München, Techn. Universiẗat, Diss., 2007.

Alginate is an exopolysaccharide secreted by Pseudomonas aeruginosa and is a major component of biofilms that infect the lungs of cystic fibrosis patients. Ten proteins have been implicated in alginate polymerization, modification and export, and are believed to assemble into a multi-protein complex that spans the cell envelope and coordinates the synthesis and secretion of alginate. AlgK is a protein encoded in the alginate biosynthetic operon, which is required for the secretion of high-molecular weight alginate. This study describes structural and functional studies of AlgK to improve our understanding of AlgK’s role in alginate biosynthesis. To shed light on the function of AlgK, C14-palmitic acid labeling and sucrose gradient fractionation studies confirmed that AlgK is an outer membrane lipoprotein. Cellular fractionation experiments also found that AlgK is involved in the proper localization of AlgE, the alginate secretion pore in the outer membrane. The structure of AlgK was determined to 2.5 Å resolution by X-ray crystallography and revealed that the protein folds into 22 alpha-helices that pack into a flexible right-handed solenoid. Closer examination of the amino acid sequence revealed that AlgK carries 9.5 tetratricopeptide repeat (TPR)-like elements. Given the role that TPR motifs generally play in protein-protein interaction and the assembly of multi-protein complexes, the presence of these motifs in AlgK suggests that it can bind to one or more proteins. Based on the results presented in this study, we propose that AlgK acts as a scaffold for the assembly of the alginate secretion complex. By mapping highly conserved residues onto the surface of our model, three putative sites of protein-protein interaction were identified. We hypothesize that the N-terminus of AlgK binds to AlgE in the outer membrane, and the C-terminus of AlgK binds to periplasmic and/or inner membrane Alg proteins, thereby acting as a linker between the inner and outer membrane components of the alginate biosynthetic complex. We further hypothesize that together AlgE and AlgK constitute a novel exopolysaccharide secretin. The alginate biosynthetic complex appears to be distinct from the canonical capsular polysaccharide systems currently described.

The mutant-prevention concentration (MPC) is a novel susceptibility measurement defined by a concentration threshold that would require cells to contain two concurrent resistance mutations for growth. Pneuococcal pneumonia, infections caused by <i> Pseudomonas aeruginosa</i>, and urinary tract infections caused by Gram-negative bacilli represent three distinct clinical situations for which fluoroquinolone-resistance occurs. MPC results were defined and measured for fluoroquinolones against clinical isolates of <i>Citrobacter freundii, Enterobacter cloacae, Escherichia. coli, Klebsiella pneumoniae, P. aeruginosa,</i> and <i> Streptococus pneumoniae</i>. Against clinical isolates of <i>S. pneumoniae</i>, MPC results for six fluoroquinolones were measured. Based on their potential for restricting the selection of resistant mutants, the six fluoroquinolones, in descending order, were found to be gemifloxacin > moxifloxacin > trovafloxacin > gatifloxacin > grepafloxacin > levofloxacin. For several compounds, 90% of clinical isolates that lacked a known resistance mutation had a MPC value that was close to or below the serum levels that could be attained with a dosing regimen recommended by the manufacturers. These data identify gemifloxacin, moxifloxacin and gatifloxacin as good candidates for determining whether MPC can be used as a guide for choosing and eventually administering fluoroquinolones to significantly reduce the development of fluoroquinolone ¡Vresistant <i>S. pneumoniae</i>. MPC90 results for 155 clinical isolates of <i>P. aeruginosa </i>against ciprofloxacin and levofloxacin were 4 and 16 Ýg/ml, respectively. Serum drug concentrations reported previously for standard doses were above MPC90 for 5.5 hr for ciprofloxacin and 0 hr for levofloxacin. These data suggest that superior clinical performance of ciprofloxacin correlates with activity against resistant mutant subpopulations measured in vitro. MPC results were compared with minimum inhibitory concentrations (MIC) measurements preformed by agar dilution, and microbroth dilution and minimal inhibitory concentrations (MBC) for 100 clinical isolates of <i>C. freundii </i> (n=20), <i>E. cloacae</i> (n=20), <i>E. coli</i> (n=20), <i>K. pneumoniae</i> (n=20), and <i>P. aeruginosa</i> (n=20) for ciprofloxacin, levofloxacin and garenoxacin. MPC results were 2-to-8 fold higher than MIC or MBC results. Ciprofloxacin MPC results for <i>E.coli, C. freundii, E. cloacae, K. pneumoniae</i>, and <i>P. aeruginosa</i> were 0.5, 2, 1, 1, and 4 Ýg/ml, respectively. Levofloxacin, MPC results were were 1, 2, 4, 1, and 16 Ýg/ml, respectively. Garenoxacin, MPC were 1, 8, >8, 4, and >32 Ýg/ml, respectively. Garenoxacin had the highest MIC and MPC results and was the least active compound tested against isolates of <i>C. freundii, E. cloacae</i>, and <i>P. aeruginosa</i>. These data support the rational use of quinolones in the treatments of urinary tract infections and suppression of resistance. Incorporation of the MPC measurement into dosing strategies may preserve the longevity of antimicrobial compounds for future infectious diseases.

