scholarly journals TheprrF-Encoded Small Regulatory RNAs Are Required for Iron Homeostasis and Virulence of Pseudomonas aeruginosa

2014 ◽  
Vol 83 (3) ◽  
pp. 863-875 ◽  
Author(s):  
Alexandria A. Reinhart ◽  
Daniel A. Powell ◽  
Angela T. Nguyen ◽  
Maura O'Neill ◽  
Louise Djapgne ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Iron Homeostasis ◽  
Opportunistic Pathogen ◽  
Wild Type ◽  
Content Type ◽  
Genes Encoding ◽  
Small Regulatory Rnas ◽  
Regulatory Rnas ◽  
Heme Regulation ◽  
Heme Binding Protein

Pseudomonas aeruginosais an opportunistic pathogen that requires iron to cause infection, but it also must regulate the uptake of iron to avoid iron toxicity. The iron-responsive PrrF1 and PrrF2 small regulatory RNAs (sRNAs) are part ofP. aeruginosa'siron regulatory network and affect the expression of at least 50 genes encoding iron-containing proteins. The genes encoding the PrrF1 and PrrF2 sRNAs are encoded in tandem inP. aeruginosa, allowing for the expression of a distinct, heme-responsive sRNA named PrrH that appears to regulate genes involved in heme metabolism. Using a combination of growth, mass spectrometry, and gene expression analysis, we showed that the ΔprrF1,2mutant, which lacks expression of the PrrF and PrrH sRNAs, is defective for both iron and heme homeostasis. We also identifiedphuS, encoding a heme binding protein involved in heme acquisition, andvreR, encoding a previously identified regulator ofP. aeruginosavirulence genes, as novel targets ofprrF-mediated heme regulation. Finally, we showed that theprrFlocus encoding the PrrF and PrrH sRNAs is required forP. aeruginosavirulence in a murine model of acute lung infection. Moreover, we showed that inoculation with a ΔprrF1,2deletion mutant protects against future challenge with wild-typeP. aeruginosa. Combined, these data demonstrate that theprrF-encoded sRNAs are critical regulators ofP. aeruginosavirulence.

scholarly journals Transcriptional Response of Mucoid Pseudomonas aeruginosa to Human Respiratory Mucus

mBio ◽  
2012 ◽  
Vol 3 (6) ◽  
Author(s):  
V. Cattoir ◽  
G. Narasimhan ◽  
D. Skurnik ◽  
H. Aschard ◽  
D. Roux ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Opportunistic Pathogen ◽  
Secretion System ◽  
Type Vi Secretion System ◽  
Transcript Levels ◽  
Content Type ◽  
Type Vi Secretion ◽  
Small Regulatory Rnas ◽  
Regulatory Rnas

ABSTRACTAdaptation of bacterial pathogens to a host can lead to the selection and accumulation of specific mutations in their genomes with profound effects on the overall physiology and virulence of the organisms. The opportunistic pathogenPseudomonas aeruginosais capable of colonizing the respiratory tract of individuals with cystic fibrosis (CF), where it undergoes evolution to optimize survival as a persistent chronic human colonizer. The transcriptome of a host-adapted, alginate-overproducing isolate from a CF patient was determined following growth of the bacteria in the presence of human respiratory mucus. This stable mucoid strain responded to a number of regulatory inputs from the mucus, resulting in an unexpected repression of alginate production. Mucus in the medium also induced the production of catalases and additional peroxide-detoxifying enzymes and caused reorganization of pathways of energy generation. A specific antibacterial type VI secretion system was also induced in mucus-grown cells. Finally, a group of small regulatory RNAs was identified and a fraction of these were mucus regulated. This report provides a snapshot of responses in a pathogen adapted to a human host through assimilation of regulatory signals from tissues, optimizing its long-term survival potential.IMPORTANCEThe basis for chronic colonization of patients with cystic fibrosis (CF) by the opportunistic pathogenPseudomonas aeruginosacontinues to represent a challenging problem for basic scientists and clinicians. In this study, the host-adapted, alginate-overproducingPseudomonas aeruginosa2192 strain was used to assess the changes in its transcript levels following growth in respiratory CF mucus. Several significant and unexpected discoveries were made: (i) although the alginate overproduction in strain 2192 was caused by a stable mutation, a mucus-derived signal caused reduction in the transcript levels of alginate biosynthetic genes; (ii) mucus activated the expression of the type VI secretion system, a mechanism for killing of other bacteria in a mixed population; (iii) expression of a number of genes involved in respiration was altered; and (iv) several small regulatory RNAs were identified, some being mucus regulated. This work highlights the strong influence of the host environment in shaping bacterial survival strategies.

