Student Theses and Dissertations

Date of Award

2013

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Hang Laboratory

Abstract

The emergence of antibiotic-resistant bacterial pathogens and the discovery of new bacterial pathogens have motivated the development of novel antibacterials. One recently proposed strategy is to target pathogenic bacteria specifically by inhibiting virulence mechanisms as opposed to killing bacteria indiscriminately, which includes commensal strains. Due to the increased appreciation for the role commensal bacteria play in the immune response and the importance for maintaining a healthy microbiota, specifically targeting pathogenic bacteria is a desirable goal to attain. Genetic and biochemical studies have highlighted type III secretion systems (T3SSs) as essential components for infection of host cells by Gram-negative bacterial pathogens. Small molecules that target type III protein secretion may therefore represent a new class of antibacterial agents and provide a platform for evaluating an anti-virulence approach. The salicylidene acylhydrazides (SAHs) are a class of compounds that prevent secretion of bacterial effector proteins through the T3SS and attenuate infection from various species of Gram-negative pathogens; however, the molecular target(s) of these compounds remains unknown, and the potency of these compounds is not optimal. To discover the molecular target(s) of the SAHs in Salmonella typhimurium and determine their mechanism of action, I synthesized several alkynyl SAH analogs and employed bioorthogonal labeling techniques for proteomic analysis of their protein-binding partners. Through structure-activity relationship (SAR) analysis of the alkynyl analogs, I discovered important features for the inhibitory activity of the SAHs and observed that they covalently modify many S. typhimurium proteins; however, the protein targets responsible for the inhibitory activity of SAHs remains to be determined. Repurposing chemical inhibitors to target host enzymes required for infection has emerged as an alternative approach to subvert rapid antibiotic resistance in bacterial pathogens. Towards this goal, the isoquinolinesulfonamide H-89 was reported to limit Salmonella replication in macrophages through inhibition of Akt, a host kinase that is activated during infection. However, more potent Akt-specific inhibitors are less effective at inhibiting bacterial replication, suggesting an alternative mechanism of action for H- 89. I discovered that H-89 does not target Akt in host cells to restrict bacterial replication, but more likely prevents bacterial replication by inhibiting the expression of S. typhimurium T3SS components and effector proteins required for bacterial invasion and replication in host cells. As H-89 does not interfere with bacterial growth in liquid culture, these results highlight isoquinolinesulfonamides as a new class of lead compounds for targeting bacterial virulence.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

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