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Disarming and Sensing the Bacterial Type III Secretion System

  • Author(s): Duncan, Miles Christopher
  • Advisor(s): Stone, Victoria A
  • et al.
Abstract

The aims of my dissertation research were to determine how a bacterial virulence factor, the type III secretion system (T3SS), is sensed by the innate immune system and to identify small molecule T3SS inhibitors for use as biochemical tools and as potential therapeutics. The T3SS is a needle-like apparatus used by dozens of bacterial pathogens to inject effector proteins inside target host cells. This prevalent virulence factor allows bacterial survival and replication within the host, and T3SS- positive pathogens collectively cause more than 200 million illnesses each year. These T3SSs are highly conserved between bacterial genera, making them important for study and a potential drug target.

The first half of this work focuses on discovering T3SS inhibitors. In collaboration with the Linington lab and Chemical Screening Center, I developed a high-throughput screen (HTS) to identify small molecules that block T3SS formation or its proper function. This HTS was based on the host NF-κB response to the T3SS, and it uncovered piericidins as a novel family of T3SS inhibitors. These molecules are not toxic to bacteria or eukaryotic cells, and prevented type III secretion in vitro and T3SS-mediated effector translocation into host cells.

The second goal of my dissertation was to determine how the T3SS triggers host immune responses, in particular the proinflammatory transcription factor NF-κB.

I carried out a chemical genetics screen to identify host-targeted small molecules that blocked the ability of eukaryotic cells to respond to the T3SS. I identified multiple pathways essential for this response, including five that prevented normal T3SS activity on host cells. As a result of this screen, I hypothesized and demonstrated that the T3SSs of intracellular and extracellular bacteria activate different immune responses. Specifically, intracellular bacteria trigger non-canonical NF-κB, an outcome not typically associated with innate immune sensing of pathogens. This finding could have broad implications for infections by intracellular pathogens that utilize a T3SS, as hyper-induction of non-canonical NF-κB is associated with rheumatoid arthritis and an increased cancer risk.

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