Background
The complement system is an essential branch of our humoral immune system that is responsible for clearing invading bacteria. Upon recognition of bacteria by complement proteins, bacteria are labelled with proteolytic enzymes called convertases. These convertases can cleave complement component C5 into C5b which together with C6, C7, C8 and multiple copies of C9 initiates the assembly of the Membrane Attack Complex (MAC). The MAC is a large pore-forming protein complex that can directly kill Gram-negative bacteria and is therefore essential in preventing bacterial infections.
Research aim
Although it has long since been established that the MAC is essential in killing bacteria, the precise molecular mechanism by which it kills bacteria remains unclear. This study aims to characterize the molecular steps of MAC assembly to understand how the MAC kills bacteria.
Methodology & results
E. coli have been labelled with convertases in a purified manner to measure pore formation and killing by flow cytometry. We show that the formation of bactericidal pores requires the MAC precursor C5b6 to be retained at the target surface. Although C5b6 that is released from the surface can still form a pore that permeates the bacterial outer membrane, this pore lacks ability to damage the inner membrane and kill the bacterium. Co-elution of convertases shows that they are in complex with C5b6 and thus retain C5b6 at the target surface. Finally, we show that these locally generated pores are more stably inserted into the membrane by atomic force microscopy and are resistant to cleavage by trypsin, implying these pores are also more deeply inserted into the membrane.
Conclusion
In conclusion, we show that the role of convertases extends beyond the cleavage of C5. Local formation of C5b6 by surface-bound convertases properly inserts MAC pores into the bacterial membrane to ensure killing of Gram-negative bacteria.