scholarly journals The Pseudomonas aeruginosa PrrF Small RNAs Regulate Iron Homeostasis during Acute Murine Lung Infection

2017 ◽  
Vol 85 (5) ◽  
Author(s):  
Alexandria A. Reinhart ◽  
Angela T. Nguyen ◽  
Luke K. Brewer ◽  
Justin Bevere ◽  
Jace W. Jones ◽  
...  
Keyword(s):  
Gene Expression ◽  
Pseudomonas Aeruginosa ◽  
Small Rnas ◽  
Iron Homeostasis ◽  
Critical Role ◽  
Opportunistic Pathogen ◽  
Lung Infection ◽  
Content Type ◽  
The Individual

ABSTRACT Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that requires iron for virulence. Iron homeostasis is maintained in part by the PrrF1 and PrrF2 small RNAs (sRNAs), which block the expression of iron-containing proteins under iron-depleted conditions. The PrrF sRNAs also promote the production of the Pseudomonas quinolone signal (PQS), a quorum sensing molecule that activates the expression of several virulence genes. The tandem arrangement of the prrF genes allows for expression of a third sRNA, PrrH, which is predicted to regulate gene expression through its unique sequence derived from the prrF1-prrF2 intergenic (IG) sequence (the PrrHIG sequence). Previous studies showed that the prrF locus is required for acute lung infection. However, the individual functions of the PrrF and PrrH sRNAs were not determined. Here, we describe a system for differentiating PrrF and PrrH functions by deleting the PrrHIG sequence [prrF(ΔHIG)]. Our analyses of this construct indicate that the PrrF sRNAs, but not PrrH, are required for acute lung infection by P. aeruginosa. Moreover, we show that the virulence defect of the ΔprrF1-prrF2 mutant is due to decreased bacterial burden during acute lung infection. In vivo analysis of gene expression in lung homogenates shows that PrrF-mediated regulation of genes for iron-containing proteins is disrupted in the ΔprrF1-prrF2 mutant during infection, while the expression of genes that mediate PrrF-regulated PQS production are not affected by prrF deletion in vivo. Combined, these studies demonstrate that regulation of iron utilization plays a critical role in P. aeruginosa's ability to survive during infection.

scholarly journals Loss of RNA Chaperone Hfq Unveils a Toxic Pathway in Pseudomonas aeruginosa

2019 ◽  
Vol 201 (20) ◽  
Author(s):  
Ian T. Hill ◽  
Thomas Tallo ◽  
Matthew J. Dorman ◽  
Simon L. Dove
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Opportunistic Pathogen ◽  
Toxic Effects ◽  
Growth Defect ◽  
Wild Type ◽  
Rna Chaperone ◽  
Small Protein ◽  
Content Type ◽  
Transcription Regulator ◽  
Mutant Cells

