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.

Iron limitation by transferrin promotes simultaneous cheating of pyoverdine and exoprotease in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a primary bacterial model to study cooperative behaviors because it yields exoproducts such as siderophores and exoproteases that act as public goods and can be exploited by selfish nonproducers behaving as social cheaters. Iron-limited growth medium, mainly casamino acids medium supplemented with transferrin, is typically used to isolate and study nonproducer mutants of the siderophore pyoverdine. However, using a protein as the iron chelator could inadvertently select mutants unable to produce exoproteases, since these enzymes can degrade the transferrin to facilitate iron release. Here we investigated the evolutionary dynamics of pyoverdine and exoprotease production in media in which iron was limited by using either transferrin or a cation chelating resin. We show that concomitant loss of pyoverdine and exoprotease production readily develops in media containing transferrin, whereas only pyoverdine loss emerges in medium treated with the resin. Characterization of exoprotease- and pyoverdine-less mutants revealed loss in motility, different mutations, and large genome deletions (13-33 kb) including Quorum Sensing (lasR, rsal, and lasI) and flagellar genes. Our work shows that using transferrin as an iron chelator imposes simultaneous selective pressure for the loss of pyoverdine and exoprotease production. The unintended effect of transferrin uncovered by our experiments can help to inform the design of similar studies.

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.

Characterization of FlgP, an Essential Protein for Flagellar Assembly in Rhodobacter sphaeroides.

The flagellar lipoprotein FlgP has been identified in several species of bacteria, and its absence provokes different phenotypes. In this study, we show that in the alphaproteobacterium Rhodobacter sphaeroides, a ΔflgP mutant is unable to assemble the hook and the filament. In contrast, the membrane/supramembrane (MS) ring and the flagellar rod appear to be assembled. In the absence of FlgP a severe defect in the transition from rod to hook polymerization occurs. In agreement with this idea, we noticed a reduction in the amount of intracellular flagellin and the chemotactic protein CheY4, both encoded by genes dependent on σ28 This suggests that in the absence of flgP the switch to export the anti-sigma factor, FlgM, does not occur. The presence of FlgP was detected by Western blot in samples of isolated wild-type filament basal bodies, indicating that FlgP is an integral part of the flagellar structure. In this regard, we show that FlgP interacts with FlgH and FlgT, indicating that FlgP should be localized closely to the L and H rings. We propose that FlgP could affect the architecture of the L ring, which has been recently identified to be responsible for the rod-hook transition.IMPORTANCE Flagellar based motility confers a selective advantage on bacteria by allowing migration to favorable environments or in pathogenic species to reach the optimal niche for colonization. The flagellar structure has been well established in Salmonella However, other accessory components have been identified in other species. Many of these have been implied in adapting the flagellar function to enable faster rotation, or higher torque. FlgP has been proposed to be the main component of the basal disk located underlying the outer membrane in Campylobacter jejuni and Vibrio fischeri Its role is still unclear, and its absence impacts motility differently in different species. The study of these new components will bring a better understanding of the evolution of this complex organelle.