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), ¿ESX-1, and the complemented strain ¿ESX-1::ESX-1 to directly determine how ESX-
1 affects intracellular trafficking and phagosomal damage. I will inhibit phagosome trafficking during neutrophil
infection with ¿ESX-1 and assess bacterial survival to determine if intracellular trafficking is required to control
¿ESX-1 Mtb. Additionally, I will use bulk RNA sequencing following neutrophil infection with WT, ¿ESX-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, ¿ESX-1, and ¿ESX-
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 ¿ESX-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.