Author's School

School of Medicine

ORCID

https://orcid.org/0000-0002-9207-7237

Author's Department/Program

Biology and Biomedical Sciences

Language

English (en)

Date of Award

8-15-2026

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Deborah J. Veis

Committee Members

Gabriel Mbalaviele, Yousef Abu-Amer, James E. Cassat, Jennifer A. Philips, Elisha D. Roberson

Abstract

Bone infection is most commonly caused by the gram-positive bacterium Staphylococcus aureus. It has myriad ways in which it can circumvent or subvert killing by the host or antibiotics. Intracellular infection is one such niche that is protective from immune and antibiotic attack. In 2019, Krauss and colleagues discovered that infected osteoclasts (OCs) allow S. aureus to replicate intracellularly. There they found that the ability of S. aureus to grow inside the cell was tied to OC differentiation. The magnitude of overnight bacterial expansion positively correlated with OC maturity and duration cultured with the osteoclastogenic factor RANK ligand (RANKL). Bacterial growth was prevented by knocking out osteoclastogenic genes, identifying that OC differentiation promotes intracellular S. aureus growth. There are many cellular changes that occur during differentiation: NF-κB signaling alters to further promote differentiation, gene expression profiles shift, uptake of glucose and glutamine increase, iron uptake increases, cells generate more mitochondria, the cell undergoes cytoplasmic rearrangements, and cells fuse together to create polykaryons.

Here we sought to identify which of these changes are relevant to intracellular S. aureus growth. We conducted dual species RNA-sequencing on a time course of infected OC cultures and compared them to infections of their precursor cells, Bone Marrow Macrophages (BMMs). Transcriptomic analysis identified that intracellular S. aureus gene expression is predominantly determined by the host cell type they infected. Murine samples also primarily clustered by cell type but secondarily clustered by infection timepoint. Pathway analysis of differentially expressed S. aureus genes revealed bacteria in OCs are enriched for metabolic gene expression compared to those in BMMs. Infecting OCs with S. aureus metabolism and nutrient transport mutants revealed that S. aureus require glycolytic pathways and aspartate biosynthesis to grow intracellularly. These manipulations also identified that glutamine transport by S. aureus is crucial for intracellular growth. These data suggest that nutrient abundance in OCs is one facet that supports S. aureus expansion.

OCs did not clear metabolic mutants, even if they did not grow. Similarly, pathway analysis of differentially expressed host genes indicated a delayed defense response in OCs. These data inform how OCs fail to respond to infection effectively. Examination of bacterial stress response pathway gene expression reveal that S. aureus in OCs activate PerR, which responds to peroxidative stress. However, we did not see activation of the SOS response, which would indicate that oxidative or other general stress responses were overwhelmed. These data suggest S. aureus in OCs experience oxidative stress but not enough to trigger an emergency cellular damage response. When we measured the redox level of OCs we found that inoculation with S. aureus can durably reduce the OC culture’s oxidative state. These data may indicate that S. aureus can effectively fight reactive oxygen and nitrogen species produced by OCs.

In contrast, S. aureus in BMMs show lower expression of the PerR regulon than in OCs, with exception of iron sequestration genes that are also regulated by Fur, a transcriptional regulator of the iron starvation response. Additionally, S. aureus in BMMs show greater activation of the SOS pathway, compared to those in OCs, suggesting damage accumulation in the bacterium. Examination of the Fur regulon showed greater activation of iron stress genes by S. aureus in BMMs. These data indicate that S. aureus in BMMs may be starved for iron.

We found that S. aureus in OCs, compared to those in BMMs, activated few of the iron stress response genes regulated by Fur. Deletion of several iron acquisition genes regulated by Fur had no impact on bacterial growth in OCs. However, the mutants tested impaired one or two iron acquisition pathways at a time and cannot rule out possible compensation from other pathways. Preliminary experiments limiting host access to iron indicate that this condition may enhance S. aureus’ ability to grow intracellularly.

These studies investigated the basic factors which OCs support intracellular S. aureus growth. Taken together, our results paint a picture of OCs as a nutrient rich environment with an inability to fight infection.

DOI

https://doi.org/10.48765/4048-rk76

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