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The aim of this thesis has been to obtain a P. aeruginosa strain able to successfully overtake a wildtype population in structured environments. Previous approaches have focused on saving the cost of producing pyoverdine but in this case, I decided to focus on altering the benefits, under the premise that fitter strains would be able to succeed given that receptors are the actual carriers of the benefit (iron in this case). Contrary to previous studies, the novelty of this strategy comes given by the fact that mutants for pyoverdine receptors are still able to produce pyoverdine, allowing them to grow in the absence of a wildtype strain, resulting in no limitations imposed by structure.
I performed competition experiments in three different environments: unstructured, semi-structured and in vivo (G. mellonella). The main reason for selecting these conditions was that most of the previous work describe unstructured environments although structure is a key element facilitating cooperative strategies. The results obtained highlight a complex scenario and, in particular, more information would be required about the dynamics of bacterial growth in an animal host for its use as an alternative treatment within the premises of evolutionary medicine.
Previously published results vary depending on the animal model considered. In this sense, it is important to decipher the relevance of the public good under study in the host chosen. As demonstrated in this dissertation, the lack of virulence variability does not imply that social dynamics are absent. As such, public goods with negligible impact on virulence can still be used as gates for Trojan horses. In addition, there are a number of limitations in previous studies: many of them use strains labelled as PAO1, but after circulation in multiple labs for decades, the genotypes are likely altered. Equally, the defective strains used in several publications were generated by random mutagenesis using exposure to UV light, assuming that no mutagenesis happened in other loci. Off- target effects could be avoided by producing clean directed deletions, and where possible,
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in the same genes, given that is not the same to mutate a pyoverdine synthesis gene than pvdS, a key regulatory element affecting multiple traits.
The characterisation of monocultures indicates that only under stress-induced situations (presence of gentamicin), the pyoverdine system can be used as a gateway to alter the dynamics of the population. This happens by the differential investments in pyoverdine generated by the presence of the antibiotic, which is higher in the wildtype strain compared to the mutants in the receptor proteins. The competition experiments revealed that the lack of FpvB was fundamental for the success in the three environments when gentamicin was present, given that all ferripyoverdine enters the cell through the primary receptor, which is arguably more efficient and linked to pyoverdine production.
Based on the data collected with synthetically improved transcriptional fusions, a probable switch between secondary receptor and primary receptor takes place in the wildtype strain, likely affecting the time required to reach an optimal cost/benefits ratio. An in-depth analysis of the mechanisms of this switch requires further research in terms of transcriptional regulation and translation. Indeed, further research on thee FpvB receptor is required, given that nothing relevant has been added to the literature since its discovery. Some of the results indicate that FpvB could act as a gate for toxins, altering the results of the competition studies, potentially being part of the growth limiting factors for primary receptor mutants. These results also demonstrate that it is possible to manipulate the cost/benefit ratio by altering the expression of secondary receptors in a primary receptor mutant strain, which translates in a better growth and a lower pyoverdine production in the presence of an environmental stressor. This poses an initial advantage in competition studies compared to its parental strain.
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Unlike previous studies, I have demonstrated that these dynamics take place even when the strains are resistant to gentamicin and not only when sublethal doses of the antibiotic are used on sensitive strains. This paves the way for novel antimicrobial therapies, in which antibiotics and optimized strains with reduced virulence and/or specific antimicrobial sensitivity are combined. Further research will be required to understand whether this is a common effect of all antibiotics, or it is only limited to gentamicin and aminoglycosides, as well as the mechanisms leading to it. It will be important to determine the relevance of the type of resistance mechanism, since this could affect the outcome.
This work demonstrates that the role of receptors pyoverdine is of key relevance for growth dynamics. It demonstrates for the first time that pyoverdine receptors can be used as tools to optimize bacterial growth even in animal models in which the public good is not essential for invasion. Additional research is required to understand if this conclusion can be generalised to other public goods or other pathogens. These could include other iron uptake systems or diffusible public goods, like QS molecules, perhaps by overexpressing the regulatory/sensory proteins binding the signalling molecules, allowing to trigger an earlier quorum response.
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