Abstract

Cytolethal distending toxin (CDT) is a virulence factor produced by many Gram-negative bacteria, including Haemophilus ducreyi. This fastidious pathogen is the causative agent of genital cancroid. CDT is a heterotrimeric toxin with an AB2 structure consisting of a cell-binding "B" domain (CdtA + CdtC) and a catalytic "A" domain (CdtB) that has DNase activity. This toxin assembles in the bacterial periplasm that lacks ATP and is secreted into the extracellular environment. After cell binding, CDT is internalized by endocytosis and travels through the endosomes and Golgi before arriving in the endoplasmic reticulum (ER). CdtA is lost from the holotoxin before reaching the Golgi, and CdtB separates from CdtC in the ER. CdtB is then transported into the nucleus, inducing cell cycle arrest and apoptosis. Using disassembly of the AB5 pertussis toxin as a model, we explore that ATP, which is present in the ER lumen but not in the endosomes or Golgi, will cause dissociation of the CdtB/CdtC heterodimer. We have cloned and purified the three individual subunits of the H. ducreyi CDT. When combined, the subunits form a lethal holotoxin. Examining the individual toxin subunits, only CdtB binds with ATP but does not function as an ATPase. CdtB's binding to ATP also does not cause global changes to its secondary structure. After isolating the CdtB/CdtC heterodimer, we have shown the addition of ATP causes CdtC to dissociate from CdtB. The work presented in this Thesis provides a molecular basis for why the CdtB/CdtC heterodimer disassembles after reaching the ER and confirms the novel two-stage disassembly mechanism for CDT, a first in the AB toxin field.

Notes

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Graduation Date

2019

Semester

Summer

Advisor

Teter, Kenneth

Degree

Master of Science (M.S.)

College

College of Medicine

Department

Biomedical Sciences

Degree Program

Biotechnology

Format

application/pdf

Identifier

CFE0007657

URL

http://purl.fcla.edu/fcla/etd/CFE0007657

Language

English

Release Date

August 2020

Length of Campus-only Access

1 year

Access Status

Masters Thesis (Campus-only Access)

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