Protein Purification of Shigella dysenteriae Transcription Factor VirF

by Brian Li (VI), Arjun Sen (VI), Maddie Humphreys (V), Daniela Karnaugh (V), Dr. Pousont, Dr. D’Ausilio

Abstract

Shigella is a pathogen that is a leading cause of bacterial foodborne illness worldwide. The virulence cascade of shigellosis requires one of the AraC superfamily transcription factors, the protein VirF. Other members of this superfamily, such as ToxT, which is involved in the virulence cascade of Vibrio cholerae, show inhibition of DNA by binding a small fatty acid. We wish to determine whether or not VirF can be inhibited in a similar manner, and are therefore working to express, purify, and ultimately solve the crystal structures of VirF. Solving the structures will help to reveal the mechanism of transcriptional regulation in S. dysenteriae and could lead to development of novel treatments for Shigella related illnesses.

Introduction

The Shigella bacterium causes sh igellosis, specifically mammalian gastroenteritis. An estimated 80-165 million cases of gastroenteritis due to Shigella species occur globally each year. Symptoms vary from inflammation of the intestines causing diarrhea to nausea and vomiting. Shigella also kills over 600,000 people annually. Infection begins with the ingestion of contaminated food or water. Many of the genes required for intestinal penetration, invasion of host cells, and intestinal and diarrheal disease are carried out by the major regulatory protein VirF located on the PINV as part of the regulatory cascade. This member of the AraC protein family is a DNA-binding protein that activates toxicity factors icsA and virB genes. These genes are then able to activate other downstream effectors involving invasion of the cell wall and host infection. Thus, inhibiting VirF would inhibit the whole virulence cascade of shigellosis.

Methods

Our team will be working at the laboratory at the Pingry School under the guidance of Dr. Jennifer Pousant and Dr. Morgan D’Ausilio. We received the VirF plasmid dried on filter paper from Dartmouth’s Kull Laboratory in January 2021. We rehydrated the plasmid in distilled water, and proceeded to conduct bacterial transformation to replicate the plasmid. Then, through a miniprep, we isolated the plasmid from the bacteria and sent the plasmid to a lab for sequencing. We then analyzed the sequencing/plasmid map to confirm there are no mutations in our sample.

Future Steps

Our goal is to isolate the VirF proteins which will help to understand the mechanism of transcriptional regulation in S. dysenteriae. Understanding how to possibly inhibit these structures could lead to development of novel treatments for Shigella related illnesses. Our next step is to run an autoinduction to express our target protein. We will then purify our protein through affinity chromatography.