Investigation into the dysregulation of pattern recognition receptor signalling in Leishmania mexicana infected THP-1 cells

Barker, Rebecca and Unterholzner, Leonie and Bates, Paul (2023) Investigation into the dysregulation of pattern recognition receptor signalling in Leishmania mexicana infected THP-1 cells. PhD thesis, Lancaster University.

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Abstract

Leishmaniasis is a neglected tropical disease caused by parasites of the Leishmania genus and is spread to humans by sandfly bite. Leishmaniasis is the second largest parasitic killer after Malaria and manifests a wide variety of symptoms from disfiguring skin lesions to fatal liver damage. Despite approximately 1 billion people living in endemic areas, with 1 million new cases occurring each year, treatment options for the disease remain limited, and a successful vaccine is yet to be developed. The lack of successful control of Leishmaniasis can be partially attributed to an incomplete knowledge of how Leishmania establishes infection. The parasites are unusual as they infect and multiply inside macrophages; specialised innate immune cells that usually detect and destroy invading pathogens. Therefore to replicate, Leishmania successfully evades the hosts innate immune response in order to infect macrophages and persist within them. Leishmania express numerous virulence factors to aid infectivity by inhibiting host signalling pathways, some of which are downstream of pathogen recognition receptors (PRRs). After recognition of pathogens by the various PRRs, downstream signalling can activate host microbicidal and pro-inflammatory responses to eliminate the infection. By combining parasite cell culture and molecular biology techniques, we examined how Leishmania mexicana may disrupt the pathogen sensing and communication pathways of the human macrophage. We focused on parasite interactions with PRRs, especially the cytoplasmic nucleic acid sensors as there has been little investigation concerning them in the context of Leishmania infection. After establishing a successful in vitro L. mexicana infection within human THP-1 macrophages, we discovered that these cells had an enhanced response to transfected dsDNA following L. mexicana infection. This was observed by the increased activation of a key adaptor protein stimulator of interferon genes (STING), in the cGAS-STING DNA sensing pathway and its downstream targets, in addition to an increased cytokine response to dsDNA. Through protein analysis we also observed that STING and other key components of the DNA sensing pathway are modified during infection. Through further experiments we identified the modification of STING as a cleavage event resulting in partial loss of its C-terminal tail. While cleavage to this form is highly upregulated during infection, we have suggested that this alternative form of STING is a naturally occurring variant and may have a regulatory role under normal conditions that Leishmania parasites could exploit for their own benefit. As the initial immune response to Leishmania infection has a significant impact on the course of the disease, identifying new host targets of parasite virulence factors will be crucial to increasing understanding of Leishmaniasis and may provide future therapeutic targets. Therefore, investigation of STING and other components of the cGAS-STING DNA sensing pathway is necessary to fully understand their role during Leishmania infection and thus identify therapeutic targets.