Recent estimates suggest that over 65% of microbial infections in the body are biofilm related. In many biofilm-forming bacteria, carbohydrate polymers or exopolysaccharides are key components of the biofilm matrix. Exopolysaccharides act as a structural support, an immunoprotector, as well as a barrier against antibiotics.
A homopolymer of partially deacetylated b-1,6-linked N-acetylglucosamine (dPNAG) residues has emerged as a key biofilm matrix exopolysaccharide in Gram-positive bacterium Staphylococcus epidermidis. The genes encoding production of exopolysaccharide in S. epidermidis are organized in the icaADBC operon. IcaA and IcaD are membrane proteins and required for GlcNAc polymerization. IcaC is predicted to be a protein responsible for the export of the growing polymer through the membrane. IcaB is a surface-attached protein, which involved in the introduction of positive charges into polymer by de-N-acetylation of ~15% of GlcNAc moieties. Transposon mutagenesis studies showed that DicaB strains are unable to form biofilms in vitro. However, the activity of IcaB enzyme has not been yet confirmed in vitro.
To study the function of IcaB enzyme, the recombinant PNAG de-N-acetylase from S. epidermidis was overexpressed in E. coli. The biochemical properties of the purified protein were studied in detail. Our results clearly indicate that IcaB is a metal dependent de-N-acetylase, which is capable of de-N-acetylating GlcNAc oligomeric pseudo-substrates as well as de-O-acetylation of a non-carbohydrate substrate.
In an attempt to more precisely study the mechanism of IcaB deacetylase, we designed and synthesized phosphonamidate based inhibitors, as structural mimics of a tetrahedral intermediate formed during deacetylation by IcaB. Additionally, since de-N-acetylation by IcaB is essential for the biofilm formation, the inhibition of IcaB is a potential therapeutic strategy for the treatment of biofilm-associated bacterial infections. The inhibitory activities of the synthesized compounds were studied in both in vitro assay with purified protein and bacterial biofilm assay.
