Global Regulatory Pathways and Cross-talk Control Pseudomonas aeruginosa Environmental Lifestyle and Virulence Phenotype

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

Characterization of a Type IV Pilus Biogenesis Operon in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic bacterial pathogen and frequent cause of nosocomial infections. Its intrinsic antibiotic resistance makes it challenging to treat. Additionally, P. aeruginosa is the dominant pathogen responsible for chronic pulmonary infection in individuals with cystic fibrosis (CF). Type IV pili (Tfp) are retractile surface appendages that promote P. aeruginosa virulence by mediating i) bacterial adherence to host tissue, ii) twitching motility (TM), a form of surface-associated bacterial translocation that aids in bacterial dissemination, and iii) formation of biofilm communities. In P. aeruginosa, Tfp fibers are primarily composed of a single repeating subunit termed pilin, which is encoded by the pilA gene. In addition, there are several less abundant proteins associated with the fiber that play key structural and functional roles. The five `pilin-like' proteins (FimU, PilV, PilW, PilX, PilE), which share the highly conserved N-terminal -helical region of pilin, are encoded by an operon (fimU-pilVWXY1Y2E (fimU)) that also encodes the structurally distinct protein, PilY1. The pilin-like proteins and PilY1 are incorporated into the mature Tfp fiber and are required for Tfp biogenesis; however, PilY1 has additional roles in antagonizing pilus retraction and mediating attachment to host epithelial cells. Here we describe the regulatory mechanisms that control expression of the fimU Tfp biogenesis operon. Specifically, we identified two linked, but independent networks involving the transcriptional regulators AlgR and Vfr that converge to control fimU promoter activity. Additionally, we revealed a positive feedback mechanism that results in fimU activation when either the pilin-like proteins or PilY1 is not expressed. We determined that enhanced fimU expression involves upregulation of the TCS AlgZ/R. Based on the recently solved PilY1 crystal structure, which revealed an EF-hand-like calcium-binding site within the C-terminus of the protein; we investigated the role of calcium binding in PilY1 function. Both in vitro and in vivo studies demonstrated the importance of calcium binding and release in the control of pilus extension/retraction. Together, these studies detail the regulatory mechanisms involved in controlling both fimU transcription and PilY1 function and provide an enhanced understanding of Tfp biogenesis and regulation.Doctor of Philosoph

A Hierarchical Cascade of Second Messengers Regulates Pseudomonas aeruginosa Surface Behaviors

Biofilms are surface-attached multicellular communities. Using single-cell tracking microscopy, we showed that apilY1 mutant of Pseudomonas aeruginosa is defective in early biofilm formation. We leveraged the observation that PilY1 pro- tein levels increase on a surface to perform a genetic screen to identify mutants altered in surface-grown expression of this pro- tein. Based on our genetic studies, we found that soon after initiating surface growth, cyclic AMP (cAMP) levels increase, depen- dent on PilJ, a chemoreceptor-like protein of the Pil-Chp complex, and the type IV pilus (TFP). cAMP and its receptor protein Vfr, together with the FimS-AlgR two-component system (TCS), upregulate the expression of PilY1 upon surface growth. FimS and PilJ interact, suggesting a mechanism by which Pil-Chp can regulate FimS function. The subsequent secretion of PilY1 is dependent on the TFP assembly system; thus, PilY1 is not deployed until the pilus is assembled, allowing an ordered signaling cascade. Cell surface-associated PilY1 in turn signals through the TFP alignment complex PilMNOP and the diguanylate cyclase SadC to activate downstream cyclic di-GMP (c-di-GMP) production, thereby repressing swarming motility. Overall, our data support a model whereby P. aeruginosa senses the surface through the Pil-Chp chemotaxis-like complex, TFP, and PilY1 to reg- ulate cAMP and c-di-GMP production, thereby employing a hierarchical regulatory cascade of second messengers to coordinate its program of surface behaviors

A Hierarchical Cascade of Second Messengers Regulates Pseudomonas aeruginosa Surface Behaviors

ABSTRACT Biofilms are surface-attached multicellular communities. Using single-cell tracking microscopy, we showed that a pilY1 mutant of Pseudomonas aeruginosa is defective in early biofilm formation. We leveraged the observation that PilY1 protein levels increase on a surface to perform a genetic screen to identify mutants altered in surface-grown expression of this protein. Based on our genetic studies, we found that soon after initiating surface growth, cyclic AMP (cAMP) levels increase, dependent on PilJ, a chemoreceptor-like protein of the Pil-Chp complex, and the type IV pilus (TFP). cAMP and its receptor protein Vfr, together with the FimS-AlgR two-component system (TCS), upregulate the expression of PilY1 upon surface growth. FimS and PilJ interact, suggesting a mechanism by which Pil-Chp can regulate FimS function. The subsequent secretion of PilY1 is dependent on the TFP assembly system; thus, PilY1 is not deployed until the pilus is assembled, allowing an ordered signaling cascade. Cell surface-associated PilY1 in turn signals through the TFP alignment complex PilMNOP and the diguanylate cyclase SadC to activate downstream cyclic di-GMP (c-di-GMP) production, thereby repressing swarming motility. Overall, our data support a model whereby P.aeruginosa senses the surface through the Pil-Chp chemotaxis-like complex, TFP, and PilY1 to regulate cAMP and c-di-GMP production, thereby employing a hierarchical regulatory cascade of second messengers to coordinate its program of surface behaviors. IMPORTANCEBiofilms are surface-attached multicellular communities. Here, we show that a stepwise regulatory circuit, involving ordered signaling via two different second messengers, is required for Pseudomonas aeruginosa to control early events in cell-surface interactions. We propose that our studies have uncovered a multilayered “surface-sensing” system that allows P.aeruginosa to effectively coordinate its surface-associated behaviors. Understanding how cells transition into the biofilm state on a surface may provide new approaches to prevent formation of these communities

Pseudomonas aeruginosa PilY1 Binds Integrin in an RGD- and Calcium-Dependent Manner

PilY1 is a type IV pilus (tfp)-associated protein from the opportunistic pathogen Pseudomonas aeruginosa that shares functional similarity with related proteins in infectious Neisseria and Kingella species. Previous data have shown that PilY1 acts as a calcium-dependent pilus biogenesis factor necessary for twitching motility with a specific calcium binding site located at amino acids 850–859 in the 1,163 residue protein. In addition to motility, PilY1 is also thought to play an important role in the adhesion of P. aeruginosa tfp to host epithelial cells. Here, we show that PilY1 contains an integrin binding arginine-glycine-aspartic acid (RGD) motif located at residues 619–621 in the PilY1 from the PAK strain of P. aeruginosa; this motif is conserved in the PilY1s from the other P. aeruginosa strains of known sequence. We demonstrate that purified PilY1 binds integrin in vitro in an RGD-dependent manner. Furthermore, we identify a second calcium binding site (amino acids 600–608) located ten residues upstream of the RGD. Eliminating calcium binding from this site using a D608A mutation abolished integrin binding; in contrast, a calcium binding mimic (D608K) preserved integrin binding. Finally, we show that the previously established PilY1 calcium binding site at 851–859 also impacts the protein's association with integrin. Taken together, these data indicate that PilY1 binds to integrin in an RGD- and calcium-dependent manner in vitro. As such, P. aeruginosa may employ these interactions to mediate host epithelial cell binding in vivo