Kingella kingae est un pathogène important en pédiatrie responsable d'infections ostéo-articulaires, de bactériémies, et d'endocardite, principalement chez les enfants entre 6 et 36 mois, et survenant soit de façon sporadique ou épidémique dans les crèches. L'endocardite à K. kingae est une infection sévère qui, lorsqu'elle survient chez un enfant en crèche, devrait impérativement conduire à une investigation épidémiologique pour identifier d'éventuels cas suspects, prendre en charge rapidement les cas épidémiques, et prévenir la survenue de nouveaux cas.L'analyse génomique du genre Kingella a conduit à la description taxonomique de K. negevensis; au développement de nouveaux tests diagnostiques et de génotypage pour K. kingae et K. negevensis. Le développement du premier test par PCR en temps réel spécifique de K. negevensis a ainsi révélé que cette bactérie pouvait être occasionnellement pathogène chez l'enfant, voire mortelle chez le patient immunodéprimé. La découverte d'un locus RTX dans le génome de K. negevensis homologue à celui de K. kingae a ensuite permis de réajuster les stratégies diagnostiques des infections à K. kingae et K. negevensis.... / Kingella kingae is an important paediatric pathogen responsible for bone and joint infections, bacteremia, and endocarditis. Invasive infections affect primary children aged 6-36 months, and may occur either sporadically, or break out in daycare centers. Kingella kingae endocarditis is a serious infection, which should prompt the search for an outbreak of K. kingae infections when it occurs in a day-care attendee and the prevention of new cases.The genomic analysis of the Kingella genus conducted to the taxonomic description of K. negevensis. The design of the first K. negevensis-specific real-time PCR test allowed to disclose that this organism may occasionally be invasive in infants, and even fatal in immunosuppressed patients. The discovery of an homologuous Kingella RTX locus within the K. negevensis genome allowed to refine diagnostic strategies in clinical microbiology for the molecular detection of K. kingae and K. negevensis....

<p>Kingella kingae is a bacterial pathogen that is increasingly recognized as an etiology of septic arthritis, osteomyelitis, bacteremia, and endocarditis in young children. The pathogenesis of K. kingae disease starts with bacterial adherence to the respiratory epithelium of the posterior pharynx. Previous work has identified type IV pili and a trimeric autotransporter protein called Knh (Kingella NhhA homolog) as critical factors for adherence to human epithelial cells. Additional studies established that the presence of a polysaccharide capsule interferes with Knh-mediated adherence. Given the inhibitory role of capsule during adherence we sought to uncover the genes involved in capsule expression to understand how capsule is elaborated on the cell surface. Additionally, this work aimed to further characterize capsule diversity among K. kingae clinical isolates and to investigate the relationship between capsule type and site of isolation. </p><p>We first set out to identify the carbohydrates present in the K. kingae capsule present in the prototype strain 269-492. Glycosyl composition and NMR analysis of surface extractable polysaccharides demonstrated two distinct polysaccharides, one consisting of GalNAc and Kdo with the structure →3)-β-GalpNAc-(1→5)-β-Kdop-(2→ and the other containing galactose alone with the structure →5)-β-Galf-(1→. </p><p>To discern the two polysaccharides we disrupted the ctrA gene required for surface localization of the K. kingae polysaccharide capsule and observed a loss of GalNAc and Kdo but no effect on the presence of Gal in bacterial surface extracts. In contrast, deletion of the pamABCDE locus involved in production of a reported galactan exopolysaccharide eliminated Gal but had no effect on the presence of GalNAc and Kdo in surface extracts. These results established that K. kingae strain KK01 produces a polysaccharide capsule with the structure →3)-β-GalpNAc-(1→5)-β-Kdop-(2→ and a separate exopolysaccharide with the structure →5)-β-Galf-(1→. </p><p>Having established that K. kingae produces a capsule comprised of GalNAc and Kdo, we next set out to identify the genetic determinants of capsule through a transposon mutagenesis screen. In addition to the previously identified ctrABCD operon, lipA, lipB, and a putative glycosyltransferase termed csaA (capsule synthesis region A gene A) were found to be essential for the production of surface-localized capsule. The ctr operon, lipA, lipB, and csaA were found to be present at unlinked locations throughout the genome, which is atypical for gram-negative organisms that elaborate a capsule dependent on an ABC-type transporter for surface localization. Through examining capsule localization in the ctrA, lipA, lipB, and csaA mutant strains, we determined that the ctrABCD, lipA/lipB, and csaA gene products respectively function in capsule export, assembly, and synthesis, respectively. The GalNAc transferase and Kdo transferase domains found in CsaA further support its role in catalyzing the synthesis of the GalNAc-Kdo capsule in the K. kingae prototype strain.