Chronic microbial infections result from persistent host colonization that is not cleared via the immune response or therapeutics. Within the host, microbes can undergo adaptive evolution, whereby beneficial traits promoting persistence arise due to selection; these traits can therefore affect disease outcomes and treatment strategies. The Gram-negative opportunistic pathogen Pseudomonas aeruginosa is the primary cause of chronic, fatal respiratory infections in individuals with the heritable disease cystic fibrosis (CF). The goal of this dissertation is to identify adaptations that allow P. aeruginosa to persist in the host during chronic CF lung infection. To achieve this goal, P. aeruginosa was chronologically sampled from 3 CF patients, ranging from the first infecting bacterium (the ancestor) to ~40,000 generations post-infection. By comparing gene expression profiles of ancestral and evolved isolates sampled from multiple patients, I identified 24 parallel gene expression changes that occurred over time within each lineage, suggesting that these traits are beneficial to the bacterium. Because most of these traits had unknown physiological roles, I sought to characterize their biological significance. I used a gain-of-function genetic screen and discovered that a subset of these genes enhance biofilm formation, a sessile mode of growth proposed to be important during chronic CF lung infection. I showed that enhanced biofilm formation is due to increased production of the exopolysaccharide Psl, which is traditionally viewed as less critical for maintaining chronic infections than other virulence factors. Lastly, I demonstrated that a majority (~72%) of chronic P. aeruginosa isolates produce more Psl than their corresponding ancestor, suggesting that this exopolysaccharide is important during chronic infection and an adaptive trait. / text

Constipation and diarrhea are common in autistic patients. Antibiotic treatment against bacteria appears to partially alleviate autistic-related symptoms. The bacterium Clostridium bolteae has been shown to be overabundant in the intestinal tract of autistic children suffering from gastric intestinal ailments, and as such is an organism that could potentially aggravate gastrointestinal symptoms. Investigation of the cell-wall polysaccharides of C. bolteae was employed in order to evaluate their structure and immunogenicity. Exploration revealed that C. bolteae produces a conserved specific capsular polysaccharide comprised of rhamnose and mannose units: [->3)-α-D-Manp-(1->4)-β-D-Rhap-(1->], which is immunogenic in rabbits. This is the first described immunogen of C. bolteae and indicates the prospect of using this polysaccharide as a vaccine to reduce or prevent colonization of the intestinal tract in autistic patients, and as a diagnostic marker for rapid detection. This diagnostic target can be used in a multivalent vaccine, which may potentially include Sutterella and Desulfovibrio.

Campylobacter jejuni bacterial pathogen is among the primary causes of food-borne acute gastroenteritis in North America and the world. It has also been linked to severe post-infection sequelae such as Guillain-Barré syndrome. Previous studies identified C. jejuni surface capsular polysaccharide (CPS) as a target for creation of a carbohydrate based vaccine in which the CPS is conjugated to a carrier protein. In this thesis, following sample purification, aspects of C. jejuni HS:5 CPS structure were characterized using numerous analytical techniques such as NMR and GC-MS. CPS is comprised of α-DD-Heptoses linked at C2 to the anomeric carbons of glucose. The α-Glucose molecules are linked though C4 to the α-DD-Heptose anomeric carbon. The α-DD-Heptose structure also has an occasional ring structured amino acid modification. Following characterization the CPS was oxidized and developed into a prototype glycoconjugate vaccine using TEMPO oxidation and EDC-CRM197 coupling methods. / The Natural Sciences and Engineering Research Council of Canada (NSERC)

In this project, Cytophaga hutchinsonii, an aerobic gliding bacterium with cellulose-degrading ability, was studied, since its cellulase system was unknown and might be very different from those of other cellulose-degrading species. Only ß-1,4- endoglucanases and non-specific ß-glucosidases were found in the C. hutchinsonii genome sequence, whereas specific exoglucanases were apparently absent. Almost all putative cellulases were composed of catalytic domains only, without carbohydrate-binding modules. Samples from C. hutchinsonii cultures were analyzed by using TLC and colorimetric assays. Glucose was detected in the cellobiose grown culture, but not in cellulose-grown cultures, suggesting that cellobiose is hydrolyzed extracellularly rather than being directly assimilated, and that cellulose may not be degraded via cellobiose. Also, cellobiose-based cultures caused greater acidification of the medium than glucose or cellulose grown cultures. Nine putative cellulases were expressed in four bacterial strains. In some cases, expression was toxic to host cells. The crude lysates were tested for endoglucanase, specific exoglucanase or nonspecific ß-glucosidase activity. CHU_1280 and CHU_1842 showed apparent endoglucanase activity when expressed in Citrobacter freundii. Four putative GH family 3 ß-glucosidases with similar conserved domains were expressed in Escherichia coli JM109 and E. coli BL21(DE3)pLysS. One of these, CHU_2268, was found to possess MUC-degrading ability. This suggests that CHU_2268 may be the 'missing' exoglucanase in C. hutchinsonii. Another two ß-glucosidases, CHU_2273 and CHU_3784, possessed only MUG-degrading activity.

