Mycobacterial resistance to neutrophil oxidants

Neutrophils are one of the first immune cells to respond when lungs are infected with Mycobacterium tuberculosis – the world’s leading infectious killer that results in millions of deaths every year. Normally, neutrophils ingest pathogens and generate bactericidal oxidants including hypochlorous acid (HOCl). Previous studies have suggested that mycobacteria are more resistance to neutrophil oxidants than other bacteria, though this has not been extensively investigated. In this project I have investigated neutrophil phagocytosis, oxidant production and killing of Mycobacterium smegmatis – an environmental bacterium closely related to M. tuberculosis. Bacteria were added to neutrophils, and at various times samples were taken to investigate the bacterial viability. A slow speed centrifugation was used to separate extracellular bacteria from neutrophils with their ingested bacteria. The loss of bacteria from the extracellular media was used to quantify phagocytosis, while killing was quantified by the lysis of neutrophils and measuring the surviving intracellular bacteria. I was able to show that M. smegmatis were being phagocytosed at a rate three times slower than S. aureus and killed at a rate two times slower than S. aureus. annexin/ propidium iodide staining of neutrophils exposed to higher ratios of M. smegmatis confirmed that neutrophils were not dying, and therefore not disrupting any results. To confirm that phagocytosis was occurring, bacteria were labelled with FITC probe and incubated with neutrophils. Assessment by flow cytometry showed fluorescence associated with neutrophils even after quenching any extracellular bacteria with trypan blue; indicating that neutrophils had internalised the bacteria. There was a similar increase in oxidation of the redox-sensitive probe dihydrorhodamine 123 (DHR) in neutrophils phagocytosing M. smegmatis at a ratio of one to one compared to S. aureus at a ratio of one neutrophil to ten bacteria, indicating a strong oxidative presence. Interestingly, the HOCl-sensitive probe R19S showed lower levels of HOCl in response to M. smegmatis than S. aureus at the same ratios seen in the DHR assay, though significant HOCl production was still occurring. The slow killing of M. smegmatis is therefore likely to be due to reduced levels of HOCl in the neutrophil phagosome and innate resistance of this bacterium towards oxidants. In the future, attempts at finding targets to sensitise M. smegmatis to neutrophil oxidants could help to identify targets in M. tuberculosis and reduce the impact tuberculosis has on the world