Investigating the role of RNA binding proteins in a neglected tropical disease

Abstract

Burkholderia pseudomallei is a gram-negative bacterium that is commonly found in soil and is endemic to tropical regions such as Thailand and northern Australia. This pathogen causes melioidosis, a bacterial infection that affects both humans and animals. Transmission occurs primarily through inhalation of soil, dust or water droplets, or through contact with contaminated soil or water. Once inside a host, B. pseudomallei can cause localized infections or disseminate throughout the body, resulting in a wide range of symptoms depending on the infection site including abscesses, headaches, muscle/joint pain, weight loss, and fever.

In this study, we investigated the role of the RNA-binding protein Tex as a potential virulence factor contributing to the growth and survival of B. pseudomallei. For these investigations, we used Burkholderia thailandensis, a closely related but non-pathogenic species, as a model organism. Previous studies have demonstrated that Tex influences the survival of B. thailandensis, and mutant strains deficient in Tex exhibit attenuated phenotypes.

We generated a tagged strain of B. thailandensis expressing the Tex allele by creating a new complementation construct by cloning an allele of the tex gene containing His and Flag tags into a Burkholderia expression vector (pPumbi).

We then transformed a B. thailandensis tex knockout mutant (ΔTex) with pPumbi to produce a strain which only expresses the tagged allele in the native organism. Successful expression of the Tex allele in B. thailandensis was confirmed through Western blot analysis. As Tex has previously been shown to be secreted by B. pseudomallei, we next examined bacterial supernatants for the presence of extracellular Tex. Our findings revealed high intracellular expression of the Tex protein, although it was not secreted, contrary to previous reports.

Finally, as Tex has been shown to be important for bacterial survival inside host macrophages, we assessed the sensitivity of wild type, ΔTex, and complemented Tex strains to reactive oxygen species (ROS) and reactive nitrogen species (RNS). Our results support the hypothesis that Tex is important for the survival of B. thailandensis within host cells and plays a crucial role in its pathogenesis. We have generated a powerful new tool to be used in further studies to elucidate the specific mechanisms by which Tex contributes to bacterial virulence.