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Identifying Chemical Cues in the Polymer-Mediated Engineering of Microorganisms

Periodic Reporting for period 1 - PolyBact (Identifying Chemical Cues in the Polymer-Mediated Engineering of Microorganisms)

Reporting period: 2019-02-01 to 2021-01-31

Understanding and controlling the interaction of polymers and microorganisms such as bacteria is of critical importance because the binding of microorganisms to polymers and surfaces has major consequences in healthcare, biotechnology and industry. On one hand, we need to understand how to inhibit adhesion and thus prevent unwanted fouling of surfaces, which results in infection, or in damage to industrial equipment. On the other hand, biofilms provide a unique platform for biocatalysis, where bacteria make chemicals for a variety of purposes. This way the toughness and resistance of biofilms to external damage is exploited to prepare high end value products. An underlying limitation that is compromising the development of polymers for these applications, is our lack of understanding of how these materials are affecting microbial physiology. Whilst we have a basic understanding of what chemistries need to be used for each application, for example to induce cell death, or inhibit the adhesion to host or surfaces, little is known about how bacteria respond and adapt to the presence of these polymers: we do not know how complex chemical and physical cues affect downstream cellular signalling following interaction with these materials, to affect phenotypical changes such as biofilm production or virulence factor expression. As a consequence, many candidate polymers are not translated to application because eventually they do not induce the expected effect. Therefore, the overall objectives of the project was to identify the roles of different types of polymer-bacteria interactions in bacterial behaviour.
To this end, we synthesized a series of polymers bearing different functionalities and, using Vibrio cholerae as a model organism, explored the responses of bacteria to these nanomaterials.
We discovered that cationic and hydrophobic functionalities were able to induce formation of cell clusters whereas polymers containing sugars demonstrated only short-lived interaction with cells and did not form stable clusters. Then we found that cationic polymers with high charge density and hydrophobic polymers induced biofilm formation, this effect was dependent on pH and polymer concentration. A decrease in biofilm production was observed only for polymers showing high cytotoxicity. At the same time, none of the tested polymers had significant effect on cholera toxin production.
We synthesized a series of polymers bearing different functionalities by attaching various chemical modulators to the reactive polymer precursor. Within the project, we optimized the polymerisation conditions that allowed for a robust and efficient method of the synthesis of the precursor polymer.
Having identified limitations of the originally proposed strategy based on the coupling of various chemical modulators to the parent polymer, we exploited the co-functionalisation approach. To obtain a series of polymers with a gradual change in a certain property, we were coupling two modulators taken at different ratios to the precursor polymer at the same time. In this case, the ratio between the modulators determines the property of the resulting polymer. In particular, we synthesized cationic, hydrophobic, and sugar-containing polymers.
Using Vibrio cholerae as a model organism, we explored different types of bacterial responses to the obtained polymeric nanomaterials. To identify the role of charge, hydrophobicity, and selective binding to sugars, we tested the ability of each group of polymers to affect bacterial viability, to induce formation of cell clusters and biofilms. We also explored how cells sense the polymers in terms of gene regulation.
The obtained data suggests that cationic polymers with high charge density at non-toxic concentrations cluster bacteria regardless of the specific chemistry of the charged group. This results in increased biofilm formation. At the same time, polycations with low charge density do not induce biofilm formation.
Our results emphasize the importance of hydrophobic interactions in bacterial binding to substrates and strong response of bacteria to such nanomaterials. Importantly, the revealed ability of hydrophobic polymers to promote biofilm formation was observed for both V. cholerae and E .coli species. However, the mechanisms underlying this effect requires further studies.
For the sugar-containing polymers, we observed short-lived interactions without formation of stable clusters and any significant effect on biofilm formation and regulation of genes associated with virulence and biofilms.
The results were disseminated in a scientific journal that ensures open access and in multiple national and international conferences in relevant areas. Owing to the interdisciplinary nature of this project, the results were presented to different audiences, including scientific community, industry representatives, and journal editors in polymer science, microbiology, biomaterials, chemical biology, as well as general public.
Our results allowed for a preliminary comparison of the responses of different species (Vibrio cholerae and Escherichia coli) to the same polymers and contributed towards understanding fundamental rules governing polymer-bacteria interactions that can have implications in healthcare, biotechnology, and industry.
Overall, the observed regularities are of great importance for creating novel antimicrobial polymers and nanomaterials and developing new strategies that would allow for the control over microbial biofilms and could be tailored to specific microorganisms without loss in efficiency. Importantly, the obtained results can provide useful insights for creating strategies against antimicrobial resistance (AMR) which, according to WHO, is a global health and development threat that requires urgent multisectoral action.
However, the achieved results can be exploited for non-pathogenic microorganisms with the potential impact in biotechnology and relevant socio-economic effects.
PolyBact -- Project overview