To cheat or not to cheat: cheatable and non-cheatable virulence factors in Pseudomonas aeruginosa.

Important bacterial pathogens such as Pseudomonas aeruginosa produce several exoproducts such as siderophores, degradative enzymes, biosurfactants, and exopolysaccharides that are used extracellularly, benefiting all members of the population, hence being public goods. Since the production of public goods is a cooperative trait, it is in principle susceptible to cheating by individuals in the population who do not invest in their production, but use their benefits, hence increasing their fitness at the expense of the cooperators' fitness. Among the most studied virulence factors susceptible to cheating are siderophores and exoproteases, with several studies in vitro and some in animal infection models. In addition to these two well-known examples, cheating with other virulence factors such as exopolysaccharides, biosurfactants, eDNA production, secretion systems, and biofilm formation has also been studied. In this review, we discuss the evidence of the susceptibility of each of those virulence factors to cheating, as well as the mechanisms that counteract this behavior and the possible consequences for bacterial virulence.

The bright side of social cheaters: potential beneficial roles of "social cheaters" in microbial communities.

Cooperation in microbial communities via production of public goods is susceptible to social cheating, since selfish individuals that do not contribute to their synthesis but benefit from their production thrive in the presence of cooperators. This behavior has been observed in the laboratory using bacterial and yeast models. Moreover, growing evidence indicates that cheating is frequent in natural microbial communities. In the laboratory, social cheating can promote population collapse or "tragedy of the commons" when excessive. Nevertheless, there are diverse mechanisms that counteract cheating in microbes, as well as theoretical and experimental evidence that suggests possible beneficial roles of social cheaters for the microbial populations. In this mini review manuscript we compile and discuss such possible roles.

Rhamnolipids stabilize quorum sensing mediated cooperation in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is one of the main models to study social behaviors in bacteria since it synthesizes several exoproducts, including exoproteases and siderophores and release them to the environment. Exoproteases and siderophores are public goods that can be utilized by the individuals that produce them but also by non-producers, that are considered social cheaters. Molecularly exoprotease cheaters are mutants in regulatory genes such as lasR, and are commonly isolated from chronic infections and selected in the laboratory upon serial cultivation in media with protein as a sole carbon source. Despite that the production of exoproteases is exploitable, cooperators have also ways to restrict the growth and selection of social cheaters, for instance by producing toxic metabolites like pyocyanin. In this work, using bacterial competitions, serial cultivation and growth assays, we demonstrated that rhamnolipids which production is regulated by quorum sensing, selectively affect the growth of lasR mutants and are able to restrict social cheating, hence contributing to the maintenance of cooperation in Pseudomonas aeruginosa populations.

Seeding Public Goods Is Essential for Maintaining Cooperation in Pseudomonas aeruginosa.

Quorum sensing in Pseudomonas aeruginosa controls the production of costly public goods such as exoproteases. This cooperative behavior is susceptible to social cheating by mutants that do not invest in the exoprotease production but assimilate the amino acids and peptides derived by the hydrolysis of proteins in the extracellular media. In sequential cultures with protein as the sole carbon source, these social cheaters are readily selected and often reach equilibrium with the exoprotease producers. Nevertheless, an excess of cheaters causes the collapse of population growth. In this work, using the reference strain PA14 and a clinical isolate from a burn patient, we demonstrate that the initial amount of public goods (exoprotease) that comes with the inoculum in each sequential culture is essential for maintaining population growth and that eliminating the exoprotease in the inoculum leads to rapid population collapse. Therefore, our results suggest that sequential washes should be combined with public good inhibitors to more effectively combat P. aeruginosa infections.

Pyocyanin Restricts Social Cheating in Pseudomonas aeruginosa.

Quorum sensing (QS) in Pseudomonas aeruginosa coordinates the expression of virulence factors, such as exoproteases and siderophores, that are public goods utilized by the whole population of bacteria, regardless of whether they invested or not in their production. These public goods can be used by QS defective mutants for growth, and since these mutants do not contribute to public goods production, they are considered social cheaters. Pyocyanin is a phenazine that is a toxic, QS-controlled metabolite produced by P. aeruginosa. It is a redox-active compound and promotes the generation of reactive oxygen species; it also possesses antibacterial properties and increases fitness in competition with other bacterial species. Since QS-deficient individuals are less able to tolerate oxidative stress, we hypothesized that the pyocyanin produced by the wild-type population could promote selection of functional QS systems in this bacterium. Here, we demonstrate, using competition experiments and mathematical models, that, indeed, pyocyanin increases the fitness of the cooperative QS-proficient individuals and restricts the appearance of social cheaters. In addition, we also show that pyocyanin is able to select QS in other bacteria such as Acinetobacter baumannii.

Selection of Functional Quorum Sensing Systems by Lysogenic Bacteriophages in Pseudomonas aeruginosa.

Quorum sensing (QS) in Pseudomonas aeruginosa coordinates the expression of virulence factors, some of which are used as public goods. Since their production is a cooperative behavior, it is susceptible to social cheating in which non-cooperative QS deficient mutants use the resources without investing in their production. Nevertheless, functional QS systems are abundant; hence, mechanisms regulating the amount of cheating should exist. Evidence that demonstrates a tight relationship between QS and the susceptibility of bacteria against the attack of lytic phages is increasing; nevertheless, the relationship between temperate phages and QS has been much less explored. Therefore, in this work, we studied the effects of having a functional QS system on the susceptibility to temperate bacteriophages and how this affects the bacterial and phage dynamics. We find that both experimentally and using mathematical models, that the lysogenic bacteriophages D3112 and JBD30 select QS-proficient P. aeruginosa phenotypes as compared to the QS-deficient mutants during competition experiments with mixed strain populations in vitro and in vivo in Galleria mellonella, in spite of the fact that both phages replicate better in the wild-type background. We show that this phenomenon restricts social cheating, and we propose that temperate phages may constitute an important selective pressure toward the conservation of bacterial QS.