Date of Award

Spring 1-1-2019

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Corrella S. Detweiler

Second Advisor

Shelley D. Copley

Third Advisor

Amy E. Palmer

Fourth Advisor

James D. Orth

Fifth Advisor

Lee Niswander

Abstract

Pathogens withstand extreme host environments during infection. Salmonella enterica serovar Typhimurium survives within host macrophages, immune cells intended to phagocytose and destroy pathogens. Through decades of study, we know much about how Salmonella endures this challenging niche. I leveraged this host-pathogen interface to identify small molecules that disrupt Salmonella infection of macrophages. I developed a medium-throughput fluorescence microscopy-based screening assay and image analysis pipeline to quantify intracellular bacterial load. With this platform, I identified 300 small molecules that reduce Salmonella infection of macrophages. Of the top 60 hits, I characterized three compounds that inhibit bacterial efflux pumps and sensitize Salmonella to host antimicrobial peptides. This result highlights the importance of bacterial efflux pumps in defense against host antimicrobials, and validates efflux pumps as a therapeutic target to treat infection. I also characterized the antimicrobial activity of clomipramine, a clinically used tricyclic antidepressant. I found that anti-Salmonella activity was unrelated to clomipramine’s canonical inhibition of the serotonin reuptake transporter, and that clomipramine may activate host autophagy to clear bacteria. However, clomipramine was ineffective against Salmonella infection in vivo, which limits the possibility of repurposing this drug as an antimicrobial. Together, these studies exemplify the complexity of Salmonella infection of macrophages in how many possible pathways can be modulated by drugs to disrupt this host-pathogen interface.

Recent studies have established the roles that host and bacterial heterogeneity play in the progression and outcome of infection. I identified a unique macrophage phenotype that governs the use of lipids by Salmonella. Only within pro-inflammatory amino-acid-supplemented macrophages was lipid metabolism important for Salmonella infection. Further, only a subset of bacteria utilized lipids within these macrophages, highlighting that even in a specialized macrophage, individual Salmonella employ unique nutritional strategies. Finally, I investigated the effects of co-culturing leukocytes with infected macrophages on Salmonella infection. I found co-culturing erythrocytes or T cells altered activation, iron homeostasis, and nitric oxide levels, with the net effect of increasing Salmonella replication within macrophages. Thus, the macrophage niche is highly diverse and influenced by many factors. Together, my studies illustrate the complexity and uniqueness of the extreme Salmonella-macrophage host-pathogen interface.

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