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

Infection of human mucosal tissue by Pseudomonas aeruginosa requires sequential and mutually dependent virulence factors and a novel pilus-associated adhesin

Tissue damage predisposes humans to life-threatening disseminating infection by the opportunistic pathogen Pseudomonas aeruginosa. Bacterial adherence to host tissue is a critical first step in this infection process. It is well established that P. aeruginosa attachment to host cells involves type IV pili (TFP), which are retractile surface fibers. The molecular details of attachment and the identity of the bacterial adhesin and host receptor remain controversial. Using a mucosal epithelium model system derived from primary human tissue, we show that the pilus-associated protein PilY1 is required for bacterial adherence. We establish that P. aeruginosa preferentially binds to exposed basolateral host cell surfaces, providing a mechanistic explanation for opportunistic infection of damaged tissue. Further, we demonstrate that invasion and fulminant infection of intact host tissue requires the coordinated and mutually dependent action of multiple bacterial factors, including pilus fiber retraction and the host cell intoxication system, termed type III secretion. Our findings offer new and important insights into the complex interactions between a pathogen and its human host and provide compelling evidence that PilY1 serves as the principal P. aeruginosa adhesin for human tissue and that it specifically recognizes a host receptor localized or enriched on basolateral epithelial cell surfaces

Phosphoryl Group Flow within the Pseudomonas aeruginosa Pil-Chp Chemosensory System: DIFFERENTIAL FUNCTION OF THE EIGHT PHOSPHOTRANSFERASE AND THREE RECEIVER DOMAINS

Bacterial chemosensory signal transduction systems that regulate motility by type IV pili (T4P) can be markedly more complex than related flagellum-based chemotaxis systems. In T4P-based systems, the CheA kinase often contains numerous potential sites of phosphorylation, but the signaling mechanisms of these systems are unknown. In Pseudomonas aeruginosa, the Pil-Chp system regulates T4P-mediated twitching motility and cAMP levels, both of which play roles in pathogenesis. The Pil-Chp histidine kinase (ChpA) has eight “Xpt” domains; six are canonical histidine-containing phosphotransfer (Hpt) domains and two have a threonine (Tpt) or serine (Spt) in place of the histidine. Additionally, there are two stand-alone receiver domains (PilG and PilH) and a ChpA C-terminal receiver domain (ChpArec). Here, we demonstrate that the ChpA Xpts are functionally divided into three categories as follows: (i) those phosphorylated with ATP (Hpt4–6); (ii) those reversibly phosphorylated by ChpArec (Hpt2–6), and (iii) those with no detectable phosphorylation (Hpt1, Spt, and Tpt). There was rapid phosphotransfer from Hpt2–6 to ChpArec and from Hpt3 to PilH, whereas transfer to PilG was slower. ChpArec also had a rapid rate of autodephosphorylation. The biochemical results together with in vivo cAMP and twitching phenotypes of key ChpA phosphorylation site point mutants supported a scheme whereby ChpArec functions both as a phosphate sink and a phosphotransfer element linking Hpt4–6 to Hpt2–3. Hpt2 and Hpt3 are likely the dominant sources of phosphoryl groups for PilG and PilH, respectively. The data are synthesized in a signaling circuit that contains fundamental features of two-component phosphorelays

Intrinsic and Extrinsic Regulation of Type III Secretion Gene Expression in Pseudomonas Aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen that is particularly problematic in the healthcare setting where it is a frequent cause of pneumonia, bloodstream, and urinary tract infections. An important determinant of P. aeruginosa virulence is a type III secretion system (T3SS). T3SS-dependent intoxication is a complex process that minimally requires binding of P. aeruginosa to host cells, injection of the cytotoxic effector proteins through the host cell plasma membrane, and induction of T3SS gene expression. The latter process, referred to as contact-dependent expression, involves a well-characterized regulatory cascade that activates T3SS gene expression in response to host cell contact. Although host cell contact is a primary activating signal for T3SS gene expression, the involvement of multiple membrane-bound regulatory systems indicates that additional environmental signals also play a role in controlling expression of the T3SS. These regulatory systems coordinate T3SS gene expression with many other cellular activities including motility, mucoidy, polysaccharide production, and biofilm formation. The signals to which the organism responds are poorly understood but many seem to be coupled to the metabolic state of the cell and integrated within a master circuit that assimilates informational signals from endogenous and exogenous sources. Herein we review progress toward unraveling this complex circuitry, provide analysis of the current knowledge gaps, and highlight potential areas for future studies. Complete understanding of the regulatory networks that control T3SS gene expression will maximize opportunities for the development of strategies to treat P. aeruginosa infections

