License to Kill: Bioengineering a bacterium with Antimicrobial Activity via the Exploitation of Toxin-antitoxin Systems and Conjugation

Mary Girard (2012 Goldschmidt Female Graduate Student Scholarship Recipient)

Mary’s mentor is Dr. Christophe Herman in the Departments of Molecular Virology & Microbiology and Molecular & Human Genetics, Baylor College of Medicine, Houston, TX

With the rise in antibiotic resistant bacterial infections, we are in need of new antimicrobial methods.  One potential alternative is to exploit the natural bacterial toxin-antitoxin (TA) systems. TA systems are  bacterial operons which encode a stable toxin and an unstable corresponding  antitoxin. Toxin-antitoxin gene modules are always co-expressed to alleviate toxicity as the toxin is detrimental and will kill or inhibit growth of the bacterium when the antitoxin is depleted. The role of TA systems in bacterial physiology remains unclear; one possible role for TA systems is maintenance and proper segregation of large, low-copy plasmids. If errors occur in plasmid segregation during division, the daughter cell which lacks a TA-encoding plasmid will succumb to the toxin. Accordingly, several TA systems have beenfound on conjugative plasmids, which are transferred through a sex pilus from the donor to the recipient in a process known as conjugation.

This project aims to investigate a novel antimicrobial approach that exploits conjugation and TA systems to engineer a bacterium that delivers toxins by conjugation. This strategy relies on uncoupling toxin/antitoxin gene expression by placing the toxin gene on the conjugative plasmid and the antitoxin gene on the chromosome. Upon transfer of the plasmid, only the toxins will be expressed, killing the recipient bacterium. We aim to determine whether conjugative transfer of a plasmid encoding multiple toxins will kill recipient bacteria and how this toxic effect can be targeted to specific bacterial pathogens. We are initially testing this antimicrobial strategy by performing mating assays between the engineered strain carrying a single toxin and non-pathogenic laboratory strains of E. coli, scoring for cell viability.  Preliminary data shows that this method effectively kills recipient E. coli in both planktonic and biofilm  states. Next, we will be testing different combinations of toxins to determine the effect of multiple toxins on cell viability. In the future, mating and viability assays will also be performed with a panel of bacterial pathogens. Ultimately, this work may lead to the development of this system for application in biotherapeutics or biocontainment.


Evolution of Enhanced Psl Exopolysaccharide Production in Chronic Pseudomonas aeruginosa Cystic Fibrosis Isolates

Holly K. Huse (2012 Goldschmidt Female Graduate Student Scholarship Recipient)

Holly’s mentor is Dr. Marvin Whiteley at the University of Texas at Austin, Department of Molecular Genetics and Microbiology

The Gram-negative bacterium Pseudomonas aeruginosa is a common cause of chronic respiratory infections in individuals with the heritable disease cystic fibrosis (CF). These infections can last for decades, resulting in significant morbidity and mortality. Upon establishment, P. aeruginosa infections are difficult to eradicate because the bacterium is highly persistent. This persistence has been partially attributed to P. aeruginosa’s ability to form robust biofilms. While in vitro biofilm growth is well characterized, genetic traits that evolve in vivo and contribute to biofilm formation are less understood.  Recently we identified 24 P. aeruginosa genes that were differentially expressed in chronic P. aeruginosa CF isolates compared to their isogenic progenitor strains. The goal of this study was to identify the function of these genes with the overlying hypothesis that some of these genes would promote biofilm formation in chronic P. aeruginosa strains. To test this hypothesis, we constructed strains of the laboratory bacterium P. aeruginosa PAO1 that expressed these genes at levels observed in the chronic isolates. One of these genes, phaF (PA5060), results in enhanced biofilm formation when expressed in PAO1. PhaF promotes biofilm formation via up-regulation of Psl, an exopolysaccharide essential for attachment and biofilm maintenance. PhaF regulates Psl post-transcriptionally, and the mechanism of this control is currently under investigation. Finally, we show that Psl production is enhanced in 8 of 10 chronic CF isolates compared to ancestral strains, suggesting that Psl is an important biofilm-promoting factor in vivo.


Copyright 2012 Texas Branch ASM - Design by Bryan Frederick