Biology subjects

Masson, F. M.

Publications and source records attributed to Masson, F. M..

3 recordsLinked to original sources

bioRxiv2025

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.

immunology↗

bioRxiv2024

Activity-Based Protein Profiling Identifies Klebsiella pneumoniae Serine Hydrolases with Potential Roles in Host-Pathogen Interactions

Klebsiella pneumoniae is a normal resident of the human gastro-intestinal tract and an opportunistic, critical priority pathogen that can cause a variety of severe systemic infections. Due to emerging multi-drug resistance of this pathogen, the discovery and validation of novel targets for the development of new treatment options is an urgent priority. Here, we explored the family of serine hydrolases, a highly druggable and functionally diverse enzyme family which is uncharacterized in K. pneumoniae. Using functionalized covalent fluorophosphonate inhibitors as activity-based probes we identified 10 serine hydrolases by mass spectrometry-based activity-based protein profiling, 7 of which were previously uncharacterized. Functional validation using transposon mutants deficient in either of the putative lysophospholipase PldB, esterase YjfP and patatin-like phospholipase YchK revealed severe growth defects in human colonic organoid co-culture models and reduced virulence during Galleria mellonella infection. Mutants deficient in the PldB and YjfP, but not YchK show increased susceptibility to killing by complement and the antimicrobial peptide antibiotic polymyxin B, suggesting a role in maintaining cell envelope integrity. Biochemical characterization and structural analysis of recombinant YjfP suggest this protein is a deacetylase. This study gives important insights into the molecular mechanisms underlying virulence and cell physiology of K. pneumoniae at the host-pathogen interface and it positions PldB, YjfP and YchK as potential antimicrobial or anti-virulence target candidates, inhibition of which might synergize with existing antibiotics and human immune defenses.

microbiology↗

bioRxiv2022

Colistin resistance mutations in phoQ sensitize Klebsiella pneumoniae to IgM-mediated complement killing

The Gram-negative bacterium Klebsiella pneumoniae is notorious for a strong increase of infections with antibiotic resistant strains. To treat infections with antibiotic resistant K. pneumoniae, clinicians increasingly need to use the last resort antibiotic colistin. K. pneumoniae can develop colistin resistance by modifying its membranes. During infection the membranes of Gram-negative bacteria are also targeted by the human immune system via the complement system. Gram-negative bacteria have an outer and inner membrane separated by a thin cell wall. Activation of the complement system leads to the formation of the membrane attack complex (MAC), a pore that inserts into the outer membrane, and ultimately leads to lysis of the bacterium. As both colistin and the MAC interact with the outer membrane of Gram-negative bacteria, we wondered if developing colistin resistance influences MAC-mediated killing of K. pneumoniae. Using clinical isolates that developed colistin resistance, we found that the strain Kp209_CSTR became more sensitive to MAC-mediated killing compared to the wild-type strain. MAC-mediated membrane permeabilization of Kp209_CSTR required antibody dependent activation of the classical complement pathway. Strikingly, Kp209_CSTR was bound by IgM in human serum that did not recognise the wild-type strain. Depletion of Kp209_CSTR-specific antibodies from serum prevented MAC-mediated membrane permeabilization, which was restored by adding back IgM. Genomic sequence comparison revealed that Kp209_CSTR has a deletion in the phoQ gene. RNAseq analysis suggested that this mutation locks PhoQ in a constitutively active state. These results indicate that PhoQ activation in Kp209_CSTR leads to both colistin resistance and sensitivity to MAC-mediated killing. Together, our results show that developing colistin resistance can sensitize K. pneumoniae to killing by the immune system.

microbiology↗