Excessive C5 conversion prevents C9 polymerisation and subsequent MAC-dependent killing of Klebsiella pneumoniae

Membrane Attack Complex (MAC) pores are important in the human innate immune response to directly kill pathogenic Gram-negative bacteria. MAC pores assemble when complement proteins in serum are activated on bacteria and convert complement protein C5 into C5b, which together with C6, C7, C8, and multiple copies of C9 form a pore that damages the bacterial envelope. Due to rising multidrug-resistant infections with Gram-negative pathogen Klebsiella pneumoniae (Kpn), there is interest in developing complement-activating monoclonal antibodies (mAbs) that trigger MAC-dependent killing. However, some Kpn strains resist MAC-dependent killing in serum despite potent complement activation and C5 conversion, revealing a critical gap in understanding how these strains resist MAC-dependent killing. We demonstrate that Kpn strains can resist MAC-dependent killing through a paradoxical mechanism of excessively converting C5, which limits C9 polymerisation and subsequent killing. In these strains, spiking serum with supplementary C9 restored killing. Additionally, partially inhibiting C5 conversion using complement inhibitors increased C9 polymerisation and subsequent killing. This suggests that these Kpn strains are in principle sensitive to MAC-dependent killing, but an imbalance between generated C5b and available C9 in serum limits C9 polymerisation and prevents killing. We also observed this paradoxical effect with an of excess complement-activating mAbs on Kpn strains that are typically susceptible to MAC-dependent killing in serum. Excessive C5 conversion was responsible for this reduced killing, as supplementary C9 restored killing. Lastly, in neonatal plasma, where C9 is naturally limited, complement-activating mAbs induced killing of Kpn only in the presence of supplementary C9. Our study highlights that the balance between C5 conversion and available C9 is important for MAC-dependent killing of Kpn. Additionally, absence of killing in serum does not necessarily indicate that Kpn strains are MAC-resistant. These insights are important for interpreting mAb efficacy in serum bactericidal assays and in complement-deficient populations. Author SummaryAntibiotic-resistant infections caused by Klebsiella pneumoniae represent a major global health challenge. The human immune system normally combats such bacteria by activation of complement proteins, a group of molecules in the blood that can assemble the Membrane Attack Complex (MAC). The MAC forms pores that puncture the bacterial cell envelope and kill bacteria. Here, we found that potent activation of complement proteins can paraxodically prevent killing of certain K. pneumoniae strains. Excessive conversion of MAC component C5 limits the assembly of MAC pores consisting of multiple copies of C9, which are required for killing. We show that when extra C9 was added, the bacteria were killed, demonstrating that they are not truly resistant to MAC pores but escape through this imbalance. We also observed this effect in newborn samples, where natural C9 levels are lower and when testing antibody-based therapies designed to boost complement activity. These findings reveal that the absence of bacterial killing in laboratory assays does not always reflect genuine resistance, but can result from disproportional activation. Recognising this mechanism is critical for developing effective antibody therapies, protecting vulnerable patients, and addressing the growing threat of antibiotic-resistant infections.