</p><p>To investigate the capsule diversity that exists in K. kingae we screened a panel of strains isolated from patients with invasive disease or healthy carriers for the csaA capsule synthesis locus. We discovered that Kingella kingae expresses one of 4 capsule synthesis loci (csa, csb, csc, or csd) associated with a capsule consisting of Kdo and GalNAc (type a), Kdo and GlcNAc (type b), Kdo and ribose (type c), and GlcNAc and galactose (type d), respectively. Cloning of the csa, csb, csc, or csd locus into the empty flanking gene region in a non-encapsulated mutant (creation of an isogenic capsule swap) was sufficient to produce either the type a, type b, or type c capsule, respectively, further supporting the role of these loci in expression of a specific polysaccharide linkage. Capsule type a and capsule type b accounted for 96% of invasive strains. Conversely, capsule type c and capsule type d were found disproportionately among carrier isolates, suggesting that capsule type is important in promoting invasion and dissemination. </p><p>In conclusion, we discovered that Kingella kingae expresses a polysaccharide capsule and an exopolysaccharide on its surface that require distinct genetic loci for surface localization. Further investigation into genetic determinants of encapsulation revealed the loci ctrABCD, lipA/lipB, and a putative glycosyltransferase are required for capsule expression, with the gene products having roles in capsule export, assembly, and synthesis, respectively. The putative glycosyltransferase CsaA was determined to be a bifunctional enzyme with both GalNAc-transferase and Kdo-transferase activity. Furthermore, we discovered a total of 4 capsule types expressed in clinical isolates of K. kingae, each with a distinct capsule synthesis locus. The variation in the proportion of capsule types found between invasive strains and carriage strains suggest that capsule type is important in promoting invasion and dissemination. Taken together, this work expands our knowledge of the capsule types expressed among K. kingae carrier and invasive isolates and provides insights into the common genetic determinants of capsule expression. These contributions may lead to selecting clinically relevant capsule types to develop into a capsule based vaccine to prevent K. kingae colonization.</p> / Dissertation

<p><italic>Kingella kingae</italic> is an emerging bacterial pathogen that is being recognized increasingly as an important etiology of septic arthritis, osteomyelitis, and bacteremia, especially in young children. The pathogenesis of <italic>K. kingae</italic> disease begins with bacterial adherence to respiratory epithelium in the posterior pharynx. Previous work identified type IV pili as a critical factor for adherence to human epithelial cells. However, the finding that a significant percentage of pharyngeal isolates are non-piliated suggests that <italic>K. kingae</italic> expresses additional surface factors that modulate interactions with host cells and likely play key roles in the pathogenesis of <italic>K. kingae</italic> disease. The purpose of this work was to increase our understanding of <italic>K. kingae</italic> virulence determinants, specifically focused on defining the surface factors and the mechanism involved in <italic>K. kingae</italic> adhesive interactions with epithelial cells. Additionally, this work aimed to further characterize components of the <italic>K. kingae</italic> type IV pilus system, namely the PilC proteins and PilA2. </p><p>We first set out to identify non-pilus factors that influence <italic>K. kingae</italic> interactions with human epithelial cells. Using targeted genetic approaches, we found that insertional inactivation of the gene encoding a predicted trimeric autotransporter protein called Knh (Kingella NhhA homolog) resulted in reduced adherence to human epithelial cells. In