Primary and Secondary Sequence Structure Requirements for Recognition and Discrimination of Target RNAs by Pseudomonas aeruginosa RsmA and RsmF

ABSTRACT CsrA family RNA-binding proteins are widely distributed in bacteria and regulate gene expression at the posttranscriptional level. Pseudomonas aeruginosa has a canonical member of the CsrA family (RsmA) and a novel, structurally distinct variant (RsmF). To better understand RsmF binding properties, we performed parallel systematic evolution of ligands by exponential enrichment (SELEX) experiments for RsmA and RsmF. The initial target library consisted of 62-nucleotide (nt) RNA transcripts with central cores randomized at 15 sequential positions. Most targets selected by RsmA and RsmF were the expected size and shared a common consensus sequence (CANGGAYG) that was positioned in a hexaloop region of the stem-loop structure. RsmA and RsmF also selected for longer targets (≥96 nt) that were likely generated by rare PCR errors. Most of the long targets contained two consensus-binding sites. Representative short (single consensus site) and long (two consensus sites) targets were tested for RsmA and RsmF binding. Whereas RsmA bound the short targets with high affinity, RsmF was unable to bind the same targets. RsmA and RsmF both bound the long targets. Mutation of either consensus GGA site in the long targets reduced or eliminated RsmF binding, suggesting a requirement for two tandem binding sites. Conversely, RsmA bound long targets containing only a single GGA site with unaltered affinity. The RsmF requirement for two binding sites was confirmed with tssA1 , an in vivo regulatory target of RsmA and RsmF. Our findings suggest that RsmF binding requires two GGA-containing sites, while RsmA binding requirements are less stringent. IMPORTANCE The CsrA family of RNA-binding proteins is widely conserved in bacteria and plays important roles in the posttranscriptional regulation of protein synthesis. P. aeruginosa has two CsrA proteins, RsmA and RsmF. Although RsmA and RsmF share a few RNA targets, RsmF is unable to bind to other targets recognized by RsmA. The goal of the present study was to better understand the basis for differential binding by RsmF. Our data indicate that RsmF binding requires target RNAs with two consensus-binding sites, while RsmA recognizes targets with just a single binding site. This information should prove useful to future efforts to define the RsmF regulon and its contribution to P. aeruginosa physiology and virulence

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

Vfr Directly Activates exsA Transcription To Regulate Expression of the Pseudomonas aeruginosa Type III Secretion System

ABSTRACT The Pseudomonas aeruginosa cyclic AMP (cAMP)-Vfr system (CVS) is a global regulator of virulence gene expression. Regulatory targets include type IV pili, secreted proteases, and the type III secretion system (T3SS). The mechanism by which CVS regulates T3SS gene expression remains undefined. Single-cell expression studies previously found that only a portion of the cells within a population express the T3SS under inducing conditions, a property known as bistability. We now report that bistability is altered in a vfr mutant, wherein a substantially smaller fraction of the cells express the T3SS relative to the parental strain. Since bistability usually involves positive-feedback loops, we tested the hypothesis that virulence factor regulator (Vfr) regulates the expression of exsA . ExsA is the central regulator of T3SS gene expression and autoregulates its own expression. Although exsA is the last gene of the exsCEBA polycistronic mRNA, we demonstrate that Vfr directly activates exsA transcription from a second promoter (P exsA ) located immediately upstream of exsA . P exsA promoter activity is entirely Vfr dependent. Direct binding of Vfr to a P exsA promoter probe was demonstrated by electrophoretic mobility shift assays, and DNase I footprinting revealed an area of protection that coincides with a putative Vfr consensus-binding site. Mutagenesis of that site disrupted Vfr binding and P exsA promoter activity. We conclude that Vfr contributes to T3SS gene expression through activation of the P exsA promoter, which is internal to the previously characterized exsCEBA operon. IMPORTANCE Vfr is a cAMP-dependent DNA-binding protein that functions as a global regulator of virulence gene expression in Pseudomonas aeruginosa . Regulation by Vfr allows for the coordinate production of related virulence functions, such as type IV pili and type III secretion, required for adherence to and intoxication of host cells, respectively. Although the molecular mechanism of Vfr regulation has been defined for many target genes, a direct link between Vfr and T3SS gene expression had not been established. In the present study, we report that Vfr directly controls exsA transcription, the master regulator of T3SS gene expression, from a newly identified promoter located immediately upstream of exsA