This study examined the use of phage therapy against Pseudomonas aeruginosa strain PA14 biofilms. Part 1: 24 and 96h PA14 biofilms grown in microplates were phage treated and bacterial biomass was quantified using crystal violet staining. Part 2: 24 and 96h PA14 biofilms grown in prepared root canals of human mandibular incisors were treated with phages and intra-canal samples using paper points and round burs were taken to assess phage and bacterial counts. Part 1: Two phages (JBD4 and JBD44a) were used. Treatment with phages produced significant reduction in the mean percentage of biomass in 24h (p<0.05) and 96h (p=0.08) biofilms. Part 2: In 24 and 96h biofilms in a root canal model, no significant difference was found in colony forming units after phage treatment (p>0.05). Phage application significantly reduced the biomass of 24 and 96h PA14 biofilms grown on microplates, but did not in the extracted tooth models.

This study examined the use of phage therapy against Pseudomonas aeruginosa strain PA14 biofilms. Part 1: 24 and 96h PA14 biofilms grown in microplates were phage treated and bacterial biomass was quantified using crystal violet staining. Part 2: 24 and 96h PA14 biofilms grown in prepared root canals of human mandibular incisors were treated with phages and intra-canal samples using paper points and round burs were taken to assess phage and bacterial counts. Part 1: Two phages (JBD4 and JBD44a) were used. Treatment with phages produced significant reduction in the mean percentage of biomass in 24h (p<0.05) and 96h (p=0.08) biofilms. Part 2: In 24 and 96h biofilms in a root canal model, no significant difference was found in colony forming units after phage treatment (p>0.05). Phage application significantly reduced the biomass of 24 and 96h PA14 biofilms grown on microplates, but did not in the extracted tooth models.

Alginate is an exopolysaccharide secreted by Pseudomonas aeruginosa and is a major component of biofilms that infect the lungs of cystic fibrosis patients. Ten proteins have been implicated in alginate polymerization, modification and export, and are believed to assemble into a multi-protein complex that spans the cell envelope and coordinates the synthesis and secretion of alginate. AlgK is a protein encoded in the alginate biosynthetic operon, which is required for the secretion of high-molecular weight alginate. This study describes structural and functional studies of AlgK to improve our understanding of AlgK’s role in alginate biosynthesis. To shed light on the function of AlgK, C14-palmitic acid labeling and sucrose gradient fractionation studies confirmed that AlgK is an outer membrane lipoprotein. Cellular fractionation experiments also found that AlgK is involved in the proper localization of AlgE, the alginate secretion pore in the outer membrane. The structure of AlgK was determined to 2.5 Å resolution by X-ray crystallography and revealed that the protein folds into 22 alpha-helices that pack into a flexible right-handed solenoid. Closer examination of the amino acid sequence revealed that AlgK carries 9.5 tetratricopeptide repeat (TPR)-like elements. Given the role that TPR motifs generally play in protein-protein interaction and the assembly of multi-protein complexes, the presence of these motifs in AlgK suggests that it can bind to one or more proteins. Based on the results presented in this study, we propose that AlgK acts as a scaffold for the assembly of the alginate secretion complex. By mapping highly conserved residues onto the surface of our model, three putative sites of protein-protein interaction were identified. We hypothesize that the N-terminus of AlgK binds to AlgE in the outer membrane, and the C-terminus of AlgK binds to periplasmic and/or inner membrane Alg proteins, thereby acting as a linker between the inner and outer membrane components of the alginate biosynthetic complex. We further hypothesize that together AlgE and AlgK constitute a novel exopolysaccharide secretin. The alginate biosynthetic complex appears to be distinct from the canonical capsular polysaccharide systems currently described.

