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"Studies on Biofilm Inhibition by Kingella kingae Exopolysaccharide"

by
Meriem Bendaoud
Oral Biology Program
B.S. 2006, New Jersey City University
D.S. 1990, Algiers Dental School



Thesis Advisor: Jeffrey Kaplan, Ph.D.
Associate Professor
Department of Oral Biology

Thursday, January 19, 2012
1:00 P.M., MSB C-600


Abstract

Biofilm formation for many microorganisms is an important virulence factor and a crucial step in the pathogenesis of bacterial infection. Bacteria growing attached to abiotic and biotic surfaces are protected and resistant to antibiotic treatment and antimicrobial agents, which makes them difficult to eradicate with conventional treatments. The need for new therapeutic approaches is critical as the number of infections caused by biofilm forming bacteria such as Staphylococcus aureus and Staphylococccus epidermidis has increased. In a screen for novel antibiofilm compounds, we observed potent biofilm inhibition activity in cell-free extracts prepared from bacterial lawns of the Gram-negative oral bacterium Kingella kingae. Further studies revealed that the cell-free extracts prepared from Kingella kingae colony biofilms was able to inhibit biofilm formation by Aggregatibacter actinomycetemcomitans, Klebsiella pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, Candida albicans, and K. kingae. The extracts evidently inhibited biofilm formation by modifying the physicochemical properties of the cell surface, the biofilm matrix, and the substrate. Chemical and biochemical analyses indicated that the biofilm inhibition activity in the K. kingae extract was due to polysaccharide. Structural analyses showed that the extract contained two major polysaccharides. One was a linear polysaccharide with the structure 6)--D-GlcNAcp-(15)--D-OclAp-(2, which was identical to a capsular polysaccharide produced by Actinobacillus pleuropneumoniae serotype 5. The second was a novel linear polysaccharide, designated PAM galactan, with the structure 3)--D-Galf-(16)--D-Galf-(1. Purified PAM galactan exhibited broad-spectrum biofilm inhibition activity. A cluster of three K. kingae genes designated pamABC encoding UDP-galactopyranose mutase and two putative galactofuranosyl transferases was sufficient for the synthesis of PAM galactan in Escherichia coli. Construction and characterization of two K. kingae mutant strains revealed that both pamB and pamC genes are essential for the production of PAM galactan. Both mutant strains exhibited increased sensitivity to killing by antibacterial compounds and increased biofilm formation. PAM galactan is one of a growing number of bacterial polysaccharides that exhibit antibiofilm activity. The results of this study could bring a better understanding of the pathogenesis of Kingella kingae infections and have a tremendous impact in the development of new biofilm-specific pharmacologic strategies for the treatment, control or prevention of various bacterial infections.


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