UC Riverside

Bacteria have devised various mechanisms to survive and cause disease. This study investigated two mechanisms, β-lactam resistance in Helicobacter pylori and biosynthesis of an exopolysaccharide coat, alginate, from Pseudomonas aeruginosa.

Helicobacter pylori amoxicillin resistance

Helicobacter pylori, a gastric pathogen that causes stomach ulcers is typically treated with a multi-drug regiment, which can include amoxicillin. Amoxicillin resistance is becoming more prevalent in clinically isolate cultures and has been linked to persistent infections. Using whole genome sequencing, this study was able to identify 5 mutations, in 4 genes, that contribute to amoxicillin resistance in IS5, an in vitro created strain. This study has provided evidence that the mutations in PBP1, HefC, HofH, and HopC account for a majority of the resistance observed in the IS5 strain.

Pseudomonas aeruginosa alginate biosynthesis

Pseudomonas aeruginosa is a gram negative opportunistic pathogen which significantly impacted individuals with cystic fibrosis (CF). P. Aeruginosa initially forms acute infections but after 11 years of recurrent acute infections, CF patients develop a chronic infection of alginate producing P. aeruginosa. Alginate is synthesized by a 12-gene alginate biosynthetic operon and a separately expressed algC. The goal of this study was to determine the contribution of algL and algX to alginate synthesis and transportation.

This study utilized an AlgL KO to determine the contribution of AlgL to alginate biosynthesis. Attempts to generate resulted in two distinct phenotypes when induced, alginate producing and lethal, and non-alginate producing and viable. Through rescue experiments, this study demonstrates that the alginate producing, lethal strain is the only recoverable strain and represents the true knockout phenotype. Attempt for recover the induced lethal phenotype with catalytically inactive AlgL protein results in recovered alginate production but not viability. Together, these findings support the conclusion that AlgL degrades improperly localized alginate polymers during alginate biosynthesis and that AlgL is part of the putative multi-protein biosynthetic complex that aids alginate biosynthesis in the periplasm.

Utilizing the recently published AlgX protein structure, this study attempted to determine the significance of 6 amino acids located in the hypothetical carbohydrate binding module. Alanine substitution at T398 and R406 significantly decreased the ability of AlgX to bind to immobilized alginate polymers. Conversely, alanine substitution at W400 significantly increases AlgX binding to alginate. All three amino acid significantly decrease the amount of high molecular weight (HMW) alginate polymers produced. Together these findings suggest that T398, W400 and R406 are all essential to AlgX binding to alginate in the carbohydrate binding module and AlgX binding to alginate is important to alginate biosynthesis.