The incidence of CF related diabetes is on the rise as patient life expectancy continues to improve. Sugars elevated in diabetics include glucose, fructose, and mannose. These sugars, in addition to mannitol (recently approved as an inhaled osmolyte) are the basis for this study, aimed at assessing the impact these clinically relevant sugars have on virulence in Burkholderia multivorans. B. multivorans is a member of the Burkholderia cepacia complex (Bcc), and is the most frequent cause of Bcc infection in CF patients. Using an exopolysaccharide-deficient knockout in macrophage and Galleria mellonella infection models, biofilm formation, and adhesion assays, this study has identified exopolysaccharide-dependent and -independent phenotypes. Sequencing of B. multivorans C1576, a CF outbreak isolate, identified three putative adhesins in clinical isolate C1576 but not present in the sequenced environmental strain ATCC17616. Mannitol promoted adhesion and enhanced expression of these adhesins. This study characterised these adhesins and assessed the distribution within other clinical and environmental isolates of B. multivorans and the Bcc. Additionally, transcriptomic profiling of B. multivorans assessed the sugar response and EPS regulation during growth on clinically relevant sugars. Where possible, links were made between phenotypic studies and transcriptome data. B. multivorans EPS derived from fructose and mannitol was subjected to composition analysis using mass spectrometry, and assessed for biological activity. Still relevant to CF related diabetes, the ability of some members of the Bcc to bind insulin was assessed. Results indicated that a minority of strains bound insulin. Furthermore, by using flow cytometry cell sorting and fluorescence microscopy, results also showed only a small number of cells within a given population that bound insulin. In all, this study has added to the knowledge base of B. multivorans but more work is needed to fully understand virulence strategies exploited by this CF pathogen.

Doctor Scientiae / An esterase and lipase producing Burkholderia multivorans strain was isolated by culture enrichment strategies. A shotgun library of Burkholderia multivorans genomic DNA (prepared in E. coli/pUC18) was screened for lipase and esterase activities. Three positive recombinant clones, pTEND5, pHOLA6 and pRASHI4, conferring esterolytic and lipolytic phenotypes respectively, were identified. Full-length sequencing of DNA inserts was performed using subeloning and "primer-walking" strategies. Nucleotide sequence analysis revealed that the pRASH14 plasmid DNA consisted of two open reading frames (ORPI and ORP2) encoding 356 and 350 amino acids, respectively. Database searches revealed that ORPI and ORP2 were homologous to lipases and chaperones from subfamily I.2. In the pTEND5 sequence, an open reading frame consisting of 978 bp, encoding 326 amino acids, was identified. Database searches revealed that this open reading frame was homologous to family Vesterases. Nucleotide sequence analysis revealed that pHOLA6, plasmid DNA consisted of 1194 bp encoding 398 amino acids and showed homology to family VIII esterases. The primary structures of LipA, EstEFH5 and EstBL from pRASHI4, pTEND5 and pHOLA6, respectively, showed a classical GxSxG motif, which is conserved in many serine hydrolases. In addition, EstBL also showed a consensus SxxK motif, the serine of which acts as a catalytic nucleophile in class C B-lactames and some peptidases.

A promising approach in combatting infections from antibiotic resistant bacteria/fungi is using therapeutic monoclonal antibodies (mAbs) that can bind the outer surfaces of microbes. We have developed a synthetic vaccine platform that can rapidly generate mAbs against surface glycans from desired strains, such as lipopolysaccharides and capsular polysaccharides. To create therapeutic antibodies against Ruminococcus gnavus, Burkholderia multivorans, and Staphylococcus aureus, we synthesize glycan antigens from each strain that are necessary for use in our glycoconjugate vaccine platform. Blooms of the Gram-positive bacterium Ruminococcus gnavus have been correlated with inflammatory bowel disease, and recently a polysaccharide produced by this organism was shown to stimulate release of inflammatory cytokines. This stimulation was proposed to signal through toll-like receptor 4. We have synthesized the pentasaccharide repeating unit of this polysaccharide and showed that it stimulates TNF-α and IL-6 release from bone marrow-derived dendritic cells in a TLR4-dependent manner. A related glycan does not stimulate significant cytokine release, demonstrating TLR4 selectivity in glycan recognition. Ceragenins are a class of antimicrobials that have broad-spectrum activity against many antimicrobial resistant strains of fungi, Gram-negative bacteria, and Gram-positive bacteria. Previous work has shown that ceragenins can tolerate large structural modifications without loss of activity, such as incorporation of long polyethylene glycol linkers and other domains with selective binding activities. Here we perform structure activity relationship studies of the tail domain of ceragenins to bind hydroxyapatite to combat bone infection, copper for preventing implant related infections, and as a bioresorbable antimicrobial for use in bioresorbable medical devices.