The genetically heterogeneous opportunistic gram-negative bacteria Klebsiella pneumoniae are a common cause of sepsis and bacteremia in persons of old age or with comorbidities such as a compromised immune system or urinary tract infections (Lee et al., 2006). The complement system is the first line of defence against invading microorganisms reaching the bloodstream. It is part of the human innate immune system. It consists of more than 50 circulating and membrane-bound proteins that serve as substrates, enzymes, effectors, or regulators of an extracellular proteolytic cascade, producing different mediators of inflammation, opsonisation and killing of the invading bacteria. Upon complement activation, a cascade of reactions is initiated, which may result in the formation of the membrane attack complex (MAC), which lyses gram-negative bacteria.

Activation of the complement system takes place when pathogen-associated molecular patterns (PAMP) on the bacterial surface are recognised by pattern recognition molecules (PRM). PRM in the complement system are divided into mannan-binding lectin (MBL), ficolins, and collectin-CL-K1, which can initiate the lectin pathway (LP) (Troegeler et al., 2015), or complement factor 1q (C1q), which can recognise PAMP or bacterial surface-bound multimeric antibodies and initiate the classical pathway (CP) (Bajic et al., 2015). Both pathways result in cleavage of C4 and C2, generating C4bC2b, the classical pathway C3 convertase. When C4b is formed, a conformational change results in the exposure of an internal Cys-Gln thioester that can react with alcohol or amine groups on the bacterial surface. The C3 convertase C4bC2b cleaves C3 to C3a and C3b,

but a C3 convertase is also assembled by the alternative pathway (AP) through two different mechanisms. In plasma, H2O can occasionally hydrolyse the C3 thioester group, forming C3(H2O). C3(H2O) can, with complement factor B (FB), form the proconvertase C3(H2O)FB. Subsequent cleavage of factor B to Bb by factor D results in the formation of the C3(H2O)Bb complex, an alternative pathway C3 convertase which cleaves C3 to C3b that may become covalently deposited on bacteria just like C3b deposited by the CP C3 convertase. As C3b also forms the AP C3 convertase, a strong amplification of the initial C3b deposition takes place. The alternative C3 convertase C3bBb decays with a T1/2 of 90 seconds (Pangburn and Müller-Eberhard, 1986). The complement factor properdin (CFP) prolongs the convertase half-life 5–10 times (Smith et al., 1984) due to competition with factor I (FI) and by bridging C3b and Bb in the AP C3 proconvertase and convertase (Lorentzen et al., 2022, Pedersen et al., 2019). At a threshold density of C3b on a bacterium, the two C3 convertases may change substrate specificity and become C5 convertases. The C3bC4bC2b complex (the classical pathway C5 convertase) or the (C3b)2BbP complex (the alternative pathway C5 convertase) can turn over complement C5 and trigger MAC assembly (Bayly-Jones et al., 2017).

We previously investigated K. pneumoniae isolates from the blood of sepsis patients and found that MAC was formed in only a few of the isolates despite C3 deposition in the capsule of almost all isolates (Jensen et al., 2020). All serum-sensitive isolates (Jensen et al., 2020) had MAC formation, and all had deposition of C4 on the outer membrane, indicating formation of the CP C5 convertase and thus activation by either CP or LP (Jensen et al., 2020). In contrast, the serum-resistant isolates had no C4 deposition or MAC formation, indicating a deactivation of the complement cascade system before C5 convertase activity capable of initiating MAC formation. Both the serum-resistant and serum-sensitive isolates showed deposition of inactivated C3b (iC3b) caused by regulator-assisted FI degradation of C3b in the capsule (Jensen et al., 2020).

In the present study, in high serum concentrations, mass spectrometry (MS) was used to identify interacting complement components and their cleavage sites on both a serum-resistant HA391, which belongs to the highly pathogenic sequence type (ST) 70, has the capsule type KL136 and the O-type O1v2 (Flores-Valdez et al., 2021), and the serum-sensitive HA688 K. pneumoniae isolate of ST392, KL17 and O4 (Jensen et al., 2020, Opstrup et al., 2023a), a pandemic cluster of K. pneumoniae, pathogenic for humans, dogs and cats (da Silva et al., 2022). The serum-sensitive HA688 had all the expected complement factors on their surface. In contrast, by MS of the serum-resistant HA391, only activated AP and iC3b was the predominant complement component on the surface, explaining its serum resistance. AP is only active in high concentrations of NHS, as seen in the bloodstream. Therefore, coating of iC3b on serum-resistant K. pneumoniae is essential for bacterial clearance.