Here we present the synthesis of poly(acryloyl hydrazide), a versatile scaffold for the preparation of functional polymers, and its post-polymerisation modification using a wide range of conditions.

Vibrio cholerae is a Gram-negative bacterium found in aquatic environments and a human pathogen of global significance. Its transition between host-associated and environmental lifestyles involves the tight regulation of niche-specific phenotypes such as motility, biofilm formation, and virulence. V. cholerae’s transition from the host to environmental dispersal usually involves suppression of virulence and dispersion of biofilm communities. In contrast to this naturally occurring transition, bacterial aggregation by cationic polymers triggers a unique response, which is to suppress virulence gene expression while also triggering biofilm formation by V. cholerae, an artificial combination of traits that is potentially very useful to bind and neutralize the pathogen from contaminated water. Here, we set out to uncover the mechanistic basis of this polymer-triggered bacterial behavior. We found that bacteria–polymer aggregates undergo rapid autoinduction and achieve quorum sensing at bacterial densities far below those required for autoinduction in the absence of polymers. We demonstrate this induction of quorum sensing is due both to a rapid formation of autoinducer gradients and local enhancement of autoinducer concentrations within bacterial clusters as well as the stimulation of CAI-1 and AI-2 production by aggregated bacteria. We further found that polymers cause an induction of the biofilm-specific regulator VpsR and the biofilm structural protein RbmA, bypassing the usual suppression of biofilm during autoinduction. Overall, this study highlights that synthetic materials can be used to cross-wire natural bacterial responses to achieve a combination of phenotypes with potentially useful applications.

Here we present the intracellular delivery of mRNA using polyhydrazones prepared in situ under physiological conditions, demonstrating the versatility and adaptability of this technology to more sensitive and therapeutically relevant nucleic acids.

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...

Modern medicine is under the excruciating pressure of drug resistant bacterial strains which are ever advancing with the introduction of every new class of antibiotics. Traditional bactericidal and bacteriostatic drugs, while effective in eliminating the susceptible bacterial strains, also impose a selective pressure on bacteria which often leads to the emergence of antimicrobial resistance. An alternative approach is the development of anti-virulence therapies, which aims reduce bacterial pathogenesis while avoiding the selective pressure of classical antimicrobial inhibitors, thus rendering bacteria harmless and potentiating natural elimination from the host by innate immunity defence mechanisms. We have synthesised a selection of functional polymers of poly(acryloyl hydrazide) using a panel of aldehyde functionalisation groups and evaluated their anti-virulence properties on both Mycobacterium bovis BCG and Mycobacterium smegmatis mc2 155, two surrogate organisms to study Mycobacterium tuberculosis, the etiological agent responsible for tuberculosis. Using a combination of microscopy and in vitro studies, we have shown the effectiveness of anti-virulence polymers in reducing mycobacterial phagocytosis in J774 macrophages with minimal antimicrobial activity.