Author's School

School of Medicine

ORCID

https://orcid.org/0000-0002-1945-6468

Author's Department/Program

Biology and Biomedical Sciences

Concentration/Secondary Program

Molecular Microbiology and Microbial Pathogenesis

Language

English (en)

Date of Award

12-15-2026

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Christina L. Stallings (Chair); David A. Hunstad (Co-Chair)

Committee Members

Michael G. Caparon, Regina A. Clemens, Clifford J. Luke, Jennifer A. Philips

Abstract

The World Health Organization estimates that one-fourth of the world’s population has been exposed to Mycobacterium tuberculosis (Mtb). However, only 10% of those exposed develop active tuberculosis (TB), whereas other individuals will contain the pathogen in a latent state or clear the infection. It is unknown what mechanisms govern the development of active TB, latent infection, or clearance of infection, which is a barrier to successful treatment. Neutrophils have been identified as the predominantly infected cell type in bronchoalveolar lavage fluid, resected necrotic lung tissue, and sputum of patients with active TB. Lung neutrophil infiltration is also significantly increased in several susceptible mouse models of TB, demonstrating a link between neutrophil abundance and TB disease in both murine and human hosts. In some mouse models, depletion of neutrophils decreases bacterial burden and extends the survival of these mice during infection, indicating that the overabundance of neutrophils can be enough to exacerbate disease. Although neutrophils possess a myriad of antimicrobial functions, such as degranulation, reactive oxygen species (ROS) production, and neutrophil extracellular trap (NET) release to kill pathogens, it is unclear why neutrophils cannot kill Mtb.

We first examined whether neutrophils cannot kill Mtb due to an inhibition of their effector functions, by comparing the responses of human neutrophils to Mtb and zymosan. We found that neutrophils phagocytose and respond to Mtb infection through many antimicrobial pathways, including ROS production, granule fusion with the phagosome, degranulation, and NET release. Mtb produced more ROS, dampened secondary granule release, and reduced primary granule recruitment to the phagosome compared to zymosan infection. We also discovered that Mtb permeabilizes the phagosome of neutrophils. Through unbiased clustering of flow cytometry data from infected neutrophils, we discovered a subset of neutrophils that exhibited an Mtb-specific response based on bacterial burden. Despite the induction of potent anti-microbial functions in neutrophils, the neutrophils cannot kill Mtb, suggesting that Mtb activates its own defenses within the neutrophil.

Intracellular host-pathogen interactions of Mtb have been well-characterized in macrophages. It has been shown in macrophages that Mtb expresses ESX-1, a type VI secretion system, permeabilizes the phagosome through the effector EsxA. Following permeabilization, Mtb effectors gain access to the cytosol and inhibit ROS and nitric oxide (NO) production, phagolysosome formation, LC3-associated phagocytosis, and phagosome acidification. Additionally, Mtb DNA and RNA are released from the phagosome and activate cytosolic nucleic acid sensors, leading to the production of type I IFN through cGAS/STING and RIG-I/MAVS. ESX-1 is the primary region of genes deleted from the Bacillus Calmette–Guérin (BCG) M. bovis vaccine strain and ESX-1 mutants are attenuated for growth in macrophages, demonstrating the importance of this secretion system in Mtb virulence. ESX-1 has previously been implicated in promoting survival of the bacteria in human neutrophils, therefore we tested the role of ESX-1 in dampening human neutrophil responses to Mtb. Our data revealed that ESX-1 expression in Mtb only affects global protease activity and neutrophil viability and is not required for phagosome permeabilization, dysregulation of phagosome maturation, not bacterial survival. We show that has different requirements for virulence in different cell types and models of infection.

To better understand global responses of Mtb to the neutrophil, we used a multi-omics approach, comparing the host transcriptome, Mtb transcriptome, and metabolome of primary ex vivo bone marrow neutrophils and propagated alveolar macrophages from C57BL/6 mice at 4 hours post Mtb infection. We discovered that both infected macrophages and neutrophils were glycolytic and proinflammatory, confirming that our mouse neutrophils were activated as the human neutrophils were during Mtb infection. While infected macrophages were proinflammatory, infected neutrophils also adopted anti-inflammatory characteristics such as expressing Il10 and upregulating cholesterol homeostasis and fatty acid metabolism. To profile the Mtb transcriptome during infection, we utilized SEquening after Amplicon enRiCHment for TB (SEARCH-TB) to enrich Mtb transcripts from the infection. We discovered that Mtb in macrophages upregulated the DosR regulon, triacylglyceride biosynthesis, and stringent response associated pathways, resembling a dormant state. In contrast, in neutrophils, Mtb upregulated macromolecule synthesis, metal acquisition, redox defense, and lipid catabolism pathways. Within the neutrophil, Mtb had more access to lipids based on the higher abundance of sterol compounds and long chain fatty acids identified in untargeted metabolomics of infected neutrophils compared to infected macrophages. Furthermore, we found that Mtb in neutrophils were significantly closer to lipid droplets and more associated with lipids compared to Mtb in macrophages. and staining of lipids in infected cells, we show that within the neutrophils compared to the macrophage. This work demonstrates that although Mtb encounters stressors in both the macrophage and neutrophil, the difference in composition and abundance of the stresses determines whether dormancy will be induced. This data supports a model whereby stressors like hypoxia and NO drive dormancy in Mtb in macrophages, while in neutrophils the sensing and utilization of lipids in the neutrophil phagosome induces the upregulation of specific stress responses (e.g. redox and oxidative stress responses) without the induction of dormancy. These findings shed new light on what stressors Mtb encounters within the host cell and how Mtb differentially adapts to the neutrophil and macrophage.

This work has opened several avenues of open questions concerning the mechanisms by which Mtb dampens granule trafficking, phagosome permeabilization, and protease activity in human neutrophils. Through this work, we have shifted the paradigm in our understanding of the ways in which ESX-1 contributes to virulence and how this varies between cells. Additional next steps from this work include dissecting the specific environmental cues needed for Mtb to distinguish between stressors that require dormancy and stressors that can allow for growth and the role of lipid metabolism for Mtb survival in neutrophils. We have identified several new routes to be considered for anti-tubercular therapies, particularly during the active stage of the disease that is dominated by neutrophils. Moreover, we have identified models by which Mtb survives in different host cells and how different host cells respond to Mtb, demonstrating the heterogeneity in TB pathogenesis and complexity in treating the disease. In the face of a global health threat that is gaining widespread antibiotic resistance and has only one, very ineffective, vaccine, it is important to understand the host response to infection to develop better vaccines and identify targets for host directed therapies.

DOI

https://doi.org/10.48765/9nsb-x923

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