Honors College Thesis
 

Importance of TLR2 and TLR4 signaling against M. abscessus infection of macrophages and use of mycobacteriophage genomics toward understanding some of the aspects of phage therapy

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  • Mycobacterium abscessus (MAB) is an opportunistic pathogen responsible for infections in immunocompromised or immunosuppressed patients. Even with long-term treatment with multiple antibiotics, MAB pulmonary infections have high rates of treatment failure. Within the host, MAB is phagocytosed by key innate immune cells macrophages. The TLR2 and TLR4 are two critical Toll-Like Receptors (TLRs) that can activate innate immune signaling at the plasma membrane to suppress mycobacterial survival in macrophages. However, MAB can avoid the TLR2 activation by masking the cell surface with glycopeptidolipids (GPLs). The TLRs also play an essential role in promoting signaling cascades that lead to the activation of Nuclear Factor Kappa B (NF- κB) and pro-inflammatory cytokine production. To determine the effects of TLR2 and TLR4 activation on the ability of phagocytic cells to attenuate MAB infection, THP-1 human macrophages were pre-treated or post-treated with TLR2 and TLR4 agonists, and infected with MAB. The intracellular bacterial survival was evaluated based on the colony forming unit (CFU) counts at different time points of MAB infection. The survival data determined that TLR2 and TLR4 pre-activations were the most effective conditions for significantly reducing the MAB intracellular growth. In addition, real-time quantitative PCR was performed for key regulator genes such as GILZ and NF-kB. It was found that upregulation of GILZ led to the downregulation of TLR2 response in MAB- infected macrophages. On the other hand, the activation of TLR2/TLR4 signaling was associated with the downregulation of GILZ while stimulating the high levels of NF-κB in MAB-infected cells. Results indicate that MAB infection inhibits the activation of TLR2 and TLR4 pathways. Due to the fact that GILZ can alter TLR2/TLR4 signaling and these TLRs play a critical role in oxidative stress contributing to immune resistance against MAB infection, the ROS levels were measured in TLR-activated cells. Significantly greater ROS production was recorded in TLR2/TLR4 activated macrophages during MAB infection when compared with MAB infection alone. This data demonstrates that activation of TLR2/TLR4 signaling stimulates innate immune responses beneficial for the host cells against MAB infection. In addition, in this study, the genome assembly and annotation of 30 mycobacteriophages were initiated to identify phage genes that may code for proteins involved in direct interactions with immune receptors such as TLRs. Furthermore, two MAB clinical isolates were evaluated against 30 phages to determine if phages are capable of infecting bacteria of intracellular phenotype that escaped macrophages at later time points. Selected phages (termed Ph179 and Ph338) were able to fully clear MAB in the supernatants of macrophage monolayers at day 4 of MAB infection. This could be due to either the direct killing of MAB or indirectly via activation of innate immune defenses of macrophages. The phage genomic annotation is ongoing, and this data will help in the identification of molecular mechanisms of phage-phagocyte and MAB interaction.
  • Keywords: Mycobacterium abscessus, Glucocorticoid-Induced Leucine Zipper (GILZ), Nuclear Factor Kappa B (NF-kB), Reactive Oxidative Species (ROS), Genome assembly, mycobacteriophages, phage genome
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  • Pending Publication
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  • 2022-08-25 to 2023-03-26

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