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The Pseudomonas aeruginosa PrrF1 and PrrF2 Small Regulatory RNAs Promote 2-Alkyl-4-Quinolone Production through Redundant Regulation of the antR mRNA.

ABSTRACT: Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that requires iron for growth and virulence. Under low-iron conditions, P. aeruginosa transcribes two highly identical (95%) small regulatory RNAs (sRNAs), PrrF1 and PrrF2, which are required for virulence in acute murine lung infection models. The PrrF sRNAs promote the production of 2-akyl-4(1H)-quinolone metabolites (AQs) that mediate a range of biological activities, including quorum sensing and polymicrobial interactions. Here, we show that the PrrF1 and PrrF2 sRNAs promote AQ production by redundantly inhibiting translation of antR, which encodes a transcriptional activator of the anthranilate degradation genes. A combination of genetic and biophysical analyses was used to define the sequence requirements for PrrF regulation of antR, demonstrating that the PrrF sRNAs interact with the antR 5' untranslated region (UTR) at sequences overlapping the translational start site of this mRNA. The P. aeruginosa Hfq protein interacted with UA-rich sequences in both PrrF sRNAs (Kd [dissociation constant] = 50 nM and 70 nM). Hfq bound with lower affinity to the antR mRNA (0.3 ?M), and PrrF was able to bind to antR mRNA in the absence of Hfq. Nevertheless, Hfq increased the rate of PrrF annealing to the antR UTR by 10-fold. These studies provide a mechanistic description of how the PrrF1 and PrrF2 sRNAs mediate virulence traits, such as AQ production, in P. aeruginosaIMPORTANCE The iron-responsive PrrF sRNAs play a central role in regulating P. aeruginosa iron homeostasis and pathogenesis, yet the molecular mechanisms by which PrrF regulates gene expression are largely unknown. In this study, we used genetic and biophysical analyses to define the interactions of the PrrF sRNAs with Hfq, an RNA annealer, and the antR mRNA, which has downstream effects on quorum sensing and virulence factor production. These studies provide a comprehensive mechanistic analysis of how the PrrF sRNAs regulate virulence trait production through a key mRNA target in P. aeruginosa.

SUBMITTER: Djapgne L

PROVIDER: S-EPMC5915787 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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The Pseudomonas aeruginosa PrrF1 and PrrF2 Small Regulatory RNAs Promote 2-Alkyl-4-Quinolone Production through Redundant Regulation of the antR mRNA.

Djapgne Louise L Panja Subrata S Brewer Luke K LK Gans Jonathan H JH Kane Maureen A MA Woodson Sarah A SA Oglesby-Sherrouse Amanda G AG

Journal of bacteriology 20180424 10

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that requires iron for growth and virulence. Under low-iron conditions, P. aeruginosa transcribes two highly identical (95%) small regulatory RNAs (sRNAs), PrrF1 and PrrF2, which are required for virulence in acute murine lung infection models. The PrrF sRNAs promote the production of 2-akyl-4(1H)-quinolone metabolites (AQs) that mediate a range of biological activities, including quorum sensing and polymicrob ...[more]

PMID: 29507088

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Project description:UnlabelledPseudomonas aeruginosa is a Gram-negative bacterium that is ubiquitous in the environment, and it is an opportunistic pathogen that can infect a variety of hosts, including humans. During the process of infection, P. aeruginosa coordinates the expression of numerous virulence factors through the production of multiple cell-to-cell signaling molecules. The production of these signaling molecules is linked through a regulatory network, with the signal N-(3-oxododecanoyl) homoserine lactone and its receptor LasR controlling the induction of a second acyl-homoserine lactone signal and the Pseudomonas quinolone signal (PQS). LasR-mediated control of PQS occurs partly by activating the transcription of pqsR, a gene that encodes the PQS receptor and is necessary for PQS production. We show that LasR interacts with a single binding site in the pqsR promoter region and that it does not influence the transcription of the divergently transcribed gene, nadA. Using DNA affinity chromatography, we identified additional proteins that interact with the pqsR-nadA intergenic region. These include the H-NS family members MvaT and MvaU, and CysB, a transcriptional regulator that controls sulfur uptake and cysteine biosynthesis. We show that CysB interacts with the pqsR promoter and that CysB represses pqsR transcription and PQS production. Additionally, we provide evidence that CysB can interfere with the activation of pqsR transcription by LasR. However, as seen with other CysB-regulated genes, pqsR expression was not differentially regulated in response to cysteine levels. These findings demonstrate a novel role for CysB in influencing cell-to-cell signal production by P. aeruginosa.ImportanceThe production of PQS and other 4-hydroxy-2-alkylquinolone (HAQs) compounds is a key component of the P. aeruginosa cell-to-cell signaling network, impacts multiple physiological functions, and is required for virulence. PqsR directly regulates the genes necessary for HAQ production, but little is known about the regulation of pqsR. We identified CysB as a novel regulator of pqsR and PQS production, but, unlike other CysB-controlled genes, it does not appear to regulate pqsR in response to cysteine. This implies that CysB functions as both a cysteine-responsive and cysteine-unresponsive regulator in P. aeruginosa.

