Regulatory RNAs and the HptB/RetS signalling pathways fine-tune Pseudomonas aeruginosa pathogenesis.

Bacterial pathogenesis often depends on regulatory networks, two-component systems and small RNAs (sRNAs). In Pseudomonas aeruginosa, the RetS sensor pathway downregulates expression of two sRNAs, rsmY and rsmZ. Consequently, biofilm and the Type Six Secretion System (T6SS) are repressed, whereas the Type III Secretion System (T3SS) is activated. We show that the HptB signalling pathway controls biofilm and T3SS, and fine-tunes P. aeruginosa pathogenesis. We demonstrate that RetS and HptB intersect at the GacA response regulator, which directly controls sRNAs production. Importantly, RetS controls both sRNAs, whereas HptB exclusively regulates rsmY expression. We reveal that HptB signalling is a complex regulatory cascade. This cascade involves a response regulator, with an output domain belonging to the phosphatase 2C family, and likely an anti-anti-sigma factor. This reveals that the initial input in the Gac system comes from several signalling pathways, and the final output is adjusted by a differential control on rsmY and rsmZ. This is exemplified by the RetS-dependent but HptB-independent control on T6SS. We also demonstrate a redundant action of the two sRNAs on T3SS gene expression, while the impact on pel gene expression is additive. These features underpin a novel mechanism in the fine-tuned regulation of gene expression.

A distinct QscR regulon in the Pseudomonas aeruginosa quorum-sensing circuit.

The opportunistic pathogen Pseudomonas aeruginosa possesses two complete acyl-homoserine lactone (acyl-HSL) signaling systems. One system consists of LasI and LasR, which generate a 3-oxododecanoyl-homoserine lactone signal and respond to that signal, respectively. The other system is RhlI and RhlR, which generate butanoyl-homoserine lactone and respond to butanoyl-homoserine lactone, respectively. These quorum-sensing systems control hundreds of genes. There is also an orphan LasR-RhlR homolog, QscR, for which there is no cognate acyl-HSL synthetic enzyme. We previously reported that a qscR mutant is hypervirulent and showed that QscR transiently represses a few quorum-sensing-controlled genes. To better understand the role of QscR in P. aeruginosa gene regulation and to better understand the relationship between QscR, LasR, and RhlR control of gene expression, we used transcription profiling to identify a QscR-dependent regulon. Our analysis revealed that QscR activates some genes and represses others. Some of the repressed genes are not regulated by the LasR-I or RhlR-I systems, while others are. The LasI-generated 3-oxododecanoyl-homoserine lactone serves as a signal molecule for QscR. Thus, QscR appears to be an integral component of the P. aeruginosa quorum-sensing circuitry. QscR uses the LasI-generated acyl-homoserine lactone signal and controls a specific regulon that overlaps with the already overlapping LasR- and RhlR-dependent regulons.

Multiple sensors control reciprocal expression of Pseudomonas aeruginosa regulatory RNA and virulence genes.

The opportunistic pathogen Pseudomonas aeruginosa is responsible for a wide range of acute and chronic infections. The transition to chronic infections is accompanied by physiological changes in the bacteria favoring formation of biofilm communities. Here we report the identification of LadS, a hybrid sensor kinase that controls the reciprocal expression of genes for type III secretion and biofilm-promoting polysaccharides. Domain organization of LadS and the range of LadS-controlled genes suggest that it counteracts the activities of another sensor kinase, RetS. These two pathways converge by controlling the transcription of a small regulatory RNA, RsmZ. This work identifies a previously undescribed signal transduction network in which the activities of signal-receiving sensor kinases LadS, RetS, and GacS regulate expression of virulence genes associated with acute or chronic infection by transcriptional and posttranscriptional mechanisms.

Quorum sensing negatively controls type III secretion regulon expression in Pseudomonas aeruginosa PAO1.

A systematic analysis of the type III secretion (T3S) genes of Pseudomonas aeruginosa strain PAO1 revealed that they are under quorum-sensing control. This observation was supported by the down-regulation of the T3S regulon in the presence of RhlR-C4HSL and the corresponding advanced secretion of ExoS in a rhlI mutant.

A novel two-component system controls the expression of Pseudomonas aeruginosa fimbrial cup genes.

