The role of cholesterol in the uptake and pathogenesis of Mycobacterium avium subspecies paratuberculosis in human monocytes

Crohn’s disease (CD) is a chronic inflammatory bowel disease, primarily affecting the young, which causes marked morbidity and reduced quality of life. Currently there is no cure for CD, and the causes of this disease are poorly understood. In ruminants, Johne’s disease (JD) is characterised by chronic intestinal inflammation similar to CD and is caused by the pathogen Mycobacterium avium subspecies paratuberculosis (MAP), which invades and replicates within the phagocytes of infected animals, leading to chronic disease. There is increasing molecular and microbiological evidence of Map bacteria in CD patients. However, little is known regarding the role of Map in the aetiology of CD. This thesis demonstrated that a human isolate of Map traffics through THP-1 human monocytes via a similar path to that taken by pathogenic mycobacteria. Flow cytometry demonstrated that Map are phagocytosed via a cholesterol-dependant mechanism, potentially mediated by a cell wall constituent. Once internalised, live Map reside in cholesterol-rich areas of the cell. These compartments exhibit reduced acidity compared to the compartments containing killed-Map, and have atypical retention of markers including the late endosomal marker Rab 7 and cellular TACO protein. Both of these markers were also present on phagosomes of pathogenic mycobacteria, where they interrupt fusion of the compartment with lysosomes. This was confirmed by visualisation of these proteins on phagosomes containing M. bovis,a known mycobacterial pathogen. Cholesterol depletion using simvastatin affected Map persistence in THP-1 cells at 1 and 2 weeks post infection, a finding similar to other studies with M. tuberculosis. Spheroplast-like forms were evident after long term culture of Map with THP-1 monocytes, visualised by light and electron microscopy. These were similar to forms observed in peripheral blood leukocytes from a CD patient. Collectively, these results support the hypothesis that Map may be involved in the aetiology of at least a subset of CD cases