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

Abstract

Clinical isolates of the periodontopathogen Aggregatibacter actinomycetemcomitans form matrixencased biofilms on abiotic surfaces in vitro. A major component of the A. actinomycetemcomitans biofilm matrix is 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 β(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.

Interactions between the Aggregatibacter actinomycetemcomitans secretin HofQ and host cytokines indicate a link between natural competence and interleukin-8 uptake

“…The amount of extracellular PGA in the biofilm cultures was estimated using a Congo-red-binding assay [11] as described earlier by Ahlstrand and co-workers [17]. Biofilm cultures for this assay were prepared in 48-well microtiter plates as described above.…”

“…EMs consist of proteins, extracellular DNA (eDNA) and polysaccharides, such as poly-N-acetylglucosamine (PGA), which is the major polysaccharide in A. actinomycetemcomitans biofilms [9]. However, A. actinomycetemcomitans requires eDNA for cohesive biofilm formation, efficient adhesion to surfaces [10,11], and leukotoxin attachment to the bacterial membrane [12]. …”

Plasticity of Candida albicans Biofilms

“…In the complex matrix of a bacterial biofilm, a microenvironment is established that has been shown to contain secreted enzymes for catalyzing the polysaccharide linkages and modifications that are basic to matrix architecture and function (106,144,157,158) and secreted enzymes involved in the extracellular digestion of nutrients from the host environment or from the medium in an in vitro model (21,159,160). The role of eDNA is unclear, but experiments suggest that it indeed plays a role in biofilm formation in vitro (151,161). The ECM contributes to the establishment of a microenvironment that facilitates cell-cell communication through signaling (109,162,163).…”

Dual function of the McaS small RNA in controlling biofilm formation

“…To assay the effects of McaS on the pgaABCD operon more directly, we examined the ability of wild-type and mutant McaS to stimulate PGA production by monitoring colony color on Congo red plates (Izano et al 2008). These experiments were carried out in an DrpoS strain background to ensure that Congo red binding reflected the synthesis of PGA rather than curli fibers, which are known to be expressed in a s S -dependent manner (Hammar et al 1995).…”