Weerapura, Milani2025-05-212025-05-212025-05-21http://hdl.handle.net/10393/50506https://doi.org/10.20381/ruor-31139Macrophages help eliminate bacterial pathogens that otherwise deter human health. However, certain pathogens, such as Salmonella enterica serovar Typhimurium (STm), are instead able to survive and persist within macrophages. To develop the means to prevent this phenomenon, we must further our understanding of the interplay between STm and macrophages. SIX1 (sine oculis homeobox homologue 1) is a host transcription factor that is primarily active during, and studied in the context of, embryonic development. However, there has been an increase in studies suggesting that SIX1 can regulate inflammation, which is a key antibacterial response of macrophages. Furthermore, recent evidence suggests that bacterial pathogens can upregulate SIX1 expression. Taken together, this raises the question of whether SIX1 regulates macrophage responses during bacterial infections, such as STm infection, to the host's benefit or detriment. Addressing this question could enhance our understanding of pathways that contribute to promoting or preventing the resolution of infection. However, SIX1 has not previously been studied in this context. Herein, I demonstrate that SIX1 expression modulates STm intracellular survival in a pathogen specific manner. Human THP-1 macrophages that are SIX1 overexpressing (THP-SIX1++) or knockdown (THP-SIX1) had increased or decreased STm burden respectively. I demonstrated that these differences in STm burden were due to STm intracellular survival, as opposed to being the by-product of differences in bacterial uptake or macrophage cell death. To identify factors contributing to the SIX1-dependent STm survival phenotype, I evaluated macrophage antibacterial responses including cytokine/chemokine production, reactive oxygen species (ROS) production, and autophagy. Despite previous reports wherein SIX1 downregulated inflammation, I observed only a minimal impact on the macrophages' immunological profile during STm infection and no differences in ROS or autophagy levels. Although none of those pathways appeared relevant to our phenotype, I did discover that torin-1, an inducer of autophagy, could rescue THP-SIX1++ macrophages' inability to control STm survival and replication. Aside from inducing autophagy, which I previously tested, torin-1 is also known to inhibit AKT phosphorylation, which prevents AKT activation and signalling. In testing whether torin-1's impact on AKT phosphorylation could contribute to my phenotype, I discovered that THP-SIX1++ macrophages had elevated levels of phosphorylated AKT that were abolished in the presence of torin-1. Taken together, these findings suggest that perhaps AKT signalling contributes to the observed SIX1-dependent STm survival phenotype. However, further studies are required to elucidate the precise functional mechanism responsible for SIX1's modulation of STm survival. Nonetheless, this study highlights that, in the context of macrophage infection, the transcription factor SIX1 can exhibit pathogen specific, host detrimental effects.enSIX1Salmonella enterica serovar TyphimuriumMacrophagesThesis