Polymerization of C9 enhances bacterial cell envelope damage and killing by membrane attack complex pores

Abstract

Complement proteins can form membrane attack complex (MAC) pores that directly kill Gram-negative bacteria. MAC pores assemble by stepwise binding of C5b, C6, C7, C8 and finally C9, which can polymerize into a transmembrane ring of up to 18 C9 monomers. It is still unclear if the assembly of a polymeric-C9 ring is necessary to sufficiently damage the bacterial cell envelope to kill bacteria. In this paper, polymerization of C9 was prevented without affecting binding of C9 to C5b-8, by locking the first transmembrane helix domain of C9. Using this system, we show that polymerization of C9 strongly enhanced damage to both the bacterial outer and inner membrane, resulting in more rapid killing of several Escherichia coli and Klebsiella strains in serum. By comparing binding of wildtype and ‘locked’ C9 by flow cytometry, we also show that polymerization of C9 is impaired when the amount of available C9 per C5b-8 is limited. This suggests that an excess of C9 is required to efficiently form polymeric-C9. Finally, we show that polymerization of C9 was impaired on complement-resistant E. coli strains that survive killing by MAC pores. This suggests that these bacteria can specifically block polymerization of C9. All tested complement-resistant E. coli expressed LPS O-antigen (O-Ag), compared to only one out of four complement-sensitive E. coli. By restoring O-Ag expression in an O-Ag negative strain, we show that the O-Ag impairs polymerization of C9 and results in complement-resistance. Altogether, these insights are important to understand how MAC pores kill bacteria and how bacterial pathogens can resist MAC-dependent killing.

Soluble MAC is primarily released from MAC-resistant bacteria that potently convert complement component C5

“…Deposition of C3b on the bacterial surface ( Figure 1b ) and release of C3a in the supernatant ( Figure 1—figure supplement 1b ) were similar on three strains compared to the MAC-sensitive strains, indicating that these strains activate complement efficiently. We have previously shown that MAC components bind to these strains after complement activation ( Doorduijn et al, 2021 ), suggesting that they are ‘MAC-resistant’ strains. The other five strains showed little to no deposition of C3b and were considered ‘complement-resistant’.…”

“… Escherichia. coli strains ( a ) and Klebsiella strains ( b ) were typed for the presence of LPS O-Ag via silver staining (methods described in Doorduijn et al, 2021 ). LPS-core were distinguished from LPS O-Ag based on size.…”

Molecular features underlying Pseudomonas aeruginosa persistence in human plasma

“…MAC is a multiprotein pore whose oligomerization occurs by sequential addition of C5b, C6, C7, C8 and finally C9 which forms an oligomeric ring of 18 units inserted into bacterial membranes (Bayly-Jones, Bubeck and Dunstone, 2017). Although the composition of MAC has been known for years, its contribution to bacterial cell death and the way the bacteria die upon MAC insertion is still a debate (Doorduijn, Rooijakkers and Heesterbeek, 2019), and recent work suggested that complement-resistant bacteria can specifically block polymerization of C9 (Doorduijn et al, 2021). SrgA is a protein of 7.1 kDa after predicted signal peptide cleavage, probably localized in the bacterial periplasm and/or exported to the bacterial surface.…”

Birth and Death in Terminal Complement Pathway

“…While the pores formed by the C5b-8 complex are unstable and short-lived, they can kill nucleated cells (Tegla et al, 2011). However, the relatively more stable tubular structure of the C5b-8,9n complex is required to kill gram-negative bacteria and is the target of complement evasion strategies of specific pathogens (Doorduijn et al, 2021(Doorduijn et al, , 2020Joiner, 4 1988;Zhao et al, 2014). The C9-mediated killing of bacteria in the absence of C5b-8 has also been reported (Dankert and Esser, 1987).…”