Discovering how Mycobacterium tuberculosis promotes its survival in neutrophils - PROJECT SUMMARY Mycobacterium tuberculosis (Mtb) is the causative agent of the deadliest infectious disease, tuberculosis (TB). Ten percent of immunocompetent individuals exposed to Mtb develop active TB, where others will contain the pathogen in a latent state or clear the infection. The host factors that determine the outcome of infection are poorly understood as a result of the complex relationship between Mtb and the host. Neutrophils have been identified as the most abundant and the predominantly infected cell type in bronchoalveolar lavage fluid, resected necrotic lung tissue, and sputum of patients with active TB. Despite the recruitment of neutrophils to the site of infection, there is no restriction of Mtb growth. Neutrophils quickly phagocytose and sense Mtb, leading to rapid induction of type I interferon (IFN). Mtb-infected neutrophils also degranulate and release neutrophil extracellular traps (NETs), suggesting that following infection these responses are regulated in a way that favors the patho- gen’s growth. One Mtb factor that has been implicated in promoting survival and growth in neutrophils is the Type VII Secretion System (T7SS), ESX-1. ESX-1 has been well studied in macrophages where it inhibits phag- osome maturation, causes phagosome damage, and induces production of type I IFN. This proposal will test the hypothesis that ESX-1 activity dysregulates neutrophil responses to promote Mtb growth. In Aim 1, I will monitor granule fusion, NADPH oxidase recruitment, and galectin recruitment to the phagosome during neutrophil infec- tion with wildtype (WT), ΔESX-1, and the complemented strain ΔESX-1::ESX-1 to directly determine how ESX- 1 affects intracellular trafficking and phagosomal damage. I will inhibit phagosome trafficking during neutrophil infection with ΔESX-1 and assess bacterial survival to determine if intracellular trafficking is required to control ΔESX-1 Mtb. Additionally, I will use bulk RNA sequencing following neutrophil infection with WT, ΔESX-1, and CS Mtb as an unbiased method to complement direct testing of phagosome trafficking and damage. In Aim 2, I will assess NET release and the kinetics of NETosis during neutrophil infection with WT, ΔESX-1, and ΔESX- 1::ESX-1 Mtb to determine the role of ESX-1 in NETosis. We have discovered that type I IFN signaling is required to release NETs. Therefore, this aim will also investigate the role of ESX-1 in type I IFN signaling to determine the relationship between the T7SS and NET release. To determine if NET release is sufficient for Mtb growth, I will induce NETosis during ΔESX-1 infection and measure bacterial survival. The proposed aims will elucidate the mechanism by which ESX-1 promotes Mtb survival and growth in the neutrophil, ultimately providing insight into the contributions of neutrophils to TB pathogenesis.