Topology and Organization of the Salmonella typhimurium Type III Secretion Needle Complex Components

The correct organization of single subunits of multi-protein machines in a three dimensional context is critical for their functionality. Type III secretion systems (T3SS) are molecular machines with the capacity to deliver bacterial effector proteins into host cells and are fundamental for the biology of many pathogenic or symbiotic bacteria. A central component of T3SSs is the needle complex, a multiprotein structure that mediates the passage of effector proteins through the bacterial envelope. We have used cryo electron microscopy combined with bacterial genetics, site-specific labeling, mutational analysis, chemical derivatization and high-resolution mass spectrometry to generate an experimentally validated topographic map of a Salmonella typhimurium T3SS needle complex. This study provides insights into the organization of this evolutionary highly conserved nanomachinery and is the basis for further functional analysis

The RNA Helicase DeaD Stimulates ExsA Translation To Promote Expression of the Pseudomonas aeruginosa Type III Secretion System

ABSTRACT The Pseudomonas aeruginosa type III secretion system (T3SS) is a primary virulence factor important for phagocytic avoidance, disruption of host cell signaling, and host cell cytotoxicity. ExsA is the master regulator of T3SS transcription. The expression, synthesis, and activity of ExsA is tightly regulated by both intrinsic and extrinsic factors. Intrinsic regulation consists of the well-characterized ExsECDA partner-switching cascade, while extrinsic factors include global regulators that alter exsA transcription and/or translation. To identify novel extrinsic regulators of ExsA, we conducted a transposon mutagenesis screen in the absence of intrinsic control. Transposon disruptions within gene PA2840, which encodes a homolog of the Escherichia coli RNA-helicase DeaD, significantly reduced T3SS gene expression. Recent studies indicate that E. coli DeaD can promote translation by relieving inhibitory secondary structures within target mRNAs. We report here that PA2840, renamed DeaD, stimulates ExsA synthesis at the posttranscriptional level. Genetic experiments demonstrate that the activity of an exsA translational fusion is reduced in a deaD mutant. In addition, exsA expression in trans fails to restore T3SS gene expression in a deaD mutant. We hypothesized that DeaD relaxes mRNA secondary structure to promote exsA translation and found that altering the mRNA sequence of exsA or the native exsA Shine-Dalgarno sequence relieved the requirement for DeaD in vivo . Finally, we show that purified DeaD promotes ExsA synthesis using in vitro translation assays. Together, these data reveal a novel regulatory mechanism for P. aeruginosa DeaD and add to the complexity of global regulation of T3SS. IMPORTANCE Although members of the DEAD box family of RNA helicases are appreciated for their roles in mRNA degradation and ribosome biogenesis, an additional role in gene regulation is now emerging in bacteria. By relaxing secondary structures in mRNAs, DEAD box helicases are now thought to promote translation by enhancing ribosomal recruitment. We identify here an RNA helicase that plays a critical role in promoting ExsA synthesis, the central regulator of the Pseudomonas aeruginosa type III secretion system, and provide additional evidence that DEAD box helicases directly stimulate translation of target genes. The finding that DeaD stimulates exsA translation adds to a growing list of transcriptional and posttranscriptional regulatory mechanisms that control type III gene expression