SummaryThe transcriptional regulator MvfR is required for full Pseudomonas aeruginosa virulence, the function of multiple quorum sensing (QS)-regulated virulence factors and the synthesis of 4-hydroxy-2-alkylquinolines (HAQs), including the Pseudomonas quinolone signal (PQS). Here we investigate the role of MvfR in the QS circuitry and P. aeruginosa pathogenesis. We demonstrate using a combination of biochemical and molecular approaches, including transcription profiling, that MvfR is involved in the regulation of multiple P. aeruginosa QS-controlled genes without altering the expression of lasRI / rhlRI or the production of Nacyl-L -homoserine lactone (AHL) signals. Dissection of how mvfR is interwoven into the P. aeruginosa QS circuitry reveals that the MvfR system, through the essential contribution of PqsE, positively regulates a subset of genes dependant on both LasR and RhlR. Animal studies show that MvfR contributes to P. aeruginosa virulence by controlling the transcription of genes not under RhlR regulation, and that reduced virulence of a mvfR mutant is caused by the loss of pqsE expression and not only a deficiency in HAQs/PQS production. This study provides novel insights into the unique role of the MvfR system in AHL-mediated QS and further supports its importance in P. aeruginosa pathogenesis.
SummaryMvfR (PqsR), a Pseudomonas aeruginosa LysR-type transcriptional regulator, plays a critical role in the virulence of this pathogen. MvfR modulates the expression of multiple quorum sensing (QS)-regulated virulence factors; and the expression of the phnAB and pqsA-E genes that encode functions mediating 4-hydroxy-2-alkylquinolines (HAQs) signalling compounds biosynthesis, including 3,4-dihydroxy-2-heptylquinoline (PQS) and its precursor 4-hydroxy-2-heptylquinoline (HHQ). PQS enhances the in vitro DNA-binding affinity of MvfR to the pqsA-E promoter, to suggest it might function as the in vivo MvfR ligand. Here we identify a novel MvfR ligand, as we show that HHQ binds to the MvfR ligand-binding-domain and potentiates MvfR binding to the pqsA-E promoter leading to transcriptional activation of pqsA-E genes. We show that HHQ is highly produced in vivo, where it is not fully converted into PQS, and demonstrate that it is required for MvfR-dependent gene expression and pathogenicity; PQS is fully dispensable, as pqsHmutant cells, which produce HHQ but completely lack PQS, display normal MvfR-dependent gene expression and virulence. Conversely, PQS is required for full production of pyocyanin. These results uncover a novel biological role for HHQ; and provide novel insights on MvfR activation that may aid in the development of therapies that prevent or treat P. aeruginosa infections in humans.
There is now substantial evidence that compounds released during host stress directly activate the virulence of certain opportunistic pathogens. Here, we considered that endogenous opioids might function as such compounds, given that they are among the first signals to be released at multiple tissue sites during host stress. We tested the ability of various opioid compounds to enhance the virulence of Pseudomonas aeruginosa using pyocyanin production as a biological readout, and demonstrated enhanced virulence when P. aeruginosa was exposed to synthetic (U-50,488) and endogenous (dynorphin) κ-agonists. Using various mutants and reporter strains of P. aeruginosa, we identified involvement of key elements of the quorum sensing circuitry such as the global transcriptional regulator MvfR and the quorum sensing-related quinolone signaling molecules PQS, HHQ, and HQNO that respond to κ-opioids. The in vivo significance of κ-opioid signaling of P. aeruginosa was demonstrated in mice by showing that dynorphin is released from the intestinal mucosa following ischemia/reperfusion injury, activates quinolone signaling in P. aeruginosa, and enhances the virulence of P. aeruginosa against Lactobacillus spp. and Caenorhabditis elegans. Taken together, these data demonstrate that P. aeruginosa can intercept opioid compounds released during host stress and integrate them into core elements of quorum sensing circuitry leading to enhanced virulence.