Biofilm formation by the opportunistic pathogen Pseudomonas aeruginosa requires the expression of a number of surface adhesive components. The expression of surface organelles facilitating biofilm formation is controlled by environmental signals acting through transcriptional regulatory networks. We analysed the expression of a family of P. aeruginosa adhesins encoded by three distinct fimbrial gene clusters (cupA, cupB and cupC). Using transposon mutagenesis, we have identified several regulatory loci that upregulated cupB and cupC transcription. One such locus contains three components, RocS1, RocR and RocA1, which represent a variant of a classical two-component signal transduction pathway. RocS1 is a sensor kinase, RocA1 is a DNA binding response regulator that activates cup genes, and RocR is an antagonist of RocA1 activity. Using a two-hybrid assay, we have shown that RocS1 interacts with receiver domains of both RocA1 and RocR. Expression of the Cup system in response to environmental stimuli is accomplished by a novel mechanism in which the sensor kinase activates its cognate response regulator through a phosphorelay pathway, while an additional repressor protein modulates this interaction.

Biofilm formation in Pseudomonas aeruginosa: fimbrial cup gene clusters are controlled by the transcriptional regulator MvaT.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen which poses a major threat to long-term-hospitalized patients and individuals with cystic fibrosis. The capacity of P. aeruginosa to form biofilms is an important requirement for chronic colonization of human tissues and for persistence in implanted medical devices. Various stages of biofilm formation by this organism are mediated by extracellular appendages, such as type IV pili and flagella. Recently, we identified three P. aeruginosa gene clusters that were termed cup (chaperone-usher pathway) based on their sequence relatedness to the chaperone-usher fimbrial assembly pathway in other bacteria. The cupA gene cluster, but not the cupB or cupC cluster, is required for biofilm formation on abiotic surfaces. In this study, we identified a gene (mvaT) encoding a negative regulator of cupA expression. Such regulatory control was confirmed by several approaches, including lacZ transcriptional fusions, Northern blotting, and transcriptional profiling using DNA microarrays. MvaT also represses the expression of the cupB and cupC genes, although the extent of the regulatory effect is not as pronounced as with cupA. Consistent with this finding, mvaT mutants exhibit enhanced biofilm formation. Although the P. aeruginosa genome contains a highly homologous gene, mvaU, the repression of cupA genes is MvaT specific. Thus, MvaT appears to be an important regulatory component within a complex network that controls biofilm formation and maturation in P. aeruginosa.

Type II protein secretion in Pseudomonas aeruginosa: the pseudopilus is a multifibrillar and adhesive structure.

The type II secretion pathway of Pseudomonas aeruginosa is involved in the extracellular release of various toxins and hydrolytic enzymes such as exotoxin A and elastase. This pathway requires the function of a macromolecular complex called the Xcp secreton. The Xcp secreton shares many features with the machinery involved in type IV pilus assembly. More specifically, it involves the function of five pilin-like proteins, the XcpT-X pseudopilins. We show that, upon overexpression, the XcpT pseudopilin can be assembled in a pilus, which we call a type II pseudopilus. Image analysis and filtering of electron micrographs indicated that these appendages are composed of individual fibrils assembled together in a bundle structure. Our observations thus revealed that XcpT has properties similar to those of type IV pilin subunits. Interestingly, the assembly of the type II pseudopilus is not exclusively dependent on the Xcp machinery but can be supported by other similar machineries, such as the Pil (type IV pilus) and Hxc (type II secretion) systems of P. aeruginosa. In addition, heterologous pseudopilins can be assembled by P. aeruginosa into a type II pseudopilus. Finally, we showed that assembly of the type II pseudopilus confers increased bacterial adhesive capabilities. These observations confirmed the ability of pseudopilins to form a pilus structure and raise questions with respect to their function in terms of secretion and adhesion, two crucial biological processes in the course of bacterial infections.

Global regulation in Pseudomonas aeruginosa: the regulatory protein AlgR2 (AlgQ) acts as a modulator of quorum sensing.

The Pseudomonas aeruginosa protein AlgR2 (AlgQ) was originally identified as a regulatory protein implicated in alginate production. It also regulates the synthesis of polyphosphate as well as of a variety of secretable virulence factors, upregulating neuraminidase and siderophore synthesis and downregulating rhamnolipid biosurfactant and extracellular protease synthesis. In this study, we show that the regulatory effect of AlgR2 on elastase protease synthesis is exerted at transcriptional level on the lasB gene. We also demonstrate that AlgR2 negatively modulates the expression of quorum sensing regulatory genes lasR and rhlR. Finally, results obtained from DNA retardation assays provide evidence that AlgR2 can bind specifically to the lasR and rhlR promoters. Altogether, these data provide strong support for the hypothesis that AlgR2 is a global transcriptional regulator in P. aeruginosa.

Dimerization of the quorum sensing regulator RhlR: development of a method using EGFP fluorescence anisotropy.

