Investigation of the intracellular trafficking pathways of the dermonecrotic toxin family

Repella, Tana

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Description

Title

Investigation of the intracellular trafficking pathways of the dermonecrotic toxin family

Author(s)

Repella, Tana

Issue Date

2012-02-01T00:55:21Z

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

Wilson, Brenda A.

Committee Member(s)

• Chen, Jie
• Farrand, Stephen K.
• Orlean, Peter A.

Department of Study

Microbiology

Discipline

Microbiology

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Pasteurella multocida toxin
• Cytotoxic necrotizing factor 1
• Cytotoxic necrotizing factor 2
• Cytotoxic necrotizing factor y
• serum response element (SRE)
• translocation
• endocytosis
• Arf6

Abstract

The aim of this thesis is to further define the entry and trafficking pathways of the dermonecrotic toxin family composed of related AB toxins whose members induce dermonecrosis upon injection into animal skin. The dermonecrotic toxins Pasteurella multocida toxin (PMT) from Pasteurella multocida (P. multocida), cytotoxic necrotizing factors 1, 2, and 3 (CNF1, 2 and 3) from Escherichia coli (E. coli), the cytotoxic necrotizing factor Y (CNFY) from Yersinia pseudotuberculosis (Y. pseudotuberculosis), and the dermonecrotic toxin (DNT) from Bordetella species are related by sequence similarity and share similar intracellular GTPaseprotein targets. PMT, CNF1, CNF2, CNFY, and DNT are bacterial exotoxins that are responsible for a wide range of human and zoonotic diseases. The potent mitogenic toxin from Pasteurella multocida (PMT) is the major virulence factor associated with a number of epizootic and zoonotic diseases caused by infection with this respiratory pathogen. PMT is a glutamine-specific protein deamidase that acts on its intracellular G-protein targets to increase intracellular calcium, cytoskeletal, and mitogenic signaling. PMT enters cells through receptormediated endocytosis and then translocates into the cytosol through a pH-dependent process that is inhibited by ammonium chloride (NH4Cl) or bafilomycin A1 (BafA1). However, the detailed mechanisms that govern cellular entry, trafficking, and translocation of PMT remain unclear. Co-localization studies described herein revealed that while PMT shares an initial entry pathway with transferrin (Tfn) and cholera toxin (CT), the trafficking pathways of Tfn, CT, and PMT subsequently diverge, as Tfn is trafficked to recycling endosomes, CT is trafficked retrograde to the ER, and PMT is trafficked to late endosomes. This study implicates the small regulatory GTPase Arf6 in the endocytic trafficking of PMT. Translocation of PMT from the endocytic vesicle occurs through a pH-dependent process that is also dependent on both microtubule and actin dynamics, as evidenced by inhibition of PMT activity in our SRE-based reporter assay, with nocodazole and cytochalasin D, respectively, suggesting that membrane translocation and cytotoxicity of PMT is dependent on its transfer to late endosomal compartments. In contrast, disruption of Golgi-endoplasmic reticulum (ER) trafficking with brefeldin A (BFA) increased iii PMT activity, suggesting that inhibiting PMT trafficking to non-productive compartments that do not lead to translocation, while promoting formation of an acidic tubulovesicle system more conducive to translocation, enhances PMT translocation and activity. CNF1, CNF2, and CNF3 are virulence factors of pathogenic E. coli. Pathogenic E. coli are responsible for a wide range of diseases including intestinal infections, urinary tract infections, septicemia, neonatal meningitis, pneumonia, and hemolytic-uremic syndrome. CNFY is an exotoxin produced by pathogenic Yersinia pseudotuberculosis. DNT is an exotoxin produced by Bordetella species that induces the lesions characteristic of atrophic rhinitis. The CNFs and DNT modify and activate Rho proteins with CNF1 preferentially modifying RhoA and Cdc42, CNF2 preferentially deamidating RhoA and Rac1, and CNFY acting as a selective activator of RhoA. The work reported in this thesis uses the SRE assay to compare activation of SRE signaling pathways among the CNFs and DNT. These results show that CNF2 and CNFY are the strongest activators of SRE signaling pathways. SRE activity peaks at a concentration of 100 ng/mL for CNF1 and CNF2, while concentrations of 1 μg/mL CNFY elicited the highest SRE activation. DNT elicited minimal SRE response, presumably due to paucity of DNT cellsurface receptors on HEK 293T/17. It has been previously demonstrated that the CNFs are dependent upon endosomal acidification for translocation into the cytosol. The results reported herein demonstrate that while high concentrations of agents of endosomal acidification (BafA1 and NH4Cl) inhibit translocation of CNFs, low concentrations of these inhibitors actually enhance the activity of CNF1 and CNF2. Furthermore, a region of the N-terminus, residues 199-267 of CNF1, was identified in which the pI and charge of CNF1/2 differ from that of CNF3/Y, DNT, and PMT and tertiary structural changes that occur in this region with changing pH may be responsible for the increase in CNF1/2 activity. These results also demonstrate that the CNFs translocate from the late endosomes as treatment with nocodazole inhibits their activity. Treatment with nocodazole, which inhibits the progression from early to late endosome, was able to inhibit the NH4Cl-induced increase in CNF1 activity. iv Taken together these results support a model in which the CNFs translocate from an acidified late endosome. In the case of CNF1 and CNF2, small concentrations of inhibitors of this acidification may be able to increase CNF1/2 activity by altering the pH and thereby altering tertiary structure and folding of the toxin proteins to make translocation more favorable.

Graduation Semester

2011-12

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© 2011 Tana Lynn Repella

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