The role of opsonophagocytosis in killing of Klebsiella pneumoniae in human blood.

Klebsiella pneumoniae is an opportunistic, gram-negative pathogen causing life-threatening sepsis in patients with co-morbidity. In contrast, in healthy persons, K. pneumoniae rarely causes sepsis. To elucidate how K. pneumoniae is eliminated from normal human blood, eleven K. pneumoniae sepsis isolates were analysed. Most of the isolates were serum-resistant. They solely activated the alternative pathway (AP), and only iC3b was present on their surface due to a rapid cleavage of C3b. Despite serum resistance, all isolates were killed in normal blood. To analyse the mechanism of uptake, two isolates (serum-resistant HA391 and partially resistant HA569) were transfected with a plasmid encoding red fluorescent protein, added to whole blood, analysed by flow cytometry and uptake in neutrophil granulocytes. HA391 was not phagocytosed in 50% heat-inactivated serum (HIHS), but in normal human serum (NHS), it was phagocytosed and subsequently killed. The iC3b deposited on the bacterial surface, colocalised with complement receptor 3 (CR3) and myeloperoxidase (MPO), confirming opsonophagocytosis. HA569 was rapidly phagocytosed by granulocytes in NHS but more slowly in HIHS. Thus, the complement system is essential for the elimination of serum-resistant K. pneumoniae isolates, as neutrophil granulocytes phagocytose HA391 through opsonophagocytosis, while HA569 is also phagocytosed independently of complement. AP lacks specific pattern recognition; however, it plays an essential role in the elimination of serum-resistant K. pneumoniae, as AP is activated by these bacteria, which, nonetheless, escape complement lysis by cleaving C3b to iC3b. Hereby, the bacteria are susceptible to opsonophagocytosis, an ancient function of AP that is crucial for eliminating bacteria.