Complement modulation properties of Klebsiella Pneumoniae

Abstract

Klebsiella pneumoniae er et patogen som kræver stor opmærksomhed. Udviklingen og spredningen af multiresi- stente stammer, som forårsager kritiske kredsløbs infektioner, har ført til at dødeligheden for disse infektioner er stigende. Der er derfor et kritisk behov for nye behandlingsmuligheder. Ved at opnå en dybere forståelse af det humane immunrespons mod K. Pneumoniae, nye skridt i udviklingen mod nye behandlingsmuligheder kan tages. Dette studie tager udgangspunkt i K. Pneumoniaes egenskaber til at overleve angreb fra complement system. Ved brug af state-of-the-art tandem masse spektrometri, en innovativ måde at følge den proteolytiske kaskade ved N- terminal acetylering såvel som immunoelektron mikroskopi (IEM), to kliniske K. pneumoniae isolater (et serum resistent (391) og et serum følsomt (688)) blev undersøgt for involveringen af humane komplement regulatoriske proteiner i serum resistens. Resultaterne viste at mængden af C3 bundet til begge isolater var ens, men for isolat 391 var C3 kun bundet i kapslen, hvor det hurtigt blev nedbrudt til iC3b, i en proces det ikke involverede Faktor H. På trods af den hurtige inactivering af C3b, lykkedes det alligevel for komplement systemet at samle alle kom- ponenter af MAC på isolat 391, dog i betragtelig mindre mængder end på isolat 688. Mængden af clusterin var højest på isolat 391, dog vidste IEM at clusterin primært var bundet i den ydre periferi af kapslen, og kunne derfor ikke være årsag til isolat 391’s serum resistens. IEM viste yderligere at kapslen på 391 blev afskudt, i en proces der eksponerede en overflade under kapslen, som ikke bandt komplement komponenter. Calprotectin, et metal- bindende protein med antimikrobielle egenskaber blev identificeret i store mængder på isolat 391. Calprotectin bundet til K. pneumoniae er en ny opdagelse, og effekten herf kendes derfor ikke. Helicobacter pylori kultiveret i medie supplementeret med calprotectin, viste modifikationer af Lipid A, en ændring i overfladespændingen og biofilmdannelse. Indikationer på en forskel i overfladespændingen mellem isolat 391 og 688, blev yderligere for- stærket med proteiner identiferet på isolat 688. Her blev plasminogen, Factor V og properdin fundet i større mængder en på isolat 391, de tre proteiner er kendt for at binde til negativt ladede fosfolipider. Indikationer på en ubeskrevet bakteriel C3 konvertase blev set på isolat 688 og involverede hydrolyseret C3 (C3(H2O)), Factor B and properdin. Tilstedeværelsen af denne konvertase tilføjede yderligere til hypotesen om at serum resistens opstår om følge af en ændring i overfladespændingen. Dette studie viste at C3b i kapslen på isolat 391 hurtigt blev nedbrudt til iC3b. Alle komponenter af MAC blev dog fundet sammen med clusterin på isolat 391. IEM viste dog at clusterin primært var bundet i den ydre periferi af kapslen, og ikke tæt på den ydre membran, og kunne derfor ikke redegøre for isolat 391’s serum resistens. Indi- kationer på en forskel i overfladespænding blev observeret, denne forskel vil både kunne forklare de morfologiske ændringer i kapslen samt serum resistensen. Yderligere studier skal laves, for at bekræfte denne hypotese, samt calprotectins rolle i serum resistens såvel som vigtigheden af dannelsen af en konvertase indeholdende C3(H2O)

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).

A master thesis from Aalborg University

Complement modulation properties of Klebsiella Pneumoniae

[Komplement modulerende egenskaber hos Klebsiella pneumoniae]