Abstract

Opportunistic pathogens like Pseudomonas aeruginosa become increasingly resistant to antibiotics, and therefore represent a major threat to patients. Thus, we urgently need new approaches to fight multi-resistant pathogens. It has been suggested that, instead of targeting vital cell mechanisms, virulence factors could be inhibited with so-called anti-virulence treatments. These treatments are believed to impose lower selection pressure on the pathogen and would thereby reduce the risk of resistance development. In this thesis, we aim to extend the anti-virulence approach by targeting a secreted virulence factor that is cooperatively shared between bacteria. For many pathogens, cooperation is essential to infect hosts, and is often mediated by secreted, publically shared virulence factors. Cooperating individuals can be exploited by individuals, which do not contribute to cooperation, but reap the benefits from it, so-called cheaters. By targeting a cooperatively shared virulence factor, the cooperating community becomes phenotypic cheaters and every individual, that resumes cooperation (e.g. by developing resistance), will be exploited immediately and thus resistance is not favoured by natural selection. Such a treatment can become evolution proof. We tested this idea by inhibiting the cooperatively shared virulence factor pyoverdine. Pyoverdine is the main siderophore of P. aeruginosa, which is deployed in severely iron-limited environments to assure sufficient supply of this essential nutrient. Pyoverdine facilitates pathogenic growth at the infection site. In chapter (3) we experimentally tested a promising candidate, the transition metal gallium, as an evolution proof anti-virulence treatment, that targets pyoverdine. Gallium effectively curbed the virulence of P. aeruginosa in an insect model. Moreover, while antibiotics lost their efficacy rapidly in an evolution experiment, P. aeruginosa did not show signs of resistance to gallium. Next, we tested if and how such interference with virulence factor availability (pyoverdine) feeds back on the pathogen, its regulatory network and the host (chapter 4). We found complex relationships between these variables. While the link between virulence factor availability and virulence was positive, pyoverdine availability did not correlate monotonously with pathogen growth within the host. The amount of available virulence factor influenced the expression of virulence factors, that are regulatorily linked. Additionally, it triggered differential host immune responses. These findings highlight the necessity to closely evaluate the effects of any anti-virulence drug on the pathogen and the host, in order to design effective drugs with a predictive treatment outcome. The concept of evolution proof anti-virulence treatments builds (among others) on the assumption that the targeted virulence factor is collectively shared between individuals. Although pyoverdine cooperation has been extensively studied in the last decade, almost all studies feature experiments in batch cultures. However, little is known about whether the insights from batch culture experiments can be transferred to infections. In the host, cell numbers might be lower and bacteria might interact on the micrometre-scale in a spatially structured environment where diffusion of a shared virulence factor, and thus shareability, could be limited. Therefore, we investigated pyoverdine sharing between individuals, attached to a surface, at the level of single cells by using fluorescent microscopy, and experimentally tested the physical boundaries of pyoverdine sharing (chapter 5). We found that even in highly viscous environments, pyoverdine is publically shared over a considerable distance. These findings validate the assumption that pyoverdine is cooperatively shared, even in viscous environments, such as experienced in infections, and therefore indicates that anti-virulence treatments targeting pyoverdine (e.g. via gallium), could indeed be evolution proof.