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Poly-N-acetylglucosamine mediates biofilm formation and detergent resistance in Aggregatibacter actinomycetemcomitans.

ABSTRACT: Clinical isolates of the periodontopathogen Aggregatibacter actinomycetemcomitans form matrix-encased biofilms on abiotic surfaces in vitro. A major component of the A. actinomycetemcomitans biofilm matrix is poly-beta-1,6-N-acetyl-d-glucosamine (PGA), a hexosamine-containing polysaccharide that mediates intercellular adhesion. In this report, we describe studies on the purification, structure, genetics and function of A. actinomycetemcomitans PGA. We found that PGA was very tightly attached to A. actinomycetemcomitans biofilm cells and could be efficiently separated from the cells only by phenol extraction. A. actinomycetemcomitans PGA copurified with LPS on a gel filtration column. (1)H NMR spectra of purified A. actinomycetemcomitans PGA were consistent with a structure containing a linear chain of N-acetyl-d-glucosamine residues in beta(1,6) linkage. Genetic analyses indicated that all four genes of the pgaABCD locus were required for PGA production in A. actinomycetemcomitans. PGA mutant strains still formed biofilms in vitro. Unlike wild-type biofilms, however, PGA mutant biofilms were sensitive to detachment by DNase I and proteinase K. Treatment of A. actinomycetemcomitans biofilms with the PGA-hydrolyzing enzyme dispersin B made them 3 log units more sensitive to killing by the cationic detergent cetylpyridinium chloride. Our findings suggest that PGA, extracellular DNA and proteinaceous adhesins all contribute to the structural integrity of the A. actinomycetemcomitans biofilm matrix.

SUBMITTER: Izano EA

PROVIDER: S-EPMC2253675 | biostudies-literature | 2008 Jan

REPOSITORIES: biostudies-literature

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Poly-N-acetylglucosamine mediates biofilm formation and detergent resistance in Aggregatibacter actinomycetemcomitans.

Izano Era A EA Sadovskaya Irina I Wang Hailin H Vinogradov Evgeny E Ragunath Chandran C Ramasubbu Narayanan N Jabbouri Saïd S Perry Malcolm B MB Kaplan Jeffrey B JB

Microbial pathogenesis 20070812 1

Clinical isolates of the periodontopathogen Aggregatibacter actinomycetemcomitans form matrix-encased biofilms on abiotic surfaces in vitro. A major component of the A. actinomycetemcomitans biofilm matrix is poly-beta-1,6-N-acetyl-d-glucosamine (PGA), a hexosamine-containing polysaccharide that mediates intercellular adhesion. In this report, we describe studies on the purification, structure, genetics and function of A. actinomycetemcomitans PGA. We found that PGA was very tightly attached to ...[more]

PMID: 17851029

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Project description:Autoinducer-2 (AI-2) is required for biofilm formation and virulence of the oral pathogen Aggregatibacter actinomycetemcomitans, and we previously showed that lsrB codes for a receptor for AI-2. The lsrB gene is expressed as part of the lsrACDBFG operon, which is divergently transcribed from an adjacent lsrRK operon. In Escherichia coli, lsrRK encodes a repressor and AI-2 kinase that function to regulate lsrACDBFG. To determine if lsrRK controls lsrACDBFG expression and influences biofilm growth of A. actinomycetemcomitans, we first defined the promoters for each operon. Transcriptional reporter plasmids containing the 255-bp lsrACDBFG-lsrRK intergenic region (IGR) fused to lacZ showed that essential elements of lsrR promoter reside 89 to 255 bp upstream from the lsrR start codon. Two inverted repeat sequences that represent potential binding sites for LsrR and two sequences resembling the consensus cyclic AMP receptor protein (CRP) binding site were identified in this region. Using electrophoretic mobility shift assay (EMSA), purified LsrR and CRP proteins were shown to bind probes containing these sequences. Surprisingly, the 255-bp IGR did not contain the lsrA promoter. Instead, a fragment encompassing nucleotides +1 to +159 of lsrA together with the 255-bp IGR was required to promote lsrA transcription. This suggests that a region within the lsrA coding sequence influences transcription, or alternatively that the start codon of A. actinomycetemcomitans lsrA has been incorrectly annotated. Transformation of ?lsrR, ?lsrK, ?lsrRK, and ?crp deletion mutants with lacZ reporters containing the lsrA or lsrR promoter showed that LsrR negatively regulates and CRP positively regulates both lsrACDBFG and lsrRK. However, in contrast to what occurs in E. coli, deletion of lsrK had no effect on the transcriptional activity of the lsrA or lsrR promoters, suggesting that another kinase may be capable of phosphorylating AI-2 in A. actinomycetemcomitans. Finally, biofilm formation of the ?lsrR, ?lsrRK, and ?crp mutants was significantly reduced relative to that of the wild type, indicating that proper regulation of the lsr locus is required for optimal biofilm growth by A. actinomycetemcomitans.

Dataset Information

Poly-N-acetylglucosamine mediates biofilm formation and detergent resistance in Aggregatibacter actinomycetemcomitans.

Publications

Poly-N-acetylglucosamine mediates biofilm formation and detergent resistance in Aggregatibacter actinomycetemcomitans.

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