Competition sensing: molecular mechanisms and therapeutic potential

Bacteria are social creatures living in dense and diverse communities where different strains and species compete for a limited amount of resources. However, due to a strong focus in molecular biology on studying a single species in isolation, knowledge on the molecular pathways regulating competitive behavior is limited. Therefore, in the first part of this thesis, we aim to characterize the molecular response to competition and identify the associated regulators. Moreover, these bacterial consortia are typically more virulent and tolerant than their monospeceis counterparts. As competitive interactions are predominant in these mixed-species communities, we hypothesize that competition and not cooperation underlies the enhanced virulence. Therefore, we aim to determine the role of competitive interactions in the increased virulence of consortia. If our hypothesis holds true, we will also evaluate the potential of novel antimicrobial strategies that interfere with competition and the associated molecular responses.

To achieve these goals, we first established a simple but relevant model community based around the enteropathogen Salmonella Typhimurium that is characterized by competitive interactions. We utilized the differential fluorescence induction (DFI) method to screen our focal Salmonella strain for pathways induced in the presence of competing strains via flow cytometry. This screening revealed that competition increased the expression of genes associated with biofilm matrix production (CsgD pathway), epithelial invasion (SPI1 invasion system), and antibiotic tolerance (TolC efflux pump, AadA aminoglycoside 3-adenyltransferase) and induced the associated phenotypes. These responses were regulated by the major stress response systems RpoS, PhoPQ and SoxRS as deletion of either one abolished the induction of the competitive response. These results provide direct support for the competition sensing hypothesis. This hypothesis states that traditional stress response systems are ideal candidates to detect ecological competition by directly sensing nutrient limitation or cell damage. Moreover, the inactivation of the Type VI secretion system (T6SS) of a competitor also negated the responses to competition, indicating that T6SS-derived cell damage activates these stress response systems.

As these results further indicate that competitive interactions enhance the virulence and tolerance of microbial communities, we evaluated whether inhibiting the competitive response (=competition quenching) can be exploited as an alternative antimicrobial therapy to weaken pathogens. First we determined the genericity of this competitive response by analyzing the effect of various known probiotic strains on csgD expression. However, only a limited number of Lactobacilli induced the biofilm response, indicating that this is not a general response to competition, but rather a specific response associated with distinct competitive triggers. In addition, we tested whether we can inhibit the competitive response in our model community by weakening the competitive interactions. As the strength of interactions is closely associated with community density, we reduced the density via the in-house developed motility and biofilm inhibitor agaric acid. We confirmed that agaric acid weakened the competitive interactions. Consequently, induction of RpoS no longer occurred and the competitive response was inhibited.

In conclusion, this work shows that Salmonella induces virulence-associated traits in response to competition via activation of several stress response systems. Moreover, competition quenching could be explored as a novel antimicrobial strategy to weaken pathogens, especially as preventing the induction of only one of the activated stress response systems is sufficient to limit the competitive response.