Le présent ouvrage traite d'une alternative médicale potentielle pour inhiber l'adhésion bactérienne de la bactérie Pseudomonas aeruginosa. Cette dernière possède deux lectines, dépendantes du calcium (PA-IL et PA-IlL), ayant chacune un site de reconnaissance spécifique aux galactoses et aux fucoses, respectivement. Chez les patients atteints de la fIbrose kystique, le gène CF transmembrane regulator (CFTR) a subi une mutation, ce qui occasionne une augmentation de la présence des résidus fucoses à la surface des cellules épithéliales pulmonaires. L'attachement de cette bactérie pathogène à la glycoprotéine CFTR est la principale cause de virulence de la maladie. Pour ce faire, il a été envisagé d'utiliser les dendrimères pour intervenir dans ce processus d'adhésion bactérienne. Les dendrimères sont une classe de macromolécules monodisperses dont l'architecture arborescente autour d'un coeur plurifonctionnel est souvent comparée à différentes organisations autour de nous. En modifiant l'extrémité de ces structures au moyen de différents groupes fonctionnels, il est possible de leur conférer certaines des propriétés recherchées. Plusieurs applications dans divers domaines ont démontré leur potentiel. Particulièrement du côté de la biologie, ces molécules sont souvent comparées à des protéines, de par leur taille et leur structure globulaire. Lors de l'incorporation de fonctions carbohydrates en périphérie, des recherches ont démontré leur potentiel d'action au niveau des interactions sucres-protéines dues à l'effet cluster (augmentation de l'affinité saccharide-ligand grâce à leur multivalence). Ainsi, pour tenter d'inhiber l'adhésion de la bactérie Pseudomonas aeruginosa à la glycoprotéine CFTR, des glycodendrimères fucosylés et/ou galactosylés en surface sont développés. Basées sur des voies synthétiques appropriées, ces structures sont synthétisées à partir de produits commerciaux sur lesquels des groupements azotures et propargyliques sont installés. Ces fonctions précises permettront d'utiliser la « click chemistry », très régiospécifique et versatile. Par la suite, des tests préliminaires en inhibition compétitive (tests ELLA) et de turbidimétrie ont démontré que les deux lectines de la bactérie Pseudomonas aeruginosa pouvaient interagir avec les unités carbohydrates présentes sur les dendrimères et former un précipité détectable à 490nm en fonction du temps. Ainsi, l'utilisation des dendrimères pour inhiber l'adhésion bactérienne pourrait représenter une thérapie alternative ou un complémentaire aux antibiotiques pour le traitement d'infections mortelles. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Chimie thérapeutique, Fibrose kystique, Pseudomonas aeruginosa, Glycomimétiques, L-fucose, Glycosidation, Cycloaddition 1,3-dipolaire, Métathèse croisée, Dihydroxylation asymétrique.

Les composés glycosidiques sont une classe de molécules organiques sous-estimée en chimie médicinale. En effet, la variété de protéines se liant à ce type de molécules est très grande et plusieurs de ces récepteurs sont impliqués dans des processus physiologiques importants tels que l'adhésion cellulaire, la régulation de la croissance, l'apoptose cellulaire et la xénotransplantation. Plusieurs maladies comme le cancer, la fibrose kystique, les infections virales et bactériennes utilisent les interactions glycoside-protéine dans le but d'envahir notre organisme. Le développement de molécules actives basé sur des glycomimétiques aurait l'avantage d'offrir une sélectivité accrue pour un récepteur en particulier selon la nature du glycoside greffé sur l'élément actif. La synthèse de ce type de molécules utilise des réactions de chimie organique moderne telles que des réactions catalysées par transfert de phase, des couplages au palladium (0) de type Sonogashira et Heck ainsi que des réactions spécifiques à la chimie des sucres comme la réaction de propargylation régiosélective impliquant un acétal d'étain. Deux classes d'inhibiteurs ont été synthétisées soient des S-galactosides et lactosides ainsi que des 2-désoxy-C-galactosides. De plus, le développement d'une méthodologie de synthèse de trisaccharides basée sur une réaction d'allylation stéréosélective impliquant un complexe titane-binol a été étudié. Les diverses études réalisées démontrent bien l'efficacité des molécules synthétisées face à divers récepteurs biologiques dont les galectines et la lectine PA-IL de Pseudomonas aeruginosa. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : lectines, fibrose kystique, glycosides, glycomimétigues.

