Global regulatory pathways and cross-talk control pseudomonas aeruginosa environmental lifestyle and virulence phenotype

Abstract

Pseudomonas aeruginosa is a metabolically versatile environmental bacterium and an opportunistic human pathogen that relies on numerous signaling pathways to sense, respond, and adapt to fluctuating environmental cues. Although the environmental signals sensed by these pathways are poorly understood, they are largely responsible for determining whether P. aeruginosa adopts a planktonic or sessile lifestyle. These environmental lifestyle extremes parallel the acute and chronic infection phenotypes observed in human disease. In this review, we focus on four major pathways (cAMP/Vfr and c-di-GMP signaling, quorum sensing, and the Gac/Rsm pathway) responsible for sensing and integrating external stimuli into coherent regulatory control at the transcriptional, translational, and post-translational level. A common theme among these pathways is the inverse control of factors involved in promoting motility and acute infection and those associated with biofilm formation and chronic infection. In many instances these regulatory pathways influence one another, forming a complex network allowing P. aeruginosa to assimilate numerous external signals into an integrated regulatory circuit that controls a lifestyle continuum.

Figure 1.. Model of the regulatory pathways and cross-talk that modulate P. aeruginosa lifestyles.

Lines depict direct regulatory mechanisms within given signaling pathways as well as cross-talk between regulatory systems. Arrows represent positive regulation and T-bars indicate negative regulation. Blue lines represent transcriptional regulation while purple lines represent enzymatic reactions. Orange lines depict post-translational modification events. Black lines illustrate post-translational regulation events. Dashed lines indicate unknown mechanisms of regulation. For more details see text references to this figure. A. cAMP/Vfr signaling. The Chp chemosensory system modulates the enzymatic activity of CyaB, an adenylate cyclase that synthesizes the majority of intracellular cAMP. The phosphodiesterase CpdA degrades intracellular cAMP. When present in sufficient quantities, cAMP binds to and activates the transcription factor Vfr. cAMP/Vfr regulates numerous virulence factors, primarily those associated with an acute virulence phenotype, in addition to lasR, the master regulator of the quorum sensing hierarchy. Both vfr and cpdA are transcriptionally regulated by cAMP/Vfr creating a feedback loop to maintain cAMP homeostasis. B. Quorum Sensing. Schematic representation of the AHL- and AQ-dependent QS systems in P. aeruginosa. LasR activates lasI expression to produce C12-HSL. The LasR/C12-HSL complex positively influences expression of the second AHL-dependent QS system (RhlRI/C4-HSL) and many other target genes. The AHL-dependent systems are transcriptionally and post-transcriptionally regulated by numerous regulators including two LuxR homologues (VqsR and QscR), RsmA, and the alternative sigma factor, RpoS. The RhlR/C4-HSL complex negatively regulates the AQ-dependent QS system. C. Gac/Rsm pathway. The GacS/A TCS positively regulates expression of two sRNA molecules, RsmY and RsmZ, which bind and sequester the RNA-binding protein RsmA. Activation of the HptB signaling cascade promotes chronic virulence factor expression by specifically activating rsmY expression. GacS activity is antagonized by the hybrid sensor kinase RetS and promoted by the orphan sensor kinase LadS. Free RsmA facilitates the expression of acute virulence factors, such as the T3SS, and represses expression of chronic virulence factors including the T6SS and EPS (Pel and Psl) involved in biofilm formation. D. c-di-GMP signaling. c-di-GMP levels inversely control functions involved in motility (acute) and biofilm formation (chronic). Synthesis and degradation of c-di-GMP is facilitated by diguanylate cyclases and phosphodiesterases. Multiple DGCs and PDEs encoded within the P. aeruginosa genome appear to be spatially localized to alter local c-di-GMP concentrations and influence protein function and gene expression by largely unknown mechanisms. E. MucA signaling. The anti-sigma factor MucA sequesters the sigma factor AlgU. During chronic CF infection, mucA mutation results in constitutive AlgU activation. AlgU increases expression of algR and together AlgU and AlgR activate the alginate biosynthetic operon. AlgR negatively regulates QS by inhibiting rhlR expression. AlgR inhibits vfr expression via an unknown mechanism resulting in decreased expression of many acute virulence factors. F. HptB signaling. Upon activation by an unknown signal the sensor kinsase PA2284 transfers a phosphoryl group specifically to HptB. HptB then relays the signal to the response regulator PA3346. Phosphorylated PA3346 functions as a Ser/Thr phosphatase and dephosphorylates PA3347. Phosphorylation is hypothesized to modulate the binding activity of PA3347. In the unphosphorylated state PA3347 is thought to bind an anti-sigma factor, resulting in the release of an unidentified sigma factor, which specifically regulates rsmY expression leading to expression of genes associated with swarming motility and biofilm formation.

Figure 2.. Pseudomonas aeruginosa exhibits parallel lifestyle extremes in the environment and human host.

The interplay of four global regulatory pathways (cAMP, c-di-GMP, Quorum sensing and Gac/Rsm) appears to create a phenotypic continuum that controls transition between a planktonic and sessile lifestyle within the environment; and plays an analogous role in human infection by inversely controlling acute and chronic infection phenotypes. 1. In the environment P. aeruginosa can exist as planktonic cells or small groups of free living motile organisms, providing the means to colonize new environmental niches. 2. As the population increases, cells associate as a quorum, producing QS signal molecules and exhibit social behaviors (degradative enzyme and toxin secretion), which promote nutrient acquisition and group survival among environmental predators. 3. Upon interaction with a solid surface, P. aeruginosa can attach via Tfp or flagella. 4. Following loose-attachment, P. aeruginosa may exhibit surface motility utilizing Tfp or flagella to move toward nutrients. Upon generation of an intimate surface attachment, P. aeruginosa may initiate microcolony formation. 5. Eventually P. aeruginosa becomes sessile and produces exopolysaccharides that encase the bacteria in a complex matrix, which protects the bacteria from environmental fluctuations and provides a physical barrier against predators. 6. Unknown environmental signal(s), or stochastic processes, cause members of the sessile community to become motile, leave the biofilm, and return to a planktonic lifestyle.