Abstract

Cytolethal distending toxin (CDT) is a virulence factor produced by many Gram-negative bacteria, including Haemophilus ducreyi, the causative agent of genital chancroid. CDT is a heterotrimeric toxin consisting of a cell-binding domain (CdtA + CdtC) and a catalytic domain (CdtB) that has DNase activity. After binding to the host plasma membrane, CDT undergoes endocytosis and travels through the endosomes en route to the endoplasmic reticulum (ER). Only CdtB and CdtC arrive in the Golgi before moving to the ER. Only then does CdtB move into the nucleus, causing DNA damage that induces cell-cycle arrest and apoptosis. The previous CDT trafficking model suggested that CdtA remains on the plasma membrane while the CdtB/CdtC heterodimer is transported inside the cell. This model is based on experiments that were unable to detect CdtA inside the host cell. Here, we reexamine this model and demonstrate that CDT is internalized as an intact holotoxin. Furthermore, the acidification of the endosomes induces CdtA release from the CdtB/CdtC heterodimer. Using a cell-based ELISA, we report that CdtA facilitates CDT binding to the plasma membrane and demonstrate that nearly the entire pool of surface-bound toxin is internalized from the plasma membrane within 20 minutes. As determined by Western blot, all of internalized CdtA and most of internalized CdtB and CdtC are rapidly degraded in the lysosomes. CdtA colocalized with EEA-1, an early endosomal marker, before lysosomal degradation and was destabilized by the acidic conditions found in the early endosomes (pH 6.0-6.3). This led to its release from the CDT holotoxin as determined by circular dichroism and surface plasmon resonance. The results in this dissertation demonstrate that CDT is internalized as an intact holotoxin, with the acidic environment of endosomes triggering the separation of CdtA from the CdtB/CdtC heterodimer – the first stage of CDT's novel two-stage disassembly.

Notes

If this is your thesis or dissertation, and want to learn how to access it or for more information about readership statistics, contact us at STARS@ucf.edu

Graduation Date

2022

Semester

Fall

Advisor

Teter, Kenneth

Degree

Doctor of Philosophy (Ph.D.)

College

College of Medicine

Department

Burnett School of Biomedical Sciences

Degree Program

Biomedical Sciences

Format

application/pdf

Identifier

CFE0009362; DP0027085

URL

https://purls.library.ucf.edu/go/DP0027085

Language

English

Release Date

December 2022

Length of Campus-only Access

None

Access Status

Doctoral Dissertation (Open Access)

Download

Share

COinS