Biology subjects

Doorduijn, D. J.

Publications and source records attributed to Doorduijn, D. J..

6 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↗

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↗

bioRxiv2021

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

The Membrane Attack Complex (MAC or C5b-9) is an important effector of the immune system to kill invading microbes. MAC is formed when complement enzymes on the bacterial surface convert complement component C5 into C5b. Although the MAC is a membrane-inserted complex, soluble forms of MAC (sMAC, or terminal complement complex (TCC)) are often detected in sera of patients suffering from infections. Consequently, sMAC has been proposed as a biomarker, but it remains unclear when and how it is formed during infections. Here, we studied mechanisms of MAC formation on bacteria and found that sMAC is primarily formed in human serum by bacteria resistant to MAC-dependent killing. Surprisingly, C5 was converted into C5b more potently by MAC-resistant compared to MAC-sensitive Escherichia coli strains. Both the increase in C5 conversion and sMAC generation were linked to the expression of lipopolysaccharide (LPS) O-Antigen in the bacterial outer membrane. In addition, we found that MAC precursors are released from the surface of MAC-resistant bacteria during MAC assembly. Release of MAC precursors from bacteria induced lysis of bystander human erythrocytes in the absence of other serum components. However, serum regulators vitronectin (Vn) and clusterin (Clu) can prevent this bystander lysis. Combining size exclusion chromatography with mass spectrometry profiling, we show that sMAC released from bacteria in serum is a heterogeneous mixture of complexes composed of C5b-8, up to 3 copies of C9 and multiple copies of Vn and Clu. Altogether, our data provide molecular insight into how sMAC is generated during bacterial infections. This fundamental knowledge could form the basis for exploring the use of sMAC as biomarker.

microbiology↗

bioRxiv2021

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

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, because a robust way to specifically prevent polymerization of C9 has been lacking. In this paper, polymerization of C9 was prevented without affecting the binding of C9 to C5b-8 by locking the first transmembrane helix domain of C9. We show that polymerization of C9 strongly enhanced bacterial cell envelope damage and killing by MAC pores for several Escherichia coli and Klebsiella strains. Moreover, we show that polymerization of C9 is impaired on complement-resistant E. coli strains that survive killing by MAC pores. Altogether, these insights are important to understand how MAC pores kill bacteria and how bacterial pathogens can resist MAC-dependent killing.

microbiology↗

bioRxiv2020

In vitro evolution of colistin resistance in the Klebsiella pneumoniae complex follows multiple evolutionary trajectories with variable effects on fitness and virulence characteristics.

The increasing prevalence of multidrug-resistant Gram-negative opportunistic pathogens, including Klebsiella pneumoniae, has led to a resurgence in the use of colistin as a last-resort drug. Colistin is a cationic lipopeptide antibiotic that selectively acts on Gram-negative bacteria through electrostatic interactions with anionic phosphate groups of the lipid A moiety of lipopolysaccharides (LPS). Colistin resistance in K. pneumoniae is mediated through loss of these phosphate groups, or modification with cationic groups (e.g. 4-amino-4-deoxy-L-arabinose (L-Ara4N), or phosphoethanolamine), but also hydroxylation of acyl-groups of lipid A. Here, we study the in vitro evolutionary trajectories towards colistin resistance in clinical K. pneumoniae complex strains (three K. pneumoniae sensu stricto strains and one K. variicola subsp. variicola strain) and their impact on fitness and virulence characteristics. Through population sequencing during the in vitro evolution experiment, we found that resistance develops through a combination of single nucleotide polymorphisms (SNPs), insertion and deletions (indels), and the integration of insertion sequence (IS) elements, affecting genes associated with LPS biosynthesis and modification, and capsule structures. The development of colistin resistance decreased the maximum growth rate of one K. pneumoniae sensu stricto strain, but not in the other three K. pneumoniae sensu lato strains. Colistin-resistant strains had lipid A modified through hydroxylation, palmitoylation, and L-Ara4N addition. Colistin-resistant K. pneumoniae sensu stricto strains exhibited cross-resistance to LL-37, in contrast to the K. variicola subsp. variicola strain that did not change in susceptibility to LL-37. Virulence, as determined in a Caenorhabditis elegans survival assay, was higher in two colistin-resistant strains. Our study suggests that nosocomial K. pneumoniae complex strains can rapidly develop colistin resistance de novo through diverse evolutionary trajectories upon exposure to colistin. This effectively shortens the lifespan of this last-resort antibiotic for the treatment of infections with multidrug-resistant Klebsiella. Author summaryBacteria that frequently cause infections in hospitalised patients are becoming increasingly resistant to antibiotics. Colistin is a positively charged antibiotic that is used for the treatment of infections with multidrug-resistant Gram-negative bacteria. Colistin acts by specifically interacting with the negatively charged LPS molecule in the outer membrane of Gram-negative bacteria. Colistin resistance is mostly mediated through modification of LPS to reduce its negative charge. Here, we use a laboratory evolution experiment to show that strains belonging to the Klebsiella pneumoniae complex, a common cause of multidrug-resistant hospital-acquired infections, can rapidly accumulate mutations that reduce the negative charge of LPS without an appreciable loss of fitness. Colistin resistance can lead to cross-resistance to an antimicrobial peptide of the human innate immune system, but can increase susceptibility to serum, and virulence in a nematode model. These findings show that extensively resistant K. pneumoniae complex strains may rapidly develop resistance to the last-resort antibiotic colistin via different evolutionary trajectories, while retaining their ability to cause infections.

microbiology↗

bioRxiv2019

Bacterial killing by complement requires direct anchoring of Membrane Attack Complex precursor C5b-7

An important effector function of the human complement system is to directly kill Gram-negative bacteria via Membrane Attack Complex (MAC) pores. MAC pores are assembled when surface-bound convertase enzymes convert C5 into C5b, which together with C6, C7, C8 and multiple copies of C9 forms a transmembrane pore that damages the bacterial cell envelope. Recently, we found that bacterial killing by MAC pores requires local conversion of C5 by surface-bound convertases. In this study we aimed to understand why local assembly of MAC pores is essential for bacterial killing. Here, we show that rapid interaction of C7 with C5b6 is required to form bactericidal MAC pores. Binding experiments with fluorescently labelled C6 show that C7 prevents release of C5b6 from the bacterial surface. Moreover, trypsin shaving experiments and atomic force microscopy revealed that this rapid interaction between C7 and C5b6 is crucial to efficiently anchor C5b-7 to the bacterial cell envelope and form complete MAC pores. Using complement-resistant clinical E. coli strains, we show that bacterial pathogens can prevent complement-dependent killing by interfering with the anchoring of C5b-7. While C5 convertase assembly was unaffected, these resistant strains blocked efficient anchoring of C5b-7 and thus prevented stable insertion of MAC pores into the bacterial cell envelope. Altogether, these findings provide basic molecular insights into how bactericidal MAC pores are assembled and how bacteria evade MAC-dependent killing.

immunology↗