The mutant-prevention concentration (MPC) is a novel susceptibility measurement defined by a concentration threshold that would require cells to contain two concurrent resistance mutations for growth. Pneuococcal pneumonia, infections caused by <i> Pseudomonas aeruginosa</i>, and urinary tract infections caused by Gram-negative bacilli represent three distinct clinical situations for which fluoroquinolone-resistance occurs. MPC results were defined and measured for fluoroquinolones against clinical isolates of <i>Citrobacter freundii, Enterobacter cloacae, Escherichia. coli, Klebsiella pneumoniae, P. aeruginosa,</i> and <i> Streptococus pneumoniae</i>. Against clinical isolates of <i>S. pneumoniae</i>, MPC results for six fluoroquinolones were measured. Based on their potential for restricting the selection of resistant mutants, the six fluoroquinolones, in descending order, were found to be gemifloxacin > moxifloxacin > trovafloxacin > gatifloxacin > grepafloxacin > levofloxacin. For several compounds, 90% of clinical isolates that lacked a known resistance mutation had a MPC value that was close to or below the serum levels that could be attained with a dosing regimen recommended by the manufacturers. These data identify gemifloxacin, moxifloxacin and gatifloxacin as good candidates for determining whether MPC can be used as a guide for choosing and eventually administering fluoroquinolones to significantly reduce the development of fluoroquinolone ¡Vresistant <i>S. pneumoniae</i>. MPC90 results for 155 clinical isolates of <i>P. aeruginosa </i>against ciprofloxacin and levofloxacin were 4 and 16 Ýg/ml, respectively. Serum drug concentrations reported previously for standard doses were above MPC90 for 5.5 hr for ciprofloxacin and 0 hr for levofloxacin. These data suggest that superior clinical performance of ciprofloxacin correlates with activity against resistant mutant subpopulations measured in vitro. MPC results were compared with minimum inhibitory concentrations (MIC) measurements preformed by agar dilution, and microbroth dilution and minimal inhibitory concentrations (MBC) for 100 clinical isolates of <i>C. freundii </i> (n=20), <i>E. cloacae</i> (n=20), <i>E. coli</i> (n=20), <i>K. pneumoniae</i> (n=20), and <i>P. aeruginosa</i> (n=20) for ciprofloxacin, levofloxacin and garenoxacin. MPC results were 2-to-8 fold higher than MIC or MBC results. Ciprofloxacin MPC results for <i>E.coli, C. freundii, E. cloacae, K. pneumoniae</i>, and <i>P. aeruginosa</i> were 0.5, 2, 1, 1, and 4 Ýg/ml, respectively. Levofloxacin, MPC results were were 1, 2, 4, 1, and 16 Ýg/ml, respectively. Garenoxacin, MPC were 1, 8, >8, 4, and >32 Ýg/ml, respectively. Garenoxacin had the highest MIC and MPC results and was the least active compound tested against isolates of <i>C. freundii, E. cloacae</i>, and <i>P. aeruginosa</i>. These data support the rational use of quinolones in the treatments of urinary tract infections and suppression of resistance. Incorporation of the MPC measurement into dosing strategies may preserve the longevity of antimicrobial compounds for future infectious diseases.

Pseudomonas aeruginosa-induced pneumonia is serious problem that results in severe inflammation response and high mortality in the host. Interleukin-1£] (IL-1£]) is one of the major extracellular proinflammatory cytokines thought to be involved in many acute and chronic lung diseases. To investigate the role of caspase-1, IL-1£] and macrophage inflammatory protein-2 (MIP-2) in P. aeruginosa pneumonia induced lung damage, C57BL/6 (WT) and CASP-1-/- mice were subjected to pneumonia induced by intratracheal injection of P. aeruginosa. The lung permeability, bacterial content in blood and lung, lung myeloperoxidase (MPO) activity, total cell counts and protein in bronchoalveolar lavage fluid (BALF), NF-£eB activation as well as expression of IL-1£] and MIP-2 were assayed at 8 hr after P. aeruginosa injection. The IL-1£] inhibitor, anakinra, was also used to evaluate the role of IL-1£]. P. aeruginosa injection increased the lung permeability, lung MPO activity, bacterial counts in blood, total cell counts and protein in BALF, NF-£eB activation and expression of IL-1£] and MIP-2 in WT mice; and these increases were all decreased by administration of anakinra in WT mice or in CASP-1-/- mice. Furthermore, the lung MPO activity, total protein in BALF and expression of IL-1£] and MIP-2 were decreased in CASP-1-/- ¡÷ WT but not in WT ¡÷ CASP-1-/- chimeric mice, suggesting that pneumonia induced lung damage and IL-1£] and MIP-2 expressions depend on caspase-1 signaling of the resident cells.