Macrophage infection with wild-type Mycobacterium avium or isogenic attenuated PPE mutant revealed differences in gene expression and mycobacterial vacuole proteins Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/th83m1461

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  • Mycobacterium avium is a ubiquitous environmental organism found in water and soil. It can cause disease in patients with pre-existing pulmonary conditions, immunocompromised patients with the most prevalent being AIDS patients, as well as apparently healthy people. Studies have indicated that, upon macrophage uptake, Al. avium prevents phagosome-lysosome fusion, thus avoiding its killing. PPE-PE families of genes in mycobacteria have been suggested to have a role in mycobacterial virulence in vivo. Using an M avium-attenuated PPE transposon mutant, this study seeks to examine the differences in the phagosomal proteins expressed upon macrophage infection with the wild-type and its isogenic PPE mutant. Furthermore, this study aims to investigate the differences in the transcriptional profile of macrophages infected with M avium and the attenuated PPE mutant. First, the M avium and attenuated PPE mutant phagosome protein expression were determined by mass spectrometry analysis. It was found that wild-type phagosomes express proteins different from isogenic PPE mutant phagosomes at 30 mm, 4 h and 24 h. The proteins identified in the phagosomes of wild-type and the PPE mutant were found to be involved in bacterial uptake, antigen presentation and recognition, Rab and Rab-interacting proteins, cytoskeleton and motor proteins, proteins involved in biosynthetic pathways, transcriptional regulators, signal transduction proteins, ion channels and hypothetical proteins. Fluorescent microscopy studies were carried out to confirm the protein expression in M avium-PPE mutant-infected macrophages. After performing a DNA microarray on the macrophages infected with M avium and the PPE mutant at 4 h, we observed differences in the gene expression patterns of the wild-type and the PPE mutant infected macrophages. Finally, real-time PCR was used to confirm the differential expression of three chosen genes. The genes were found to be upregulated similar to that found in the DNA array data. Through these studies, we have determined several proteins, either previously described or not reported, to be present on the phagosome. Nonetheless, the use of the PPE mutant established the possibility that one protein may have a key function in modulating the formation of the phagosome. Another interesting aspect of our findings is that there is a possible connection between macrophage genes regulated upon uptake of a bacterium and the downstream effect on vacuole formation.
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