The formation of bacterial biofilms requires an extracellular matrix to facilitate adherence of bacteria to the surface they colonize. Carbohydrate polymers, known as exopolysaccharides, form a key component of most biofilm matrices. A wide variety of medically-important biofilm forming bacterial strains, including S. epidermidis, S. aureus, E. coli, B. pertussis, and Y. pestis generate the same β-1,6-N-acetyl glucosamine (PNAG) homopolymer as a key biofilm matrix exopolysaccharide. In E. coli, as well as in the other bacterial strains, the PNAG undergoes partial enzymatic de-N-acetylation, which is essential for surface attachment and subsequent biofilm formation. In vivo studies implied that the enzyme responsible for carrying out de-N-acetylation in E. coli is PgaB, an enzyme with sequence homologues in many Gram negative species capable of forming biofilms. In this work, the first biochemical characterization of PgaB is presented. We confirmed the activity of PgaB on β-1,6-GlcNAc oligosaccharides. The activity of PgaB is specific for the β-1,6 linkage and no de-N-acetylation of β-1,4-GlcNAc oligosaccharides was detected. Enzyme activity is dependent on the degree of substrate polymerization, as the second order rate constant for pentasaccharide substrate was determined to be four times higher than that of the tetrasaccharide substrate. Oligosaccharide sequencing studies indicate that there may be a pattern in the de-N-acetylation of substrates by PgaB. The central residue is modified in mono-de-N-acetylated pentasaccharide substrate, while di-de-N-acetylated hexasaccharide substrate shows modification mainly at the third and fifth residues from the non-reducing terminus of the substrate. Activity studies revealed that PgaB is activated by Ni2+ as well as by Fe2+, which is uncommon for deacetylase enzymes. Metal coordination to active site residues His184 and His189 was confirmed by mutagenesis studies, which also indicated that the metal likely plays a catalytic role. The results of these metal dependence studies support the observed binding of nickel and iron to the active site in PgaB crystal structures. The characterization studies presented in this thesis allow us to gain a better understanding of the de-N-acetylation aspect of the PNAG biosynthetic process and will serve as a basis for enzyme inhibitor design.

The formation of bacterial biofilms requires an extracellular matrix to facilitate adherence of bacteria to the surface they colonize. Carbohydrate polymers, known as exopolysaccharides, form a key component of most biofilm matrices. A wide variety of medically-important biofilm forming bacterial strains, including S. epidermidis, S. aureus, E. coli, B. pertussis, and Y. pestis generate the same β-1,6-N-acetyl glucosamine (PNAG) homopolymer as a key biofilm matrix exopolysaccharide. In E. coli, as well as in the other bacterial strains, the PNAG undergoes partial enzymatic de-N-acetylation, which is essential for surface attachment and subsequent biofilm formation. In vivo studies implied that the enzyme responsible for carrying out de-N-acetylation in E. coli is PgaB, an enzyme with sequence homologues in many Gram negative species capable of forming biofilms. In this work, the first biochemical characterization of PgaB is presented. We confirmed the activity of PgaB on β-1,6-GlcNAc oligosaccharides. The activity of PgaB is specific for the β-1,6 linkage and no de-N-acetylation of β-1,4-GlcNAc oligosaccharides was detected. Enzyme activity is dependent on the degree of substrate polymerization, as the second order rate constant for pentasaccharide substrate was determined to be four times higher than that of the tetrasaccharide substrate. Oligosaccharide sequencing studies indicate that there may be a pattern in the de-N-acetylation of substrates by PgaB. The central residue is modified in mono-de-N-acetylated pentasaccharide substrate, while di-de-N-acetylated hexasaccharide substrate shows modification mainly at the third and fifth residues from the non-reducing terminus of the substrate. Activity studies revealed that PgaB is activated by Ni2+ as well as by Fe2+, which is uncommon for deacetylase enzymes. Metal coordination to active site residues His184 and His189 was confirmed by mutagenesis studies, which also indicated that the metal likely plays a catalytic role. The results of these metal dependence studies support the observed binding of nickel and iron to the active site in PgaB crystal structures. The characterization studies presented in this thesis allow us to gain a better understanding of the de-N-acetylation aspect of the PNAG biosynthetic process and will serve as a basis for enzyme inhibitor design.

1,3-β-glucans are a class of natural polysaccharides with unique pharmacological properties and the ability to form triple helical structures and resilient gels. Curdlan and other 1,3-β-glucans have found application pharmacologically in the treatment of cancers and acceleration of wound healing in humans and in the impartation of infection resistance in animal husbandry. Structurally, these polysaccharides have found application in food science as thermal gels, in nanostructure formation as helical scaffolds, and in drug delivery as nanocarriers for drugs and as inclusion complexes with polynucleotides. A literature review of the important work on Curdlan research reveals two streams of research: investigation of the pharmacological significance of these polymers and their application in increasing host immunocompetency and investigation of the nature of the triple helix and its application in a variety of fields from food gels to drug delivery. Two significant contributions to the field of Curdlan research have been completed including 1) The development of a Curdlan nanoparticle drug delivery platform and 2) A new multi-component liquid crystalline hydrogel providing a new route to form polynucleotide inclusion complexes with Curdlan for gene delivery. The developed nanoparticle platform exhibited high encapsulation of chemotherapeutic drugs and a 24-hour controlled release with a particle size of 109.9 nm. The liquid crystalline hydrogel exhibited homogeneous inclusion of DNA into amorphous and crystalline phases of Curdlan and delayed and triggered release of polynucleotide content. This work has been a significant demonstration of the potential of Curdlan as a new polymer for multi-functional drug delivery.

Humboldt-Universiẗat, Diss.--Berlin, 1997.

Universiẗat, Diss., 2001--Giessen.

Techn. Universiẗat, Diss., 2005--Berlin.