Of considerable interest in the biology of pathogenic bacteria are the mechanisms of intercellular signalling that can lead to the formation of persistent infections. In this article, we have examined the intracellular behaviour of a Pseudomonas aeruginosa quorum sensing regulator RhlR believed to be important in this process. We have further examined the modulation of this behaviour in response to various auto-inducers. For these measurements, we have developed an assay based on the fluorescence anisotropy of EGFP fusion proteins that we use to measure protein-protein interactions in vivo. We show that the transcriptional regulator, RhlR, expressed as an EGFP fusion protein in Escherichia coli, forms a homodimer. This homodimer can be dissociated into monomers by the auto-inducer N-(3-oxododecanoyl)-l-homoserine lactone (3O-C12-HSL) whereas N-(butanoyl)-l-homoserine lactone (C4-HSL) has little effect. These observations are of particular interest as RhlR modulation of gene expression depends on the presence of C4-HSL, whereas 3O-C12-HSL modulates the expression of genes regulated by LasR. These observations thus provide a framework for understanding the regulatory network that links the various different QS regulators in P. aeruginosa. Furthermore, the technique we have developed should permit the study of numerous protein/protein or protein/nucleic acid interactions in vivo and so shed light on natural protein function.

Interactions of the quorum sensing regulator QscR: interaction with itself and the other regulators of Pseudomonas aeruginosa LasR and RhlR.

Pseudomonas aeruginosa controls the production of many exoproteins and secondary metabolites via a hierarchical quorum sensing (QS) regulatory cascade involving the LuxR-like proteins LasR, RhlR and their cognate signal molecules N-(3-oxododecanoyl)-l-homoserine lactone (3O-C12-HSL) and N-(butanoyl)-l-homoserine lactone (C4-HSL). The finding of a third LuxR-type protein in P. aeruginosa, QscR, adds further complexity to this regulatory network. It has been shown previously that QscR represses transcription of three QS-controlled gene clusters, phz (phenazine), hcn (hydrogen cyanide) and qsc105 (Chugani, Whiteley, Lee, D'Argenio, Manoil, and Greenberg, 2001, Proc Natl Acad Sci USA 98: 2752-2757). In this study, we identify two novel QscR targets these are lasB, encoding the extracellular elastase, and the second phenazine gene cluster, both of which are downregulated by QscR. In addition, we show that QscR synthesis is regulated by the two-component response regulator GacA. Taking advantage of the in vivo fluorescence anisotropy technology that we have developed, we show that QscR can be found in several different types of association. Indeed, we identify QscR multimers in the absence of any acyl-HSL, lower order QscR oligomers associated either with C4-HSL or 3O-C12-HSL and QscR-containing heterodimers with LasR or RhlR. The formation of heterodimers between QscR and LasR or RhlR, in the absence of acyl-HSLs, is a very exciting, new result that should improve our understanding of the QscR network and its relationship to the production of P. aeruginosa virulence factors.

Identification of XcpP domains that confer functionality and specificity to the Pseudomonas aeruginosa type II secretion apparatus.

Gram-negative bacteria have evolved several types of secretion mechanisms to release proteins into the extracellular medium. One such mechanism, the type II secretory system, is a widely conserved two-step process. The first step is the translocation of signal peptide-bearing exoproteins across the inner membrane. The second step, the translocation across the outer membrane, involves the type II secretory apparatus or secreton. The secretons are made up of 12-15 proteins (Gsp) depending on the organism. Even though the systems are conserved, heterologous secretion is mostly species restricted. Moreover, components of the secreton are not systematically exchangeable, especially with distantly related microorganisms. In closely related species, two components, the GspC and GspD (secretin) family members, confer specificity for substrate recognition and/or secreton assembly. We used Pseudomonas aeruginosa as a model organism to determine which domains of XcpP (GspC member) are involved in specificity. By constructing hybrids between XcpP and OutC, the Erwinia chrysanthemi homologue, we identified a region of 35 residues that was not exchangeable. We showed that this region might influence the stability of the XcpYZ secreton subcomplex. Remarkably, XcpP and OutC have domains, coiled-coil and PDZ, respectively, which exhibit the same function but that are structurally different. Those two domains are exchangeable and we provided evidence that they are involved in the formation of homomultimeric complexes of XcpP.

A novel type II secretion system in Pseudomonas aeruginosa.

The genome sequence of Pseudomonas aeruginosa strain PAO1 has been determined to facilitate postgenomic studies aimed at understanding the capacity of adaptation of this ubiquitous opportunistic pathogen. P. aeruginosa produces toxins and hydrolytic enzymes that are secreted via the type II secretory pathway using the Xcp machinery or 'secreton'. In this study, we characterized a novel gene cluster, called hxc for homologous to xcp. Characterization of an hxcR mutant, grown in phosphate-limiting medium, revealed the absence of a 40 kDa protein found in the culture supernatant of wild-type or xcp derivative mutant strains. The protein corresponded to the alkaline phosphatase L-AP, renamed LapA, which is secreted in an xcp-independent but hxc-dependent manner. Finally, we showed that expression of the hxc gene cluster is under phosphate regulation. This is the first report of the existence of two functional type II secretory pathways within the same organism, which could be related to the high adaptation potential of P. aeruginosa.

