Aggregation ofVibrio choleraeby cationic polymers enhances quorum sensing but over-rides biofilm dissipation in response to autoinduction

Abstract

19Vibrio cholerae is a Gram-negative bacterium found in aquatic environments and a human 20 pathogen of global significance. Its transition between host-associated and environmental life 21 styles involves the tight regulation of niche-specific phenotypes such as motility, biofilm formation 22 and virulence. V. cholerae's transition from the host to environmental dispersal usually involves 23 suppression of virulence and dispersion of biofilm communities. In contrast to this naturally 24 occurring transition, bacterial aggregation by cationic polymers triggers a unique response, which 25 is to suppress virulence gene expression while also triggering biofilm formation by V. cholerae, 26 an artificial combination of traits that is potentially very useful to bind and neutralize the pathogen 27 from contaminated water. Here, we set out to uncover the mechanistic basis of this polymer-28 triggered bacterial behavior. We found that bacteria-polymer aggregates undergo rapid 29 autoinduction and achieve quorum sensing at bacterial densities far below those required for 30 autoinduction in the absence of polymers. We demonstrate this induction of quorum sensing is due 31 both to a rapid formation of autoinducer gradients and local enhancement of autoinducer 32 concentrations within bacterial clusters, as well as the stimulation of CAI-1 and AI-2 production 33 by aggregated bacteria. We further found that polymers cause an induction of the biofilm specific 34 regulator VpsR and the biofilm structural protein RbmA, bypassing the usual suppression of 35 biofilm during autoinduction. Overall, this study highlights that synthetic materials can be used to 36 cross-wire natural bacterial responses to achieve a combination of phenotypes with potentially 37 useful applications. 38 39 40 41 Polymers and quorum sensing in V. cholerae 42 Both natural and synthetic cationic macromolecules, such as cationic antimicrobial peptides, 43 cationic polymers and dendrimers, have been extensively reported as antimicrobial. 1 Due to their 44 positive charge, these polymers can efficiently bind the negatively charged envelope of Gram-45 negative and Gram-positive bacteria. 2 At high concentrations and charge densities, these molecules 46 have the potential to interfere with membrane integrity and decrease bacterial viability. 1 However, 47 antimicrobial activity is heavily dependent on the length and nature of the polymer and, more 48 importantly, on the nature of the targeted microbe. At low concentrations, cationic polymers are 49 still capable of causing bacterial aggregation by mediating electrostatic interactions, but do so 50 without significantly affecting bacterial membrane integrity and growth. 3 51 We and others have previously reported that bacteria clustered by sub-inhibitory 52 concentrations of cationic polymers display interesting phenotypes resembling those of biofilm 53 communities. 3 For instance, we have recently demonstrated that clustering of the diarrheal 54 pathogen Vibrio cholerae with methacrylamides containing primar...

Polymer-induced biofilms for enhanced biocatalysis

“…Following our previous work with V. cholerae, 31,32 for which we had seen an increase in biofilm formation upon incubation with cationic polymers, we postulate here that this polymerinduced biofilm formation can be exploited in other microorganisms for the production of biofilms for biotechnology.…”

“…4,[23][24][25][26] In our case, we focused on biocatalysis, where biofilms can protect microbial cells from harsh conditions such as extreme pH or temperature, or from the presence of detrimental chemicals such as organic solvents. In our previous work, we had exploited cationic polymers to induce clustering in a range of bacteria, including Vibrio harveyi, [33][34][35][36] V. cholerae, 31,32 Pseudomonas aeruginosa, 34,35 Staphylococcus aureus 35 and E. coli, 34,35 and, as just mentioned, we observed increased biofilm formation for V. cholerae. However, we also observed that cationic polymers were toxic to some of the E. coli strains employed.…”

“…We had already identified that this was the case for human pathogen Vibrio cholerae, that upon interaction with cationic polymers, would adopt a nonvirulent sessile lifestyle, characterized by an increase in biofilm formation and a decrease in toxin production. 31,32 In this manuscript, we show that synthetic polymers induce the nucleation of biofilms in biotechnology relevant E. coli, resulting in increased biocatalytic activity in a model biotransformation (Scheme 1). First, using a post-polymerization functionalization strategy, we prepare a set of linear polymers carrying mildly cationic, aromatic, heteroaromatic or aliphatic moieties.…”

“…Our previous work with cationic polymers indicates that changes in microbial physiology, including biofilm formation, are the result of polymer-induced clustering of bacteria. [31][32][33][34][35][36] Here, we wanted to evaluate if a similar aggregation was being induced by hydrophobic polymers. To this end, aggregation of bacteria was first evaluated monitoring changes in the optical density for E. coli MC4100 cultures in the absence and presence of these functional polymers (Fig.…”