The LysR-type transcriptional regulator MvfR (PqsR) (multiple virulence factor regulator) plays a critical role in Pseudomonas aeruginosa pathogenicity via the transcriptional regulation of multiple quorum-sensing (QS)-regulated virulence factors. LasR activates full mvfR transcription, and MvfR subsequently activates pqsA-E expression. This study identifies and characterizes the key cis-regulatory elements through which mvfR and pqsA-E transcription is regulated in the highly virulent P. aeruginosa strain PA14. Deletion and site-directed mutagenesis indicate that: (1) LasR activates mvfR transcription by binding to a las/rhl box, CTAACAAAAGACATAG, centred at "513 bp upstream of the MvfR translational start site; and (2) RhlR represses pqsA transcription by binding to a las/rhl box, CTGTGAGATTTGGGAG, centred at "311 bp upstream of the pqsA transcriptional initiation site. Furthermore, it is shown that MvfR activates pqsA-E transcription by binding to a LysR box, TTCGGACTCCGAA, centred at "45 bp relative to the pqsA transcriptional initiation site, demonstrating that this LysR box has a critical role in the physical interaction between the MvfR protein and the pqsA promoter. These results provide new insights into the regulatory relationships between LasR and mvfR, and between MvfR/RhlR and the pqs operon, and elucidate further the complex regulation of the P. aeruginosa QS circuitry.
A significant number of environmental microorganisms can cause serious, even fatal, acute and chronic infections in humans. The severity and outcome of each type of infection depends on the expression of specific bacterial phenotypes controlled by complex regulatory networks that sense and respond to the host environment. Although bacterial signals that contribute to a successful acute infection have been identified in a number of pathogens, the signals that mediate the onset and establishment of chronic infections have yet to be discovered. We identified a volatile, low molecular weight molecule, 2-amino acetophenone (2-AA), produced by the opportunistic human pathogen Pseudomonas aeruginosa that reduces bacterial virulence in vivo in flies and in an acute mouse infection model. 2-AA modulates the activity of the virulence regulator MvfR (multiple virulence factor regulator) via a negative feedback loop and it promotes the emergence of P. aeruginosa phenotypes that likely promote chronic lung infections, including accumulation of lasR mutants, long-term survival at stationary phase, and persistence in a Drosophila infection model. We report for the first time the existence of a quorum sensing (QS) regulated volatile molecule that induces bistability phenotype by stochastically silencing acute virulence functions in P. aeruginosa. We propose that 2-AA mediates changes in a subpopulation of cells that facilitate the exploitation of dynamic host environments and promote gene expression changes that favor chronic infections.
Pathogenic bacteria use interconnected multi-layered regulatory networks, such as quorum sensing (QS) networks to sense and respond to environmental cues and external and internal bacterial cell signals, and thereby adapt to and exploit target hosts. Despite the many advances that have been made in understanding QS regulation, little is known regarding how these inputs are integrated and processed in the context of multi-layered QS regulatory networks. Here we report the examination of the Pseudomonas aeruginosa QS 4-hydroxy-2-alkylquinolines (HAQs) MvfR regulatory network and determination of its interaction with the QS acyl-homoserine-lactone (AHL) RhlR network. The aim of this work was to elucidate paradigmatically the complex relationships between multi-layered regulatory QS circuitries, their signaling molecules, and the environmental cues to which they respond. Our findings revealed positive and negative homeostatic regulatory loops that fine-tune the MvfR regulon via a multi-layered dependent homeostatic regulation of the cell-cell signaling molecules PQS and HHQ, and interplay between these molecules and iron. We discovered that the MvfR regulon component PqsE is a key mediator in orchestrating this homeostatic regulation, and in establishing a connection to the QS rhlR system in cooperation with RhlR. Our results show that P. aeruginosa modulates the intensity of its virulence response, at least in part, through this multi-layered interplay. Our findings underscore the importance of the homeostatic interplay that balances competition within and between QS systems via cell-cell signaling molecules and environmental cues in the control of virulence gene expression. Elucidation of the fine-tuning of this complex relationship offers novel insights into the regulation of these systems and may inform strategies designed to limit infections caused by P. aeruginosa and related human pathogens.