Project description:The Pseudomonas quinolone signal (PQS) is an important quorum-sensing molecule in Pseudomonas aeruginosa that also mediates its own packaging and transport by stimulating outer membrane vesicle (OMV) formation. Because OMVs have been implicated in many virulence-associated behaviors, it is critical that we understand how they are formed. Our group proposed the bilayer-couple model for OMV biogenesis, where PQS intercalates into the outer membrane, causing expansion of the outer leaflet and consequently inducing curvature. In accordance with the model, we hypothesized that PQS must be transported from the cytoplasm to the outer membrane before it can initiate OMV formation. We initially examined two laboratory strains of P. aeruginosa and found significant strain-dependent differences. PQS export correlated strongly with OMV production, even though equivalent amounts of total PQS were produced by both strains. Interestingly, we discovered that poor OMV producers sequestered the majority of PQS in the inner membrane, which appeared to be the result of early saturation of the export pathway. Further analysis showed that strain-specific PQS export and OMV biogenesis patterns were stable once established but could be significantly altered by changing the growth medium. Finally, we demonstrated that the associations described for laboratory strains also held for three clinical strains. These results suggest that factors controlling the export of PQS dictate OMV biogenesis. This work provides new insight into PQS-controlled virulence in P. aeruginosa and provides important tools to further study signal export and OMV biogenesis.IMPORTANCE Bacterial secretion has been recognized as an essential facet of microbial pathogenesis and human disease. Numerous virulence factors have been found to be transported within outer membrane vesicles (OMVs), and delivery using these biological nanoparticles often results in increased potency. OMV biogenesis is an important but poorly understood process that is ubiquitous among Gram-negative organisms. Our group seeks to understand the biochemical mechanisms behind the formation of OMVs and has developed a model of small-molecule-induced membrane curvature as an important driver of this process. With this work, we demonstrate that PQS, a known small-molecule OMV inducer, must be exported to promote OMV biogenesis in both lab-adapted and clinical strains of Pseudomonas aeruginosa In supporting and expanding the bilayer-couple model of OMV biogenesis, the current work lays the groundwork for studying environmental and genetic factors that modulate OMV production and, consequently, the packaging and delivery of many bacterial factors.

Project description:Pseudomonas aeruginosa is an opportunistic pathogen that is frequently associated with both acute and chronic infections. P. aeruginosa possesses a complex regulatory network that modulates nutrient acquisition and virulence, but our knowledge of these networks is largely based on studies with shaking cultures, which are not likely representative of conditions during infection. Here, we provide proteomic, metabolic, and genetic evidence that regulation by iron, a critical metallonutrient, is altered in static P. aeruginosa cultures. Specifically, we observed a loss of iron-induced expression of proteins for oxidative phosphorylation, tricarboxylic acid (TCA) cycle metabolism under static conditions. Moreover, we identified type VI secretion as a target of iron regulation in P. aeruginosa cells under static but not shaking conditions, and we present evidence that this regulation occurs via PrrF small regulatory RNA (sRNA)-dependent production of 2-alkyl-4(1H)-quinolone metabolites. These results yield new iron regulation paradigms in an important opportunistic pathogen and highlight the need to redefine iron homeostasis in static microbial communities.IMPORTANCE Host-mediated iron starvation is a broadly conserved signal for microbial pathogens to upregulate expression of virulence traits required for successful infection. Historically, global iron regulatory studies in microorganisms have been conducted in shaking cultures to ensure culture homogeneity, yet these conditions are likely not reflective of growth during infection. Pseudomonas aeruginosa is a well-studied opportunistic pathogen and model organism for iron regulatory studies. Iron homeostasis is maintained through the Fur protein and PrrF small regulatory sRNAs, the functions of which are highly conserved in many other bacterial species. In the current study, we examined how static growth affects the known iron and PrrF regulons of P. aeruginosa, leading to the discovery of novel PrrF-regulated virulence processes. This study demonstrates how the utilization of distinct growth models can enhance our understanding of basic physiological processes that may also affect pathogenesis.

Dataset Information

The Pseudomonas aeruginosa PrrF1 and PrrF2 Small Regulatory RNAs Promote 2-Alkyl-4-Quinolone Production through Redundant Regulation of the antR mRNA.

Publications

The Pseudomonas aeruginosa PrrF1 and PrrF2 Small Regulatory RNAs Promote 2-Alkyl-4-Quinolone Production through Redundant Regulation of the <i>antR</i> mRNA.

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