Date of Award

2016

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Cell & Molecular Biology

First Advisor

Igor E. Brodsky

Abstract

Multicellular organisms constantly encounter microbes, ranging from beneficial to pathogenic. In order to mount appropriate immune responses that allow the host to clear pathogens while maintaining a balance with nonpathogenic microbes, the innate immune system must discriminate between pathogens and commensals. Through the recognition of virulence structures and activities, innate immune cells can distinguish pathogens from commensals. One such virulence structure, the type III secretion system (T3SS), translocates effector proteins into target cells in order to disrupt or modulate host cell signaling pathways and establish replicative niches. Over 25 species of pathogenic gram-negative bacteria depend upon T3SSs to cause productive infection. However, recognition of T3SS activity by cytosolic Pattern Recognition Receptors (PRRs) of the Nucleotide-Binding Domain Leucine Rich Repeat (NLR) family, either through detection of translocated products or membrane disruption, induces assembly of multiprotein complexes known as inflammasomes. Yersinia pseudotuberculosis (Yptb) is an ideal model for inflammasome recognition of the T3SS as Yptb expresses an archetypal T3SS and is a genetically tractable, natural rodent pathogen. Investigation of the interaction between the inflammasomes and the T3SS could reveal important mechanistic and cell biological information about the inflammasomes themselves as well as a potential target for treating T3SS expressing bacteria. Although effectors of Yptb has been shown to actively inhibit inflammasome activation, until this work very little was known about what inflammasome is actually activated by the T3SS, what activity of the T3SS is recognized, and how Yersinia’s YopK protein inhibits inflammasome activation. Therefore, we investigated the bacterial and host interactions required for inflammasome activation and the mechanism by which YopK inhibits inflammasome activation. To dissect the contribution of the different consequences of T3SSs, pore-formation and translocation, to inflammasome activation, we took advantage of variants of YopD and LcrH that separate these functions. Our findings indicated that inflammasome activation required hyper-translocation of YopB/D. Using macrophages deficient in caspase-1, caspase-11, or certain guanylate binding proteins, we characterized the host pathways activated by hyper-translocation of YopD/B. Finally, using mutations in YopK, we characterized how YopK prevents inflammasome activation. Overall, our findings help define how bacterial virulence activities activate innate immune responses.

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