The expression of gene products in bacteria can be inhibited by the use of RNA external guide sequences (EGSs) that hybridize to a target mRNA. Endogenous RNase P cleaves the mRNA in the complex, making it inactive. EGSs participate in this biochemical reaction as the data presented here show. They promote mRNA cleavage at the expected site and sometimes at other secondary sites. Higher-order structure must affect these reactions if the cleavage does not occur at the defined site, which has been determined by techniques based on their ability to find sites that are accessible to the EGS oligonucleotides. Sites defined by a random EGS technique occur as expected. Oligonucleotides made up primarily of defined or random nucleotides are extremely useful in inhibiting expression of the gyrA and rnpA genes from several different bacteria or the cat gene that determines resistance to chloramphenicol in Escherichia coli. An EGS made up of a peptidephosphorodiamidate morpholino oligonucleotide (PPMO) does not cleave at the same site as an unmodified RNA EGS for reasons that are only partly understood. However, PPMO-EGSs are useful in inhibiting the expression of targeted genes from Gram-negative and Gram-positive organisms during ordinary growth in broth and may provide a basis for broad-spectrum antibiotics.drug resistance ͉ Gram-positive and Gram-negative bacteria ͉ peptide-phosphorodiamidate morpholino oligonucleotide ͉ RNase P T he utility of bacterial transformation for therapeutic purposes has been limited by the number of species that will undergo transformation and the frequency with which that event happens. To accommodate new therapies that involve small nucleic acids, a means has to be developed to enable bacterial species to take up these nucleic acids with relative ease. The covalent linkage of arginine-rich peptides to the ends of chemically-modified RNAs facilitates the uptake of the RNA analog (1, 2) and other similar molecules (4,5). This methodology in combination with an effective means of inactivating gene expression has to be developed to make it useful for therapeutic agents. There are other processes that function in bacteria to inhibit gene expression (3, 6), but the external guide sequence (EGS) technology (7,8) that is mediated by RNase P cleavage of the target RNA seems optimal in this regard.RNAi and siRNA (ref. 9 and references therein) are not useful tools for the transformation of bacterial species because these RNAs rely on an intracellular complex, the Dicer complex (9) in particular, to release ssRNA that will base-pair with the target mRNA. EGS technology, which is just as effective as siRNA in mammalian cells in tissue culture (10), is very effective in Escherichia coli (11,12) and Salmonella typhimurium (13). Bacterial cells can be altered from drug resistance to drug sensitivity with the methods generally described here (11), and a similar method has also been reported (14). Essential genes can also be inactivated in terms of their expression. The EGS method will allow 3-bp mismatche...
A method of inhibiting the expression of particular genes by using external guide sequences (EGSs) has been improved in its rapidity and specificity. Random EGSs that have 14-nt random sequences are used in the selection procedure for an EGS that attacks the mRNA for a gene in a particular location. A mixture of the random EGSs, the particular target RNA, and RNase P is used in the diagnostic procedure, which, after completion, is analyzed in a gel with suitable control lanes. Within a few hours, the procedure is complete. The action of EGSs designed by an older method is compared with EGSs designed by the random EGS method on mRNAs from two bacterial pathogens.S election of accessible sites in target RNAs is critical for efficient mRNA inactivation strategies. Many of the target sequences in cellular RNAs are inaccessible because of the secondary or tertiary structures of the RNA or the binding of proteins to the target RNA in vivo. Several approaches for the mapping of accessible sites in target RNAs have been reported. These approaches range from analyses in silico to cleavage by complex ribozyme constructs (e.g., 1-6), but they are timeconsuming and are not reliable in terms of their efficacy.In Gram-negative bacteria, down-regulation of gene expression at the RNA level has been achieved by directing external guide sequences (EGSs) to pair with complementary regions in mRNAs from individual genes. The EGS will hydrogen-bond to the target RNA and generate an RNA-RNA stem structure mimicking the natural precursor tRNA (ptRNA) cleavage site (7-9). The target RNA in the complex is cleaved by RNase P (10-14).EGSs have been designed to successfully alter several bacterial phenotypes (11)(12)(13)(14). In mammalian cells, RNase P RNA has been specifically directed to destroy tumor-specific fusion genes created as a result of chromosome abnormalities (15) and to inhibit viral gene expression and growth in cell cultures (16,17).Here we describe a rapid method to map directly accessible and cleavable sites in target RNA by the Escherichia coli RNase P holoenzyme and a random EGS (rEGS) library. ResultsDesign of the rEGSe and rEGSx Libraries. The rEGSe and rEGSx libraries were constructed by using a partially randomized oligonucleotide as a template for PCR (Fig. 1). This oligo contains the T7 promoter sequence upstream of the random 14-nt sequence (N 14 ). The length of the randomized region could be varied, but 14 nts were selected because 13-to 16-nt EGSs were shown to work well in previous studies in bacteria (10)(11)(12)(13)(14). In addition, the oligonucleotide contains a cytosine that will base pair to a guanosine in the target RNA sequence and a 3Ј-ACCA sequence. A guanosine is the preferred nucleotide immediately 3Ј of the natural ptRNA cleavage site, and a 3Ј-ACCA sequence in ptRNA is important for cleavage in vivo by E. coli RNase P. The only difference between the rEGSe and rEGSx libraries is that the former contains all of the nucleotides after the BstNI site (Fig. 1), whereas the latter was digested by Bst...
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