Controlling microbial multicellular behaviors with saccharide derivatives

Microbial multicellular behaviors like biofilm formation and swarming motility are known to increase their tolerance against antimicrobials. From microbial standpoint, nonmicrobicidal agents that do not impede growth are tolerable and therefore, there is a lower propensity to develop resistance against such agents as compared to microbicidal ones (antibiotics). This study describes a new antibiofilm approach of using nonmicrobicidal saccharide derivatives for controlling the multicellular behaviors of gram-negative bacteria, Pseudomonas aeruginosa and fungus, Candida albicans. Pseudomonas aeruginosa is known to secrete rhamnolipids, a class of biosurfactants that plays an important role in maintaining the architecture of its biofilm and promoting its swarming motility. Here we show the ability of certain synthetic nonmicrobicidal disaccharide derivatives (DSDs) to mimic the biofunctions of rhamnolipids. The rhlA mutant of P. aeruginosa is incapable of synthesizing rhamnolipids and also unable to swarm on semi-solid agar gel. When the natural ligands, rhamnolipids were externally added into the semi-solid agar gel in a concentration dependent manner, the swarming of the rhlA mutant reactivated at lower concentrations (10 μM) and then at relatively higher concentrations (15 μM), the swarming reactivation was reversed. When some active synthetic DSDs were tested on the rhlA mutant, the bacterial swarming first reactivated and then the activation reversed at higher DSD concentrations, similar to the effect of externally added rhamnolipids. Previously, a known bacterial signalling molecule has been shown to exhibit a similar concentration dependent activation and then activation reversal for light simulation by Vibrio fischeri. Some DSDs having disaccharide stereochemistries (cellobiose or maltose) and a bulky aliphatic tail (3, 7, 11trimethyl-dodecanyl) caused swarming reactivation of the rhlA mutant at concentrations lower than that caused by the externally added rhamnolipids. The synthetic nonmicrobicidal DSDs were also very effective at inhibiting the adhesion of P. aeruginosa to polystyrene surface, and at inhibiting the bacterial biofilm formation. These DSDs were also potent dispersers of pre-formed biofilm of P. aeruginosa. The potent antibiofilm (inhibition and dispersion) activities were observed for those DSDs that possessed a disaccharide (cellobiose or maltose) stereochemistry and a bulky aliphatic chain such as 3, 7, 11-trimethyldodecanyl. These potent DSDs had half-maximal inhibitory concentrations for biofilm inhibition (IC50) and dispersion (DC50) comparable to those of known potent antibiofilm agents against P. aeruginosa. Gene-reporting assays indicate that the mechanism of action of such DSDs is not via the known las or rhl quorum sensing systems of P. aeruginosa but that the adhesin potein, pilin maybe a likely target of such molecules. Biofilms formed under natural settings are usually formed by both bacteria and fungus that co-reside in the same microenvironment. Therefore, agents that can prevent mixed biofilms are desirable from a therapeutic standpoint. Despite being nonmicrobicidal to both fungal blastospores and hyphae, the synthetic DSDs were able to inhibit the biofilm formation of fungus Candida albicans. Microscopic evaluation showed that most DSDs did not prevent the blastospores-to-hyphae morphogenesis. The DSDs were effective at inhibiting biofilm formation of Candida albicans when applied within five minutes of seeding the test surface with fungal cells. Using a surface based assay it was shown that one DSD dramatically reduced the surface adhesion of Candida albicans hyphae. The antibiofilm activity of such DSDs against Candida albicans is probably due to their ability to prevent hyphae surface adhesion. Controlling Microbial Multicellular Behaviors With Saccharide Derivatives by Nischal Singh M. Phil. (Chemistry), Syracuse University, 2011 M.Sc. (Chemistry), University of Mumbai, Mumbai, India, 2008 B.Sc. (Chemistry), University of Mumbai, Mumbai, India 2006 Dissertation Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemistry Syracuse University August 2015 Copyright © Nischal Singh 2015 All Rights Reserved