Studies on the mode of action of dermonecrotic toxins: Effects on adipogenic signaling pathways and delivery of c-terminal cargo into the cytosol

Bannai, Yuka

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Description

Title

Studies on the mode of action of dermonecrotic toxins: Effects on adipogenic signaling pathways and delivery of c-terminal cargo into the cytosol

Author(s)

Bannai, Yuka

Issue Date

2014-05-30T17:07:27Z

Director of Research (if dissertation) or Advisor (if thesis)

Wilson, Brenda A.

Doctoral Committee Chair(s)

Wilson, Brenda A.

Committee Member(s)

• Tapping, Richard I.
• Shisler, Joanna L.
• Farrand, Stephen K.

Department of Study

Microbiology

Discipline

Microbiology

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• bacterial protein toxin
• Gαq
• Gα12/13
• Rho-associated protein kinase (Rho/ROCK)
• Wnt
• differentiation
• CAATT enhancer-binding protein α (C/EBPα)
• peroxisome-proliferator-activated receptor γ (PPARγ)
• membrane translocation
• cargo delivery
• Pasteurella multocida toxin
• dermonecrotic toxin
• Bordetella dermonecrotic toxin
• E. coli cytotoxic necrotizing factor
• receptor binding
• receptor-mediated endocytosis
• deamidation
• intracellular trafficking
• endosomal vesicles
• endosomal acidification
• modular toxin
• delivery vehicle
• intoxication
• bacterial pathogen
• zoonotic disease
• G-protein
• atrophic rhinitis
• bone resoption
• dermonecrosis
• structure-function
• pasteurellosis
• functional domains
• cytosolic fractions
• subcellular fractionation

Abstract

The dermonecrotic toxin family consists of the dermonecrotic toxin from Bordetella spp. (DNT), the cytotoxic necrotizing factors from Escherichia coli (CNF1, CNF2 and CNF3) and Yersinia pseudotuberculosis (CNFY), and the mitogenic toxin from Pasteurella multocida (PMT). These relatively large (100-160 kDa), single polypeptide protein toxins share some regions of homology with one another in their protein sequence, which may account for some of their similar biological activities. Though much effort has been directed toward understanding the mode of toxin entry and their molecular determinants, we still lack sufficient evidence to fully comprehend the intoxication process. The focus of this thesis is therefore to address two major aspects of the toxin’s mode of action: (1) The involvement of the dermonecrotic toxin in adipogenesis and the effects on adipocyte differentiation, and (2) PMT-mediated delivery of its cargo. In this work we demonstrate the involvement of dermonecrotic toxins in adipogenesis. We show that treatment with any one of the dermonecrotic toxins blocked adipogenesis and inhibited adipocyte differentiation through Rho/ROCK-dependent pathway, suggesting an important role of the Rho/ROCK pathway in the regulation of adipocyte differentiation and adipogenesis. CNF1- and DNT- mediated inhibition of Rho/ROCK signaling displayed stronger effects compared to that of PMT, indicating that additional pathways mediate the action of PMT. Knockdown studies confirm that PMT-mediated downregulation of Notch1 does not occur through Rho/ROCK or Gαq but through G12/13 signaling. We present herein a better-defined trafficking pathway of PMT and mechanism by which the toxin delivers the activity domain into host cell. Using an HA-tagged PMT (PMT-HA), we show the subcellular localization and nature of the protein after internalization in Swiss 3T3 cells. Our experiments reveal an internalized intact moiety corresponding to the activity domain, providing the first evidence that delivery of C-terminus takes place from late endosomes and involves the release of endogenous cargo. We also probe the early steps of intoxication using an N-terminal mutant of PMT (1-568) fused to GFP and the later stages of the process using a biotinylated full-length toxin and truncated mutants. This investigation reveals the initial localization of the toxin in early endosomes and its subsequent localization to other subcellular vesicular compartments, such as lysosomes. Moreover, upon the delivery of cargo, the N-terminal fragment of the toxin remains in the vesicle. Both full-length and N-terminal toxins transfer most of the cargo (GFP, PMT-C) intact into the cytosol, and the N-terminal portion of the toxin gets further processed into multiple, smaller fragments, which remain associated with vesicles. Taken together, we outline a mechanism whereby after uptake and trafficking to endosomes, PMT is proteolytically processed and subsequently translocates and releases its C-terminal cargo (endogenous or exogenous) as an intact moiety into the cell cytosol. This cargo would then presumably target the plasma membrane via its membrane localization subdomain to bring the glutamine deamidase domain to the membrane-bound heterotrimeric G-protein substrate. This thesis provides new insights in toxin translocation and the understanding of the toxin mode of action.

Graduation Semester

2014-05

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Copyright 2014 Yuka Bannai

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