punching a hole into itself.

“We now know that the immune role of MPEG1 is similar to that of other proteins in this family, but that it only begins to form pores through the process of acidification. This is crucial as it shows us a novel mechanism by which macrophages can control and contain pore-forming proteins like MPEG1 whilst keeping itself safe.” said Prof James Whisstock, Director of the ARC Imaging Centre of Excellence.

The researchers achieved this by purifying MPEG1 from cells and structurally characterising the molecules using advanced imaging techniques, such as cutting edge cryo-electron microscopy and atomic force microscopy.

“What was fascinating was really a simple question. How does a cell keep itself from harm while harbouring such a dangerous molecular weapon? Cells are delicate, imagine a balloon that has a pin inside it, that it needs for protection, but at the same time it has to ensure that it isn’t harmed by the pin itself. This is the same problem our immune system has had to face in the case of MPEG1. How to kill intracellular bacteria without harming the immune cell itself.” said Mr Charles Bayly-Jones, a PhD Student in Professor Whisstock’s team.

“This may be able to help us understand why some bacteria might be resistant to destruction by macrophages. This will then give us a better idea of how to keep the immune system functioning, or what may be occurring to prevent it from functioning at its optimal ability.” said Dr Siew Siew Pang, a Postdoctoral Researcher from the ARC Imaging Centre of Excellence.

“In real-world terms, this knowledge can help us understand why MPEG1 mutations would play a role in patients suffering from certain bacteria, such as pulmonary nontuberculous mycobacterial infections. said Mr Bayly-Jones.

What remains to be shown is whether the MPEG1 structure stays attached to the phagolysosome, while creating pores in the engulfed bacteria. It may detach from the phagolysosome due to some unknown process, before behaving much like other pore-forming proteins would. These are the questions the ARC Scientists may tackle next.

ENDS

The work was a collaboration of ARC Centre of Excellence in Advanced Molecular Imaging, Biomedicine Discovery Institute (Monash University), Thermo Fischer Scientific, London Centre for Nanotechnology (University College London), Institute of Structural and Molecular Biology (University College London), Department of Physics and Astronomy (University College London), Peter MacCallum Cancer Centre, Department of Genetics (The University of Melbourne), School of Medical Science (University of New South Wales), EMBL Australia and ACRF Department of Cancer Biology and Therapeutics (John Curtin School of Medical Research).

About the ARC Centre of Excellence in Advanced Molecular Imaging
The $39 million ARC-funded Imaging CoE develops and uses innovative imaging technologies to visualise the molecular interactions that underpin the immune system. Featuring an internationally renowned team of lead scientists across five major Australian Universities and academic and commercial partners globally, the Centre uses a truly multi-scale and programmatic approach to imaging to deliver maximum impact.

The Imaging CoE is headquartered at Monash University with four collaborating organisations – La Trobe University, the University of Melbourne, University of New South Wales and the University of Queensland.

www.imagingcoe.org

About the Monash Biomedicine Discovery Institute
Committed to making the discoveries that will relieve the future burden of disease, the newly established Monash Biomedicine Discovery Institute at Monash University brings together more than 120 internationally-renowned research teams. Our researchers are supported by world-class technology and infrastructure, and partner with industry, clinicians and researchers internationally to enhance lives through discovery.

Written by Aimee Sanderson
Contact: Juliana Villa-Ortiz, 0437 119 498, juliana.villa-ortiz@monash.edu

Image by Charles Bayly-Jones. Depicting the proposed mechanism by which MPEG1 acts to kill bacteria within the macrophage phagolysosome. MPEG1 orientation prevents damaging the macrophage membrane.