Advancing the quorum in Pseudomonas aeruginosa: MvaT and the regulation of N-acylhomoserine lactone production and virulence gene expression.

Pseudomonas aeruginosa regulates the production of many exoproteins and secondary metabolites via a hierarchical quorum-sensing cascade through LasR and RhlR and their cognate signal molecules N-(3-oxododecanoyl)-L-homoserine lactone (3O-C12-HSL) and N-(butanoyl)-L-homoserine lactone (C4-HSL). In this study, we found that transcription of the quorum sensing-regulated genes lecA (coding for PA-IL lectin), lasB (coding for elastase), and rpoS appeared to be growth phase dependent and their expression could not be advanced to the logarithmic phase in cells growing in batch culture by the addition of exogenous C4-HSL and 3O-C12-HSL. To identify novel regulators responsible for this growth phase dependency, a P. aeruginosa lecA::lux reporter strain was subjected to random transposon mutagenesis. A number of mutants affected in lecA expression were found that exhibited altered production of multiple quorum sensing-dependent phenotypes. While some mutations were mapped to new loci such as clpA and mvaT and a putative efflux system, a number of mutations were also mapped to known regulators such as lasR, rhlR, and rpoS. MvaT was identified as a novel global regulator of virulence gene expression, as a mutation in mvaT resulted in enhanced lecA expression and pyocyanin production. This mutant also showed altered swarming ability and production of the LasB and LasA proteases, 3O-C12-HSL, and C4-HSL. Furthermore, addition of exogenous 3O-C12-HSL and C4-HSL to the mvaT mutant significantly advanced lecA expression, suggesting that MvaT is involved in the growth phase-dependent regulation of the lecA gene.

Identification of XcpZ domains required for assembly of the secreton of Pseudomonas aeruginosa.

Most of the exoproteins secreted by Pseudomonas aeruginosa are transported via the type II secretion system. This machinery, which is widely conserved in gram-negative bacteria, consists of 12 Xcp proteins organized as a multiprotein complex, also called the secreton. We previously reported that the mutual stabilization of XcpZ and XcpY plays an important role in the assembly of the secreton. In this study, we engineered variant XcpZ proteins by using linker insertion mutagenesis. We identified three distinct regions of XcpZ required for both the stabilization of XcpY and the functionality of the secreton. Interestingly, we also demonstrated that another component of the machinery, XcpP, can modulate the stabilizing activity of XcpZ on XcpY.

RpoS-dependent stress tolerance in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is able to persist during feast and famine in many different environments including soil, water, plants, animals and humans. The alternative sigma factor encoded by the rpoS gene is known to be important for survival under stressful conditions in several other bacterial species. To determine if the P. aeruginosa RpoS protein plays a similar role in stationary-phase-mediated resistance, an rpoS mutant was constructed and survival during exposure to hydrogen peroxide, high temperature, hyperosmolarity, low pH and ethanol was investigated. Disruption of the rpoS gene resulted in a two- to threefold increase in the rate of kill of stationary-phase cells. The rpoS mutant also survived less well than the parental strain during the initial phase of carbon or phosphate-carbon starvation. However, after 25 d starvation the remaining population of culturable cells was not significantly different. Stationary-phase cells of the RpoS-negative strain were much more stress resistant than exponentially growing RpoS-positive cells, suggesting that factors other than the RpoS protein must be associated with stationary-phase stress tolerance in P. aeruginosa. Comparison of two-dimensional PAGE of the rpoS mutant and the parental strain showed four major modifications of protein patterns associated with the rpoS mutation.

Mutual stabilization of the XcpZ and XcpY components of the secretory apparatus in Pseudomonas aeruginosa.

Protein secretion in gram-negative bacteria is often dependent on the general secretory pathway (GSP). In Pseudomonas aeruginosa, this system requires at least 12 Xcp (Gsp) proteins, which are proposed to constitute a multiprotein complex localized in the bacterial envelope. Hitherto, little was known about the mutual interactions between Xcp proteins. In this study, mutants affected in the xcpZ gene encoding a bitopic inner-membrane protein were analysed to investigate the role of this protein in the architecture of the secretory machinery. The absence of XcpZ resulted in a decreased amount of XcpY. Reciprocally, XcpZ was not detectable in a xcpY mutant, demonstrating a mutual stabilization of these two proteins. These results strongly suggest that XcpZ and XcpY interact within the functional secretory apparatus.