Graduate Thesis Or Dissertation
 

Establishment of an in vitro System for Studying Mycobacterium avium Protein Secretion

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  • Cases of pulmonary diseases caused by Mycobacterium avium complex (MAC) have increased over the years and have become a major health concern in Europe, Asia, and the United States. MAC, comprised of M. avium species and M. intracellulare, are found everywhere in the environment: in water sources and the soil. Their abilities to form biofilm in municipal water sources and their resistance to antimicrobial agents increase the risk of being exposed to this environmental pathogen and render treatment to MAC infections difficult, respectively. Upon ingestion or inhalation of contaminated water droplets, these opportunistic pathogens colonize the host and might cause nontuberculous mycobacterial (NTM) lung infection in patients with pre-existing lung pathology and disseminated disease in individuals with severe immunosuppression. Like Mycobacterium tuberculosis, M. avium survives and replicates within phagosomes. It has evolved different mechanisms to evade host immune responses like blocking phagosome-lysosome fusion, and altering the uptake of iron inside phagosomes. Iron and other metals like copper are required for bacterial growth and protein synthesis. One defense mechanism that host cells use to block the replication of intracellular pathogens inside phagosomes is to restrict the availability of nutrients, iron and other metal ions inside the vacuole or to accumulate certain metal that becomes toxic to the microorganisms. Sensing host environmental changes is key for the pathogen intracellular survival.  Many environmental cues including the single metal element are factors known to influence bacterial gene expression and virulence gene in pathogenic microorganisms like Salmonella, Shigella, or Yersinia. In Yersinia, calcium modulates the expression of the type III secretion system, which is responsible for the delivery of Yop effector proteins directly into the host cell cytosol. It is well known that bacterial secreted proteins have the ability to manipulate or modify host cells signaling pathways. Here, we were interested in identifying M. avium secreted protein inside the phagosome. Yet studying protein secretion inside host cells can be technically challenging and complex. For these reasons, an in vitro acellular system, established by Early and colleagues, which mimics the vacuole environment (metal element concentration and pH) of the phagosome at 24 hours post-infection (24 hrs elemental mixture), was used to identify M. avium secreted proteins. Fifty-five secreted proteins were identified from the M. avium culture supernatant. In this work, we aim (1) to confirm the secretion of 10 out of 55 secreted proteins identified from the M. avium culture supernatant using the beta-lactamase assay, and (2) to determine whether M. avium exposure to the elemental mixture induced gene expression or just triggered a secretory mechanism. By confirming the secretion of these 10 selected proteins inside macrophages, we will establish an in vitro system in studying M. avium protein secretion. Moreover, knowing whether the elemental mixture induces the gene expression of these 10 selected proteins or is just triggering a secretory mechanism will provide a better understanding on the effect the elemental mixture may have on M. avium.
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