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.

Biofilm is the virulence factor that is responsible for chronic infection in diseases such as Cystic Fibrosis (CF) and chronic wounds. In this thesis, we examine the role of AlgX, a required protein for alginate biosynthesis in P. aeruginosa. We show that the absence of AlgX resulted in the loss of mucoidy and in silico studies demonstrated that AlgX binds alginate. Alanine mutations demonstrated that K396, T398, W400, and R406 are important for alginate binding. Alginate rescue assays confirm the importance of these amino acid residues for alginate biosynthesis and acetylation. This is the first functional demonstration of AlgX role in alginate biofilm biosynthesis and acetylation.

Biofilm-producing bacteria and redox imbalance are the leading factors that turn acute wounds into chronic wounds. We demonstrated that LIGHT^-/- mouse wounds contain elevated levels of reactive oxygen species (ROS). To see whether chronic wounds can be generated, we increased the redox imbalance in the LIGHT^-/- wounds and infecting the wounds with biofilm-forming bacteria. We demonstrated that by using these conditions, we could induce chronic wounds in the LIGHT^-/- mouse model 100% of the time. These wounds do not re-epithelialize, contain high bacterial burden, and sustained multi-species bacterial infections that are biofilm-forming and antibiotic-resistant.

We also demonstrated that increasing the redox imbalance was sufficient to turn db/db wounds into chronic wounds. These wounds sustained spontaneous biofilm- producing bacterial infections. To verify that redox imbalance is critical for chronicity, we treated chronic wounds with antioxidants and found that oxidative stress was highly reduced, biofilm production was decreased, and bacteria became more sensitive to antibiotics. This is the first demonstration that chronic wounds can be generated and reversed by manipulating the wound's redox microenvironment in an animal model.

Norspermidine has been shown to negatively affect the structure of extracellular polymeric substances (EPS). Using the bacteria that colonized db/db chronic wounds, we demonstrated that 2 mM norspermidine reduces the amount of adherent biofilms produced by exudate bacterial communities. Furthermore, application of norspermidine increases the bactericidal effect of gentamicin. We believe that norspermidine could be used in combination with bactericidal antibiotics to control biofilm infection in dermal chronic wounds.

Helicobacter pylori is a major cause of peptic ulcer disease and potentially stomach cancer. Treatment of patients with gastritis is warranted and normally done with a combination of antibiotics (such as amoxicillin, clarithromycin or others) and a proton pump inhibitor. Resistance to most of these antibiotics in H. pylori has been seen worldwide and is constantly increasing.

The purpose of this thesis was to more systematically characterize the mechanisms of amoxicillin resistance in H. pylori. We used both a clinical isolate strain B258, the first US amoxicillin resistant strain to be examined in this way, and in vitro selected amoxicillin resistant strains. In strain B258 we determined that all

of the amoxicillin resistance was due to the presence of 2 amino acid substitutions in Penicillin Binding Protein 1(PBP1). To explain how these amino acids could increase amoxicillin resistance, we did homology modeling of PBP1 to map the location of these amino acids and their relation to the putative amoxicillin binding cleft.

To better appreciate the role of single amino acid substitutions in amoxicillin resistance, we made site-directed mutants of the most commonly found amino acids in PBP1 from amoxicillin-resistant strains. and transformed a sensitive strain with each to monitor its effect on amoxicillin resistance. We also used homology modeling and affinity binding studies using biotinylated amoxicillin to examine the role of these PBP1 mutations in amoxicillin resistance.

Finally, we examined a series of 6 in vitro selected amoxicillin resistant strains each with slightly increased MICs to amoxicillin, in order to track both the evolution of resistance as well as identify other possible novel resistance mechanisms. Whole genome sequencing of the most resistant isolate when compared to the parental sensitive strain, identified 12 genes as likely candidates affecting amoxicillin resistance. Changes in PBP1, PBP 2 and various outer membrane proteins were identified and their role in amoxicillin resistance is being systematically examined in each of the 6 isolates. Although studies are on-going, results to date have shown that in addition to PBP1, changes in outer membrane permeability are required to get high level amoxicillin resistance.

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