Salicylic acid-based poly(anhydride esters) for control of biofilm formation in Salmonella enterica serovar Typhimurium.

AIMS: Bacterial biofilms generally are more resistant to stresses as compared with free planktonic cells. Therefore, the discovery of antimicrobial stress factors that have strong inhibitory effects on bacterial biofilm formation would have great impact on the food, personal care, and medical industries. METHODS AND RESULTS: Salicylate-based poly(anhydride esters) (PAE) have previously been shown to inhibit biofilm formation, possibly by affecting surface attachment. Our research evaluated the effect of salicylate-based PAE on biofilm-forming Salmonella enterica serovar Typhimurium. To remove factors associated with surface physical and chemical parameters, we utilized a strain that forms biofilms at the air-liquid interface. Surface properties can influence biofilm characteristics, so the lack of attachment to a solid surface eliminates those constraints. The results indicate that the salicylic acid-based polymers do interfere with biofilm formation, as a clear difference was seen between bacterial strains that form biofilms at the air-liquid interface (top-forming) and those that form at the surface-liquid interface (bottom-forming). CONCLUSION: These results lead to the conclusion that the polymers may not interfere with attachment; rather, the polymers likely affect another mechanism essential for biofilm formation in Salmonella. SIGNIFICANCE AND IMPACT OF THE STUDY: Biofilm formation can be prevented through controlled release of nature-derived antimicrobials formulated into polymer systems.

Pentameric concatenated (alpha4)(2)(beta2)(3) and (alpha4)(3)(beta2)(2) nicotinic acetylcholine receptors: subunit arrangement determines functional expression.

BACKGROUND AND PURPOSE: alpha4 and beta2 nicotinic acetylcholine (ACh) receptor subunits expressed heterologously in Xenopus oocytes assemble into a mixed population of (alpha4)(2)(beta2)(3) and (alpha4)(3)(beta2)(2) receptors. In order to express these receptors separately in heterologous systems, we have engineered pentameric concatenated (alpha4)(2)(beta2)(3) and (alpha4)(3)(beta2)(2) receptors. EXPERIMENTAL APPROACH: alpha4 and beta2 subunits were concatenated by synthetic linkers into pentameric constructs to produce either (alpha4)(2)(beta2)(3) or (alpha4)(3)(beta2)(2) receptors. Using two-electrode voltage-clamp techniques, we examined the ability of the concatenated constructs to produce functional expression in Xenopus oocytes. Functional constructs were further characterized in respect to agonists, competitive antagonists, Ca2+ permeability, sensitivity to modulation by Zn2+ and sensitivity to up-regulation by chaperone protein 14-3-3. KEY RESULTS: We found that pentameric concatamers with a subunit arrangement of beta2_alpha4_beta2_alpha4_beta2 or beta2_alpha4_beta2_alpha4_alpha4 were stable and functional in Xenopus oocytes. By comparison, when alpha4 and beta2 were concatenated with a subunit order of beta2_beta2_alpha4_beta2_alpha4 or beta2_alpha4_alpha4_beta2_alpha4, functional expression in Xenopus oocytes was very low, even though the proteins were synthesized and stable. Both beta2_alpha4_beta2_alpha4_beta2 and beta2_alpha4_beta2_alpha4_alpha4 concatamers recapitulated the ACh concentration response curve, the sensitivity to Zn2+ modulation, Ca2+ permeability and the sensitivity to up-regulation by chaperone protein 14-3-3 of the corresponding non-linked (alpha4)(2)(beta2)(3) and (alpha4)(3)(beta2)(2) receptors respectively. Using these concatamers, we found that most alpha4beta2-preferring compounds studied, including A85380, 5I-A85380, cytisine, epibatidine, TC2559 and dihydro-beta-erythroidine, demonstrate stoichiometry-specific potencies and efficacies. CONCLUSIONS AND IMPLICATIONS: We concluded that the alpha4beta2 nicotinic ACh receptors produced with beta2_alpha4_beta2_alpha4_beta2 or beta2_alpha4_beta2_alpha4_alpha4 pentameric constructs are valid models of non-linked (alpha4)(2)(beta2)(3) and (alpha4)(3)(beta2)(2) receptors respectively.