Investigating the Molecular Regulators of Phagocytosis and Intracellular Killing to Modulate the Innate Immune System in Response to Infection

Antibiotic resistance is on the rise due to overuse and misuse of antimicrobial treatments. Drug resistant infections are therefore increasingly difficult to treat leading to over 700,000 deaths world-wide annually with the expected rise to 10 million deaths in 2050 if no alternative treatments are developed. Phagocytosis and subsequent intracellular killing of pathogens are key functions shared by professional phagocytes including neutrophils and macrophages. Although the mechanisms of these functions are well characterised, their regulators are not. Phenotypic variability in the phagocytic capacity of both neutrophils and macrophages has been described on numerous occasions. There has so far been very few studies to underpin the molecular signatures of these apparent sub-populations. The first aim of this project sought to determine if the phenotypes described are stochastic or inherent to the individual cell. Neutrophils and monocyte-derived macrophages (MDMs) both showed super-ingester phenotypes in which certain cells were more likely to phagocytose particulate material than others. FACS sorting and RNA sequencing allowed identification of differentially expressed genes (DEGs) between phagocytic and non-phagocytic cells. Only 18 DEGs were identified in neutrophils whilst over 1,000 DEGs were identified at two time-points in MDMs. The second and third aims of this project focused on enhancing intracellular killing of Streptococcus pneumoniae, a pathogen that is the leading cause of community acquired pneumonia which has developed resistance to penicillin. One method involved the development of a compound screen to assess the treatment with clinically approved drugs on the number of intracellular bacteria. 23 compounds were identified for further investigation into repurposing for treating infections. This followed on to the final aim of the development of an siRNA screen using a library of 18,096 siRNAs to identify novel regulators of intracellular killing and phagolysosome acidification by labelling S. pneumoniae with GFP and pHrodo. Over 3,500 siRNAs targets were tested, which showed 114 genes altered the number of internalised GFP positive bacteria and 82 genes altering phagosomal acidification. Considering where there is lack of knowledge, this project aims to identify and investigate molecular regulators of phagocytosis and intracellular killing as therapeutic targets to modulate the innate immune system in response to infection.