Classically targets for antimicrobials are found to be essential enzymes

Despite the fact that the 24-hr deglycosylation reduced the molecular size of the main protein bands of G207 and G208 only by 10% on SDS-PAGE, it produced significant changes in antibacterial activities of these two glycoproteins. The size of the inhibition zones produced by G208 was dependent on concentrations of glps added to the well. Deglycosylation of G208 caused a reduction of the inhibition zones at all three dilutions in comparison to glycosylated G208, although these decreases were not statistically significant. In contrast, G207 produced a zone of inhibition only at its highest concentration, and deglycosylation of G207 resulted in a significant decrease or a complete loss of the antibacterial activity of this glycoprotein. These results established that G207 displayed mostly agglutinating activity for which glycosylation was essential. In contrast, the mechanism by which G208 produced antibacterial Axitinib VEGFR/PDGFR inhibitor effect was not solely glycosylation-dependent. The unsuspected finding of this study was the identification of honey glycoproteins as active principal molecules that caused agglutination and a rapid, concentration-depended WZ8040 bactericidal effect on both Gram-negative E. coli and Gram positive B. subtilis. The presence of high mannose- type of oligosaccharides in honey glycoproteins allowed their selective isolation using resin-immobilized Concavalin A. Subsequently, we have demonstrated that only the high mannose- type glycoproteins retained by ConA-column showed growth inhibitory and bactericidal activities, while flow-through proteins devoid of mannose-rich glycans were unable to inhibit bacterial growth, reduce bacterial viability or influence bacterial cell shape. These results indicated that the high-mannose structures have a significant role in the antibacterial activity of isolated honey glycoproteins. Due to the presence of carbohydrate moiety, glps displayed lectin-like activity, agglutinating both Gram-positive B. subtilis and Gram-negative E. coli. Agglutinating specificity of honey glps was similar to that of ConA. Both, Glps and ConA had much lower lower reactivity with murine red blood cells than phytohemagglutinin, but efficiently agglutinated both E. coli and B. subtilis cells. This supports reports indicating that ConA binds mannose receptors on B. subtilis cell wall peptidoglycans and on E. coli cell envelope with high affinity, but does not recognize branched, complex-type N-glycans, containing galactose on erythrocyte membranes. The binding specificity of ConA and glps allowed the dissociation of agglutinating from hemagglutinating activities.

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