addition, using a variety of techniques, including morphological analysis, cationic ferritin staining, and thin section transmission electron microscopy, we established that <italic>K. kingae</italic> elaborates a surface-associated polysaccharide capsule that requires a predicted ABC-type transporter export operon called <italic>ctrABCD for surface presentation. Furthermore, using quantitative human epithelial cell adherence assays, we discovered that the presence of surface capsule interferes with Knh-mediated adherence by non-piliated organisms and that maximal adherence in the presence of capsule requires the predicted type IV pilus retraction machinery, PilT/PilU. Based on the data presented here, we propose a novel adherence mechanism that allows <italic>K. kingae</italic> to adhere efficiently to human epithelial cells while remaining encapsulated and more resistant to immune clearance. </p><p>Having established that <italic>K. kingae</italic> produces a capsule, a large-scale polysaccharide purification technique was developed for capsule analysis of strain 269-492. Biochemical assays determined that the purified material contained thiobarbituric and phenol-sulfuric acid reactive glycosyl residues. In collaboration with the University of Georgia Complex Carbohydrate Research Center (CCRC), mass spectrometry identified galactose, N-acetyl-galactosamine, and Kdo as the major glycosyl components of the polysaccharide preparation. NMR spectroscopy revealed that the purified material contained two distinct polysaccharides with the structures of &rarr;5)&ndash;&beta;&ndash;Gal<italic>f</italic>&ndash;(1&rarr; and &rarr;3)&ndash;&beta;&ndash;GalNAc<italic>p</italic>&ndash;(1&rarr;5)&ndash;&beta;&ndash;Kdo<italic>p</italic>&ndash;(2&rarr;. Further characterization of the polysaccharides expressed by <italic>K. kingae</italic> may have implications for disease prevention strategies. </p><p>Previous work in our lab found that two PilC-like proteins called PilC1 and PilC2 influence type IV pili expression and pilus-mediated adherence. Production of either PilC1 or PilC2 is necessary for <italic>K. kingae</italic> piliation and bacterial adherence. We set out to further investigate the role of PilC1 and PilC2 in type IV pilus-associated phenotypes. We found that PilC1 contains a functional nine amino acid calcium-binding (Ca-binding) site with homology to the <italic>Pseudomonas aeruginosa</italic> PilY1 Ca-binding site and that PilC2 contains a functional 12 amino acid Ca-binding site with homology to the human calmodulin Ca-binding site. Using targeted mutagenesis to disrupt the Ca-binding sites, we demonstrated that the PilC1 and PilC2 Ca-binding sites are dispensable for piliation. Interestingly, we show that the PilC1 site is necessary for twitching motility and adherence to Chang epithelial cells, while the PilC2 site has only a minor influence on twitching motility and no influence on adherence. These findings establish key differences in PilC1 and PilC2 function in <italic>K. kingae</italic> and provide insights into the biology of the PilC-like family of proteins.</p><p>Lastly, we set out to define the role of the PilA2 minor pilin in <italic>K. kingae</italic> strain 269-492. While previous studies indicated that PilA2 is not essential for pilus expression or adherence to epithelial cells, analysis of the pilin locus in a diverse set of clinical isolates revealed that the <italic>pilA2</italic> gene sequence is highly conserved, suggesting it serves an important function. Using targeted mutagenesis we showed that PilA2 is not essential for twitching motility and may or may not be involved in natural competence. Western blot analysis was unable to detect PilA2 in wild type pilus preparations, indicating that it is expressed at a level beneath the assay detection limit or does not localize to the pilus. Additionally, site-directed mutagenesis was used to place <italic>pilA2</italic> under control of the highly active <italic>pilA1</italic> promoter and showed that PilA2 is able to be assembled into fibers that mediate intermediate adherence to epithelial cells. </p><p>Taken together, this work expands our knowledge of the <italic>K. kingae</italic> surface factor repertoire and provides insights into the roles of type IV pilus components. The mechanism of<italic> K. kingae</italic> adherence to epithelial cells is beginning to emerge. These contributions may lead to novel strategies for the prevention of invasive <italic>K. kingae</italic> disease in young children.</p> / Dissertation