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

UNLABELLED: 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 avfr 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 (PexsA) located immediately upstream of exsA PexsA promoter activity is entirely Vfr dependent. Direct binding of Vfr to a PexsA 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 PexsA promoter activity. We conclude that Vfr contributes to T3SS gene expression through activation of the PexsA 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.

An unusual CsrA family member operates in series with RsmA to amplify posttranscriptional responses in Pseudomonas aeruginosa.

Members of the CsrA family of prokaryotic mRNA-binding proteins alter the translation and/or stability of transcripts needed for numerous global physiological processes. The previously described CsrA family member in Pseudomonas aeruginosa (RsmA) plays a central role in determining infection modality by reciprocally regulating processes associated with acute (type III secretion and motility) and chronic (type VI secretion and biofilm formation) infection. Here we describe a second, structurally distinct RsmA homolog in P. aeruginosa (RsmF) that has an overlapping yet unique regulatory role. RsmF deviates from the canonical 5 ß-strand and carboxyl-terminal α-helix topology of all other CsrA proteins by having the α-helix internally positioned. Despite striking changes in topology, RsmF adopts a tertiary structure similar to other CsrA family members and binds a subset of RsmA mRNA targets, suggesting that RsmF activity is mediated through a conserved mechanism of RNA recognition. Whereas deletion of rsmF alone had little effect on RsmA-regulated processes, strains lacking both rsmA and rsmF exhibited enhanced RsmA phenotypes for markers of both type III and type VI secretion systems. In addition, simultaneous deletion of rsmA and rsmF resulted in superior biofilm formation relative to the wild-type or rsmA strains. We show that RsmF translation is derepressed in an rsmA mutant and demonstrate that RsmA specifically binds to rsmF mRNA in vitro, creating a global hierarchical regulatory cascade that operates at the posttranscriptional level.

The AlgZR two-component system recalibrates the RsmAYZ posttranscriptional regulatory system to inhibit expression of the Pseudomonas aeruginosa type III secretion system.

Pseudomonas aeruginosa causes chronic airway infections in cystic fibrosis (CF) patients. A classic feature of CF airway isolates is the mucoid phenotype. Mucoidy arises through mutation of the mucA anti-sigma factor and subsequent activation of the AlgU regulon. Inactivation of mucA also results in reduced expression of the Vfr transcription factor. Vfr regulates several important virulence factors, including a type III secretion system (T3SS). In the present study, we report that ExsA expression, the master regulator of T3SS gene expression, is further reduced in mucA mutants through a Vfr-independent mechanism involving the RsmAYZ regulatory system. RsmA is an RNA binding protein required for T3SS gene expression. Genetic experiments suggest that the AlgZR two-component system, part of the AlgU regulon, inhibits ExsA expression by increasing the expression of RsmY and RsmZ, two small noncoding RNAs that sequester RsmA from target mRNAs. Epistasis analyses revealed that increasing the concentration of free RsmA, through either rsmYZ deletion or increased RsmA expression, partially restored T3SS gene expression in the mucA mutant. Furthermore, increasing RsmA availability in combination with Vfr complementation fully restored T3SS expression. Recalibration of the RsmAYZ system by AlgZR, however, did not alter the expression of other selected RsmA-dependent targets. We account for this observation by showing that ExsA expression is more sensitive to changes in free RsmA than other members of the RsmA regulon. Together, these data indicate that recalibration of the RsmAYZ system partially accounts for reduced T3SS gene expression in mucA mutants.

The Pseudomonas aeruginosa Vfr regulator controls global virulence factor expression through cyclic AMP-dependent and -independent mechanisms.

Vfr is a global regulator of virulence factor expression in the human pathogen Pseudomonas aeruginosa. Although indirect evidence suggests that Vfr activity is controlled by cyclic AMP (cAMP), it has been hypothesized that the putative cAMP binding pocket of Vfr may accommodate additional cyclic nucleotides. In this study, we used two different approaches to generate apo-Vfr and examined its ability to bind a representative set of virulence gene promoters in the absence and presence of different allosteric effectors. Of the cyclic nucleotides tested, only cAMP was able to restore DNA binding activity to apo-Vfr. In contrast, cGMP was capable of inhibiting cAMP-Vfr DNA binding. Further, we demonstrate that vfr expression is autoregulated and cAMP dependent and involves Vfr binding to a previously unidentified site within the vfr promoter region. Using a combination of in vitro and in vivo approaches, we show that cAMP is required for Vfr-dependent regulation of a specific subset of virulence genes. In contrast, we discovered that Vfr controls expression of the lasR promoter in a cAMP-independent manner. In summary, our data support a model in which Vfr controls virulence gene expression by distinct (cAMP-dependent and -independent) mechanisms, which may allow P. aeruginosa to fine-tune its virulence program in response to specific host cues or environments.

Activation of the Pseudomonas aeruginosa AlgU regulon through mucA mutation inhibits cyclic AMP/Vfr signaling.

Pseudomonas aeruginosa is an opportunistic pathogen that causes acute, invasive infections in immunocompromised individuals and chronic, persistent respiratory infections in individuals with cystic fibrosis (CF). The differential progression of acute or chronic infections involves the production of distinct sets of virulence factors. P. aeruginosa strains isolated from patients with acute respiratory infection are generally nonencapsulated and express a variety of invasive virulence factors, including flagella, the type III secretion system (T3SS), type IV pili (TFP), and multiple secreted toxins and degradative enzymes. Strains isolated from chronically infected CF patients, however, typically lack expression of invasive virulence factors and have a mucoid phenotype due to the production of an alginate capsule. The mucoid phenotype results from loss-of-function mutations in mucA, which encodes an anti-sigma factor that normally prevents alginate synthesis. Here, we report that the cyclic AMP/Vfr-dependent signaling (CVS) pathway is defective in mucA mutants and that the defect occurs at the level of vfr expression. The CVS pathway regulates the expression of multiple invasive virulence factors, including T3SS, exotoxin A, protease IV, and TFP. We further demonstrate that mucA-dependent CVS inhibition involves the alternative sigma factor AlgU (AlgT) and the response regulator AlgR but does not depend on alginate production. Our findings show that a single naturally occurring mutation leads to inverse regulation of virulence factors involved in acute and persistent infections. These results suggest that mucoid conversion and inhibition of invasive virulence determinants may both confer a selective advantage to mucA mutant strains of P. aeruginosa in the CF lung.

