The multiple signaling systems regulating virulence in Pseudomonas aeruginosa

Abstract

Cell-to-cell communication is a major process that allows bacteria to sense and coordinately react to the fluctuating conditions of the surrounding environment. In several pathogens, this process triggers the production of virulence factors and/or a switch in bacterial lifestyle that is a major determining factor in the outcome and severity of the infection. Understanding how bacteria control these signaling systems is crucial to the development of novel antimicrobial agents capable of reducing virulence while allowing the immune system of the host to clear bacterial infection, an approach likely to reduce the selective pressures for development of resistance. We provide here an up-to-date overview of the molecular basis and physiological implications of cell-to-cell signaling systems in Gram-negative bacteria, focusing on the well-studied bacterium Pseudomonas aeruginosa. All of the known cell-to-cell signaling systems in this bacterium are described, from the most-studied systems, i.e., N-acyl homoserine lactones (AHLs), the 4-quinolones, the global activator of antibiotic and cyanide synthesis (GAC), the cyclic di-GMP (c-di-GMP) and cyclic AMP (cAMP) systems, and the alarmones guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), to less-well-studied signaling molecules, including diketopiperazines, fatty acids (diffusible signal factor [DSF]-like factors), pyoverdine, and pyocyanin. This overview clearly illustrates that bacterial communication is far more complex than initially thought and delivers a clear distinction between signals that are quorum sensing dependent and those relying on alternative factors for their production.

Fig 1

Virulence regulation of and interactions between the two AHL quorum-sensing systems in P. aeruginosa. After a threshold concentration of 3-oxo-C₁₂-HSL is produced, the 3-oxo-C₁₂-HSL–LasR complex binds the promoter regions of multiple genes, activating or repressing their transcription. Among the genes upregulated by this complex are lasI, which enhances the production of 3-oxo-C₁₂-HSL (autoinduction effect), and rhlR, which increases the production of the rhl response regulator RhlR, activating the second AHL pathway at an earlier stage. Virulence factors regulated by each respective receptor-ligand complex are detailed on the left.

Fig 2

Structures of the two DKPs produced by P. aeruginosa.

Fig 3

Biosynthesis, autoinduction, and virulence regulation by 4-alkyl-quinolones in P. aeruginosa. Biosynthesis of PQS starts with the conversion (by the PqsABCD proteins) of anthranilate (which originates from either the kynurenine pathway or the PhnAB anthranilate synthase) into HHQ, which is finally converted into PQS by the PqsH monooxygenase. Both HHQ and PQS bind the PqsR regulator, and the complex activates the transcription of the pqsABCDE and phnAB operons, increasing the levels of PQS (autoinduction) and pyocyanin production. Additionally, transcription of the PQS operon results in an increase in the levels of PqsE, an enzyme of uncharacterized function that increases the levels of pyocyanin, lectin, HCN, and rhamnolipids.

Fig 4

The GAC system network in P. aeruginosa controls the reversible transition from acute to chronic infections. The small regulatory protein RsmA binds to the promoters of multiple genes, enhancing bacterial motility and activating the production of several acute virulence factors while repressing the production of virulence factors associated with chronic infections. GacA phosphorylation via GacS stimulates the production of the small RNAs RsmZ and RsmY, which bind to the RsmA protein, releasing the repression of virulence factors associated with chronic infections and repressing the production of acute infection-associated factors. The sensor kinase LadS works in parallel to GacS, activating RsmZ and RsmY production, while the sensor kinase RetS acts in an opposite manner to LadS and GacS, forming a protein-protein complex with GacS that blocks RsmY and RsmZ production.

Fig 5

Pyoverdines produced by P. aeruginosa. Each P. aeruginosa strain produces one type of pyoverdine exclusively. The amino acids d-Tyr and l-Glu (bottom right of each PVD) are further modified during biosynthesis to yield the final pyoverdine. d-Tyr is converted into catechol, and l-Glu into either succinyl, succinamide, ketoglutaryl, or a free acid, and is thus represented by “-R”.

Fig 6

PVD signaling pathway in P. aeruginosa. In the absence of Fe-PVD (left), the signaling system is inactive. Binding of Fe-PVD to the PVD receptor FpvA (right) initiates a signaling cascade that requires TonB1 and FpvR and stimulates the production of FpvA, PVD, ToxA, and the PrpL protease.

Fig 7

Biosynthesis and signaling system of pyocyanin. Chorismic acid is transformed via the PhzA to -G proteins into phenazine-1-carboxylic acid, which is subsequently converted into different phenazines by the enzymes PhzH, PhzS, and PhzM. The product of the latter is transformed by PhzS into pyocyanin (PYO). Next to its role as a virulence factor, PYO acts as a signaling molecule activating a limited set of genes termed the PYO stimulon. A large fraction of the PYO stimulon genes are controlled by the regulator SoxR, although the mechanism by which PYO activates SoxR, as well as the activation mechanism of SoxR-independent genes, remains unknown.

Fig 8

DSF-like fatty acids controlling cell-to-cell signaling in various Gram-negative bacteria.

Fig 9

c-di-GMP signaling mechanism. Diguanylate cyclases and phosphodiesterases regulate the bacterial lifestyle (free-living versus biofilm) by balancing the internal levels of c-di-GMP. Binding of c-di-GMP to its receptor targets stimulates biofilm formation, suppressing motility. In parallel, binding of GTP to its receptors (i.e., the allosteric site of the PDE FimX) increases the c-di-GMP-degrading activity of PDE, decreasing c-di-GMP levels, suppressing biofilm formation, and increasing motility.

Fig 10

Summary of cell-to-cell signaling systems in P. aeruginosa. Dashed lines indicate that the presence of a signal activating the system has been proposed but remains to be proven. The PVD and GAC systems use membrane-associated proteins to activate signaling in response to their respective signals.