Date of Award

5-19-2020

Document Type

Doctoral Dissertation - Restricted (NYMC/Touro only) Access

Degree Name

Doctor of Philosophy

Department

Microbiology and Immunology

First Advisor

Chandra Shekhar Bakshi, DVM, Ph.D.

Second Advisor

Raj Tiwari, Ph.D.

Third Advisor

Dana Mordue, Ph.D.

Abstract

Francisella tularensis is a highly virulent, facultative intracellular, Gram‐negative bacterium that causes fatal zoonotic disease tularemia. The inflammasome is a multiprotein complex, and its assembly occurs when cytosolic sensors, absent in melanoma 2 (Aim2) and Nucleotide‐binding and oligomerization domain 3 (Nlrp3), recognizes a specific ligand and proteolytically processes the inactive forms of pro‐IL‐1β and IL18 cytokines into their secreted mature active forms. Previous studies conducted by our lab and others have shown that in F. tularensis LVS infected macrophages, the activation of the inflammasome is repressed. On the other hand, Francisella novicida, an avirulent subspecies of Francisella, strongly activates the inflammasome in the infected macrophages. The mechanisms and signaling pathways leading to the Aim2 activation have been elegantly elucidated using F. novicida strain as a model. However, these mechanisms remain unknown for the human virulent F. tularensis strains.

The goal of specific aim 1 of this study was to understand the mechanisms of suppression of Aim2 inflammasome by F. tularensis LVS and establish that the mechanisms known for F. novicida are not operative in the virulent Francisella strains. Bone marrow‐derived macrophages (BMDMs) from the wild type and mice deficient for signaling components up‐ and downstream of Aim2 were used in this study. Our results reveal that F. tularensis LVS induces very low levels of Aim2‐dependent, but gasdermin D (GsdmD)‐independent IL‐1β as compared to those infected with F. novicida. The differences in IL‐1β levels in BMDMs infected with these two Francisella strains is not due to differences in intracellular bacterial replication. Our results also demonstrate that xvii suppression/weak activation of Aim2 is due to the suppression of the cGAS‐STING DNA‐ sensing pathway upstream of Aim2. Introduction of exogenous F. tularensis LVS DNA into the cytosol of the infected BMDMs alone or in conjunction with a priming signal provided by a potent TLR4‐agonist failed to restore IL‐1β levels similar to those observed for F. novicida. These results indicated that in addition to the bacterial DNA, the DNA coming from some other source, specifically from the damaged mitochondria might contribute to the robust IL‐1β levels observed in F. novicida infected BMDMs. Our results show that F. tularensis induces enhanced mitophagy for the removal of damaged mitochondria and thereby prevents the release of mitochondrial DNA, resulting in suppression/weak activation of the Aim2 inflammasome.

Inflammasome‐dependent cytokines IL‐1β and IL‐18, in addition to their role in innate immune responses, are also required for the generation of antigen‐specific adaptive immune responses. In specific aim 1, we observed that Aim2 inflammasome is suppressed/weakly activated in F. tularensis infected BMDMs. In specific aim 2, we investigated the role of Aim2 and Nlrp3 in vaccination‐induced protective immune responses using an in vivo mouse model of tularemia. Since Francisella causes an acute infection associated with a delayed onset of innate immune responses, the infected mice succumb to infection even before adaptive immune responses could kick‐in. We developed a vaccination‐model using a previously described emrA1 mutant of F. tularensis. Our results demonstrate that we can successfully vaccinate both the Aim2‐/‐ and Nlrp3‐/‐ mice with the emrA1 mutant. The emrA1 mutant similar to the wild type mice is cleared by both Aim2‐/‐ and Nlrp3‐/‐ mice and induce comparable antibody‐ and cell‐ xviii mediated immune responses. Moreover, similar to the wild type mice, the emrA1 vaccinated Aim2‐/‐ and Nlrp3‐/‐ mice survived the intranasal 1000xLD100 challenge dose of F. tularensis LVS.

Collectively, the results from this study demonstrate that F. tularensis has evolved complex mechanisms for the suppression of Aim2 inflammasome, and the mechanisms established for F. novicida are not operative in F. tularensis. Further, both Aim2 andNlrp3,probably due to redundant roles, are dispensable for the generation of the vaccination‐ induced protective immune response. This study furthers our understanding of the unknown immunopathogenic mechanisms of the human‐virulent strains of F. tularensis.

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