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Insights Into the Virulence Determinants of the Emerging Pathogen Kingella kingae.

Record Type:	Language materials, manuscript : Monograph/item
Title/Author:	Insights Into the Virulence Determinants of the Emerging Pathogen Kingella kingae./
Author:	Porsch, Eric Allen.
Description:	1 online resource (202 pages)
Notes:	Source: Dissertations Abstracts International, Volume: 74-04, Section: B.
Contained By:	Dissertations Abstracts International74-04B.
Subject:	Microbiology. -
Online resource:	click for full text (PQDT)
ISBN:	9781267550101

Insights Into the Virulence Determinants of the Emerging Pathogen Kingella kingae.
Porsch, Eric Allen.

Insights Into the Virulence Determinants of the Emerging Pathogen Kingella kingae. - 1 online resource (202 pages)

Source: Dissertations Abstracts International, Volume: 74-04, Section: B.

Thesis (Ph.D.)--Duke University, 2012.

Includes bibliographical references

Kingella kingae is an emerging bacterial pathogen that is being recognized increasingly as an important etiology of septic arthritis, osteomyelitis, and bacteremia, especially in young children. We first set out to identify non-pilus factors that influence K. kingae 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 K. kingae elaborates a surface-associated polysaccharide capsule that requires a predicted ABC-type transporter export operon called 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 K. kingae to adhere efficiently to human epithelial cells while remaining encapsulated and more resistant to immune clearance. Having established that K. kingae 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. 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 →5)-β-Galf-(1→ and →3)-β-GalNAcp-(1→5)-β-Kdo p-(2→. Further characterization of the polysaccharides expressed by K. kingae may have implications for disease prevention strategies. 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 K. kingae 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 Pseudomonas aeruginosa 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 K. kingae and provide insights into the biology of the PilC-like family of proteins. Lastly, we set out to define the role of the PilA2 minor pilin in K. kingae 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 pilA2 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 pilA2 under control of the highly active pilA1 promoter and showed that PilA2 is able to be assembled into fibers that mediate intermediate adherence to epithelial cells. (Abstract shortened by UMI.).

Electronic reproduction.
Ann Arbor, Mich. :
ProQuest,
2024

Mode of access: World Wide Web

ISBN: 9781267550101Subjects--Topical Terms:

591510
Microbiology.
Subjects--Index Terms:

AdherenceIndex Terms--Genre/Form:

554714
Electronic books.

Insights Into the Virulence Determinants of the Emerging Pathogen Kingella kingae.
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502 $a Thesis (Ph.D.)--Duke University, 2012.
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520 $a Kingella kingae is an emerging bacterial pathogen that is being recognized increasingly as an important etiology of septic arthritis, osteomyelitis, and bacteremia, especially in young children. We first set out to identify non-pilus factors that influence K. kingae 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 K. kingae elaborates a surface-associated polysaccharide capsule that requires a predicted ABC-type transporter export operon called 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 K. kingae to adhere efficiently to human epithelial cells while remaining encapsulated and more resistant to immune clearance. Having established that K. kingae 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. 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 →5)-β-Galf-(1→ and →3)-β-GalNAcp-(1→5)-β-Kdo p-(2→. Further characterization of the polysaccharides expressed by K. kingae may have implications for disease prevention strategies. 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 K. kingae 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 Pseudomonas aeruginosa 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 K. kingae and provide insights into the biology of the PilC-like family of proteins. Lastly, we set out to define the role of the PilA2 minor pilin in K. kingae 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 pilA2 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 pilA2 under control of the highly active pilA1 promoter and showed that PilA2 is able to be assembled into fibers that mediate intermediate adherence to epithelial cells. (Abstract shortened by UMI.).
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653 $a Adherence
653 $a Autotransporters
653 $a Capsule
653 $a Kingella kingae
653 $a Pili
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710 2 $a Duke University. $b Molecular Genetics and Microbiology. $3 1182646
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