Complement modulation properties of Klebsiella Pneumoniae

Student thesis: Master thesis (including HD thesis)

  • Mikkel Eggert Thomsen
Klebsiella Pneumoniae is a pathogen of great concern. With the emergence of multi drug resistant strains that cause serious bloodstream infections, the mortality rates for these infections are increasing. Novel therapeutics are hence in urgent need. From a better understanding of the host defense mechanisms against K. pneumoniae, new advances in the development of novel therapies can arise. This study investigated how K. pneumoniae circum- vents the first line of defense, the complement system. By state-of-the-art tandem mass spectrometry (MS) and a novel approach to track the proteolytic cascade of the complement system by N-terminal acetylation, as well as immunoelectron microscopy (IEM), two clinical isolates of K. pneumoniae (one serum resistant (391) and one se- rum sensitive (688)) were analyzed, to test the involvement of human complement regulators in resistance to serum. Results showed that C3 was equally deposited on the two isolates, but for 391 solely in the capsule, where it was rapidly inactivated to iC3b, in a Factor H independent manner. Despite the rapid inactivation, the comple- ment cascade still managed to collect all components of the membrane attack complex (MAC) on isolate 391, as identified by MS, though in significantly lower abundance than on isolate 688. The abundance of clusterin was highest on isolate 391 determined by MS, but IEM showed only clusterin in the outer periphery of the capsule, hence clusterins inhibitory mechanism towards insertion of the membrane attack complex (MAC) in the outer membrane, could not account for the serum resistance of isolate 391. IEM further showed, that the capsule was shedded partly or completely of isolate 391, exposing a surface beneath that did not bind complement components.
Calprotectin, a metal sequester with antimicrobial properties, was identified in high abundance on isolate 391. Calprotectin in association to K. pneumoniae is a novel finding, and thus its function unclear. In Helicobacter pylori calprotectin has been shown to induce Lipid A modifications, change in surface charge and biofilm formation. Ev- idences of a difference in surface charge between the two isolates were further compiled by the findings of com- ponents of the coagulation system on isolate 688, that is known to bind negatively charged phospholipids. These findings included plasminogen, Factor V and properdin. Evidence of a novel bacterial C3 convertase was observed on isolate 688, involving properdin, hydrolyzed C3 (C3(H2O)) and Factor B, which also could suggest that the complement resistance can be explained by a change in surface charge.
The study confirmed that N-terminal acetylation of peptides probes proteomics to track the proteolytic cascade of the complement system, which is a gamechanger in complement research. However, results did not manage to identify a mechanism of utilization of human complement regulatory proteins, that could account for the serum resistance seen in isolate 391, however the results showed evidence of a difference in surface charge, which both could explain the morphological changes of the capsule as well as the serum resistance. Yet, further studies have to be done, to confirm this hypothesis, calprotectins role in serum resistance of K. Pneumoniae, aswell as the importance of convertases involving C3(H2O).
Publication date2019
Number of pages36
ID: 304814657