Genetic determinants of self identity and social recognition in bacteria.

The bacterium Proteus mirabilis is capable of movement on solid surfaces by a type of motility called swarming. Boundaries form between swarming colonies of different P. mirabilis strains but not between colonies of a single strain. A fundamental requirement for boundary formation is the ability to discriminate between self and nonself. We have isolated mutants that form boundaries with their parent. The mutations map within a six-gene locus that we term ids for identification of self. Five of the genes in the ids locus are required for recognition of the parent strain as self. Three of the ids genes are interchangeable between strains, and two encode specific molecular identifiers.

Translocation of ExsE into Chinese hamster ovary cells is required for transcriptional induction of the Pseudomonas aeruginosa type III secretion system.

Transcription of the Pseudomonas aeruginosa type III secretion system (T3SS) is induced under Ca(2+)-limiting growth conditions or following the contact of the bacteria with host cells. The regulatory response to low Ca(2+) levels is initiated by the T3SS-mediated secretion of ExsE, a negative regulatory protein that prevents T3SS gene transcription. In the present study, we demonstrated that ExsE plays an analogous role in transcriptional induction following host cell contact. By using a flow cytometry assay, the host contact-dependent induction of T3SS gene expression was found to be dependent upon the presence of functional type III translocation machinery. Using three independent assays, we demonstrated that ExsE was translocated into Chinese hamster ovary cells in a T3SS-dependent manner. Deletion mapping experiments indicated that the amino terminus of ExsE is required both for secretion under Ca(2+)-limiting growth conditions and for translocation into host cells. A P. aeruginosa mutant expressing an exsE allele lacking codons 3 through 20 was deficient in ExsE secretion and translocation and showed constitutive repression of T3SS gene expression under Ca(2+)-limiting growth conditions. The mutant also failed to induce T3SS gene expression following host cell contact and demonstrated a significant reduction in T3SS-dependent cytotoxicity towards Chinese hamster ovary cells, indicating that the translocation of ExsE is required for the host contact-dependent induction of T3SS gene expression.

A secreted regulatory protein couples transcription to the secretory activity of the Pseudomonas aeruginosa type III secretion system.

The type III secretion system (T3SS) of Pseudomonas aeruginosa is an important virulence determinant. Transcription of the T3SS is highly regulated and intimately coupled to the activity of the type III secretion channel. The secretion channel is generally closed, and transcription is repressed. Inducing signals such as calcium depletion, however, open the secretion channel and derepress transcription of the T3SS. The coupling of transcription with secretion requires three previously identified cytoplasmic regulatory proteins. ExsA is a DNA-binding protein required for transcriptional activation of the entire T3SS. The second regulatory protein, ExsD, functions as anti-activator by directly binding to ExsA. Finally, ExsC functions as an anti-anti-activator by directly binding to and inhibiting ExsD. Although the regulatory roles of ExsC, ExsD, and ExsA were defined through these previous studies, the mechanism of coupling transcription to secretion was unclear. We now report the identification of ExsE as a secreted regulator of the T3SS and provide evidence that ExsE functions as a direct inhibitor of ExsC. When the secretion channel is closed, ExsE is complexed with ExsC in the cytoplasm, and transcription of the T3SS is repressed by sequestration of ExsA by ExsD. We propose that the secretion of ExsE provides an initiating signal that results in an equilibrium shift whereby ExsC becomes preferentially bound to ExsD, thus allowing liberated ExsA to activate transcription of the T3SS. The presence of ExsE homologs in the T3SSs of other bacterial species suggests that this mechanism of coupling transcription to secretion may be commonly used.

GcvA binding site 1 in the gcvTHP promoter of Escherichia coli is required for GcvA-mediated repression but not for GcvA-mediated activation.

GcvA binds to three sites in the gcvTHP control region, from base -34 to -69 (site 1), from base -214 to -241 (site 2) and from base -242 to -271 (site 3). Previous results suggested that sites 3 and 2 are required for both GcvA-dependent activation and repression of a gcvT::lacZ fusion. However, the results were less clear as to the role of site 1. To determine the role of site 1 in regulation, single and multiple base changes were made in site 1 and tested for their ability to alter GcvA-mediated activation and GcvA/GcvR-mediated repression. Several of the mutants were also tested for effects on GcvA binding to site 1 and the ability of GcvA to bend DNA at site 1. The results are consistent with site 1 playing primarily a role in negative regulation of the gcvTHP operon.

The quorum sensing negative regulators EsaR and ExpR(Ecc), homologues within the LuxR family, retain the ability to function as activators of transcription.

Most LuxR homologues function as activators of transcription during the process of quorum sensing, but a few, including EsaR and ExpR(Ecc), negatively impact gene expression. The LuxR-activated luxI promoter and LuxR binding site, the lux box, were used in artificial contexts to assess the potential for transcriptional activation and DNA binding by EsaR and ExpR(Ecc). Although the acyl-homoserine lactone responsiveness of both proteins is the opposite of that shown by most LuxR family members, EsaR and ExpR(Ecc) have preserved the ability to interact with RNA polymerase and activate transcription despite their low affinity for the lux box DNA.