Direct interaction between sensor kinase proteins mediates acute and chronic disease phenotypes in a bacterial pathogen.

The genome of the opportunistic pathogen Pseudomonas aeruginosa encodes over 60 two-component sensor kinases and uses several (including RetS and GacS) to reciprocally regulate the production of virulence factors involved in the development of acute or chronic infections. We demonstrate that RetS modulates the phosphorylation state of GacS by a direct and specific interaction between these two membrane-bound sensors. The RetS-GacS interaction can be observed in vitro, in heterologous systems in vivo, and in P. aeruginosa. This function does not require the predicted RetS phosphorelay residues and provides a mechanism for integrating multiple signals without cross-phosphorylation from sensors to noncognate response regulators. These results suggest that multiple two-component systems found in a single bacterium can form multisensor signaling networks while maintaining specific phosphorelay pathways that remain insulated from detrimental cross-talk.

Two-component regulatory systems in Pseudomonas aeruginosa: an intricate network mediating fimbrial and efflux pump gene expression.

Pseudomonas aeruginosa is responsible for chronic and acute infections in humans. Chronic infections are associated with production of fimbriae and the formation of a biofilm. The two-component system Roc1 is named after its role in the regulation of cup genes, which encode components of a machinery allowing assembly of fimbriae. A non-characterized gene cluster, roc2, encodes components homologous to the Roc1 system. We show that cross-regulation occurs between the Roc1 and Roc2 signalling pathways. We demonstrate that the sensors RocS2 and RocS1 converge on the response regulator RocA1 to control cupC gene expression. This control is independent of the response regulator RocA2. Instead, we show that these sensors act via the RocA2 response regulator to repress the mexAB-oprM genes. These genes encode a multidrug efflux pump and are upregulated in the rocA2 mutant, which is less susceptible to antibiotics. It has been reported that in cystic fibrosis lungs, in which P. aeruginosa adopts the biofilm lifestyle, most isolates have an inactive MexAB-OprM pump. The concomitant RocS2-dependent upregulation of cupC genes (biofilm formation) and downregulation of mexAB-oprM genes (antibiotic resistance) is in agreement with this observation. It suggests that the Roc systems may sense the environment in the cystic fibrosis lung.

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.

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.

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.

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.

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.