One of the ways by which our immune systems attack and kill bacteria in the blood is by assembling protein complexes—think of them as nanomachines—that effectively punch minute holes in the bacterial cell membranes. Scientists at University College London (UCL) in the U.K. have now used rapid atomic force microscopy (AFM) to film how these membrane attack complexes (MACs) are formed and create the miniscule, 11-nm diameter pores in bacterial cell membranes.

Reported today in Nature Communications (“Single-molecule kinetics of pore assembly by the membrane attack complex,”) the studies also showed when in the process a failsafe mechanism kicks in to prevent MACs from harming the body’s own cells. The researchers hope that the new insights may help in the development of immune system-based strategies against antibacterial infections and potentially against cancer.

membrane attack complexes
So-called membrane attack complexes—imaged on the back of a bacterium. The scale bars correspond to 800 (left) and 30 (right) nm. [EMBO Journal]

The formation of MAC-generated pores in bacterial membranes occurs through activation of the complement system, a key component of the immune system that scans the body for pathogenic bacteria, and effectively “complements” the ability of white blood cells to kill these invading microorganisms, the authors explained.

Studies have linked dysregulation of MAC formation with the development of disease, while treatments that are designed to control complement proteins are being developed for cancer immunotherapy. “Understanding how complement proteins assemble from innocuous soluble monomers into killer transmembrane pores can, therefore, contribute to developing strategies for treating human disease where the MAC is implicated, and for repurposing the complement system as a potent immunotherapeutic,” the researchers continued.

A video sequence of the formation of a hole in a bacterial surface, recorded at 6.5 seconds per frame. The scale bar (see first frame) corresponds to 30 nanometres. Credit: Edward S. Parsons et al.
A video sequence of the formation of a hole in a bacterial surface, recorded at 6.5 seconds per frame. The scale bar (see first frame) corresponds to 30 nm. [Edward S. Parsons et al.]

MAC assembly occurs through a complex, stepwise process by which soluble complement proteins are sequentially recruited, bind, and undergo structural rearrangements to form the transmembrane MAC pore. Seven different polypeptides are involved, designated C5b, C6, C7, C8 (a hetero-trimer comprised of C8α, C8β, and C8γ), and C9. The final stage involves the recruitment of 18 copies of C9, which complete the pore.

Although prior crystallographic studies and other analyses have identified roles for the different polypeptides, what hasn’t been clear is which are the rate-limiting steps in the assembly pathway, the UCL scientists noted. The body’s own cells produce a cell surface receptor, CD59, which effectively stops the MACs from assembling and punching holes in host cells, but when in the process this failsafe kicks in hasn’t been understood. “The kinetics of MAC formation must allow a temporal window such that inhibitory factors can interfere at appropriate stages in the assembly pathway,” the team noted.

To gain further insights into the molecular mechanisms and kinetics that underpin the process of MAC-generated pore formation, the UCL team, and colleagues at the Swiss Federal Institute of Technology in Lausanne (Switzerland), and the University of Leeds (U.K.) used rapid AFM imaging to effectively film the formation of single pores in model bacterial membranes.

They found that the MAC formation process involves the construction of a complex of complement proteins that binds to the bacterial membrane, and then recruits each of the 18 C9 proteins sequentially to form the pore structure.

By tracking each step researchers showed that the pore-forming assembly line is temporarily halted as the first of the C9 proteins binds to the C5b-8 MAC precursor and is inserted into the bacterial membrane. This is the rate-limiting step that “allows a maximum temporal window for the mechanism by which human cells are protected from autoimmune attack by the MAC,” the investigators wrote. Effectively, it’s at this point that CD59 can step in to block continued pore formation, although the structural basis by which CD59 inhibits the complement proteins isn’t clear, the team noted.

“It appears as if these nanomachines wait a moment, allowing their potential victim to intervene in case it

is one of the body’s own cells instead of an invading bug, before they deal the killer blow,” explained lead study author Edward Parsons, PhD, at the UCL London Centre for Nanotechnology. “It is the insertion of the first protein of the membrane attack complex which causes the bottleneck in the killing process, added corresponding author Bart Hoogenbom, PhD, professor of biophysics at UCL’s department of physics & astronomy. “Curiously, it coincides with the point where hole formation is prevented on our own healthy cells, thus leaving them undamaged.”

The findings also support what’s known about the activity of the anticancer immunotherapies including rituximab. “When CD59-mediated inhibition is overcome by antibody-based drugs such as rituximab that facilitate MAC-induced killing of chronic lymphocytic leukemia B cells, cell death follows at the ~100 s time scale,” the authors stated.

They suggest that their results effectively define the kinetic basis for MAC assembly, and provide “insight into how human cells are protected from bystander damage by the cell surface receptor CD59, which is offered a maximum temporal window to halt the assembly at the point of C9 insertion … These assembly kinetics govern how MAC kills bacteria and how our body’s self-defense mechanism prevents membrane damage, which may also be relevant for complement dependent cytotoxicity in cancer immunotherapy.”

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