Factors influencing macrophage phagocytosis and killing of Gram negative bacteria

Thesis

Gram negative infections pose a significant burden on human health. Escherichia coli causes blood stream infection and sepsis, while Neisseria gonorrhoeae is a leading causes of pelvic inflammatory disease, infertility and ectopic pregnancy. New ways to understand immune evasion and to target these bacteria are needed. My work aimed to investigate the mechanisms of interaction of these Gram negative bacteria with macrophages and potentially identify ways to increase bacterial killing.

Previous research demonstrated that gonococcal lipooligosaccharide (LOS) is a variable structure. Methods to characterise LOS remain challenging, even though LOS is involved in host:pathogen interactions, and is a proposed target of vaccines. I investigated methods for nucleotide sequencing and flow cytometry to determinate LOS structures using isogenic deletion strains and clinical isolates. Several insights into LOS biology were found, including the dominance of expression of the α chain over alternative α chain.

Bacterial factors influencing gonococcal interactions with human macrophages have not been studied extensively. I investigated the impact of gonococcal LOS glycans on uptake of bacteria by macrophages. I found that loss of the α chain decreases phagocytosis of bacteria, while removal of the core oligosaccharide decreased bacterial attachment. I found no evidence of intracellular replication of the gonococcus in macrophages, contrary to prior literature.

GPR84 is an orphan receptor that increases phagocytosis of bacteria. I found that GPR84 agonists decreased recovery of E. coli but not N. gonorrhoeae from human macrophages in a dose dependent manner. This was caused by increased bacterial killing. This effect was rapid and sustained. The mechanism was dependent on a decrease in cAMP. I showed that the effect of the GPR84 agonist 6-OAU was GPR84 specific in murine macrophages; in human macrophages, all three novel agonists tested were GPR84 specific.

My findings provide novel tools and insights into factors involved in the interaction of N. gonorrhoeae and E. coli with macrophages, and suggest how macrophage receptors could be exploited to enhance bacterial killing for immune adjuvant therapy of infection.

Authors: Kurzyp, K

• DOI: 10.5287/ora-bmaz4gmvj
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English