ABSTRACT Hfq is an RNA chaperone that serves as a master regulator of bacterial physiology. Here we show that in the opportunistic pathogen Pseudomonas aeruginosa, the loss of Hfq can result in a dramatic reduction in growth in a manner that is dependent upon MexT, a transcription regulator that governs antibiotic resistance in this organism. Using a combination of chromatin immunoprecipitation with high-throughput sequencing and transposon insertion sequencing, we identify the MexT-activated genes responsible for mediating the growth defect of hfq mutant cells. These include a newly identified MexT-controlled gene that we call hilR. We demonstrate that hilR encodes a small protein that is acutely toxic to wild-type cells when produced ectopically. Furthermore, we show that hilR expression is negatively regulated by Hfq, offering a possible explanation for the growth defect of hfq mutant cells. Finally, we present evidence that the expression of MexT-activated genes is dependent upon GshA, an enzyme involved in the synthesis of glutathione. Our findings suggest that Hfq can influence the growth of P. aeruginosa by limiting the toxic effects of specific MexT-regulated genes. Moreover, our results identify glutathione to be a factor important for the in vivo activity of MexT. IMPORTANCE Here we show that the conserved RNA chaperone Hfq is important for the growth of the opportunistic pathogen Pseudomonas aeruginosa. We found that the growth defect of hfq mutant cells is dependent upon the expression of genes that are under the control of the transcription regulator MexT. These include a gene that we refer to as hilR, which we show is negatively regulated by Hfq and encodes a small protein that can be toxic when ectopically produced in wild-type cells. Thus, Hfq can influence the growth of P. aeruginosa by limiting the toxic effects of MexT-regulated genes, including one encoding a previously unrecognized small protein. We also show that MexT activity depends on an enzyme that synthesizes glutathione.

scholarly journals Scnn1b-Transgenic BALB/c Mice as a Model of Pseudomonas aeruginosa Infections of the Cystic Fibrosis Lung

2020 ◽  
Vol 88 (9) ◽  
Author(s):  
Kristen J. Brao ◽  
Brendan P. Wille ◽  
Joshua Lieberman ◽  
Robert K. Ernst ◽  
Mark E. Shirtliff ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Immune Cell ◽  
Opportunistic Pathogen ◽  
Sodium Absorption ◽  
Lung Pathology ◽  
Wild Type ◽  
Content Type ◽  
Lung Infections ◽  
Content Modeling

ABSTRACT The opportunistic pathogen Pseudomonas aeruginosa is responsible for much of the morbidity and mortality associated with cystic fibrosis (CF), a condition that predisposes patients to chronic lung infections. P. aeruginosa lung infections are difficult to treat because P. aeruginosa adapts to the CF lung, can develop multidrug resistance, and can form biofilms. Despite the clinical significance of P. aeruginosa, modeling P. aeruginosa infections in CF has been challenging. Here, we characterize Scnn1b-transgenic (Tg) BALB/c mice as P. aeruginosa lung infection models. Scnn1b-Tg mice overexpress the epithelial Na+ channel (ENaC) in their lungs, driving increased sodium absorption that causes lung pathology similar to CF. We intranasally infected Scnn1b-Tg mice and wild-type littermates with the laboratory P. aeruginosa strain PAO1 and CF clinical isolates and then assessed differences in bacterial clearance, cytokine responses, and histological features up to 12 days postinfection. Scnn1b-Tg mice carried higher bacterial burdens when infected with biofilm-grown rather than planktonic PAO1; Scnn1b-Tg mice also cleared infections more slowly than their wild-type littermates. Infection with PAO1 elicited significant increases in proinflammatory and Th17-linked cytokines on day 3. Scnn1b-Tg mice infected with nonmucoid early CF isolates maintained bacterial burdens and mounted immune responses similar to those of PAO1-infected Scnn1b-Tg mice. In contrast, Scnn1b-Tg mice infected with a mucoid CF isolate carried high bacterial burdens, produced significantly more interleukin 1β (IL-1β), IL-13, IL-17, IL-22, and KC, and showed severe immune cell infiltration into the bronchioles. Taken together, these results show the promise of Scnn1b-Tg mice as models of early P. aeruginosa colonization in the CF lung.

scholarly journals Identification of OprF as a Complement Component C3 Binding Acceptor Molecule on the Surface of Pseudomonas aeruginosa

2015 ◽  
Vol 83 (8) ◽  
pp. 3006-3014 ◽  
Author(s):  
Meenu Mishra ◽  
Adam Ressler ◽  
Larry S. Schlesinger ◽  
Daniel J. Wozniak
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Opportunistic Pathogen ◽  
Gram Negative Bacteria ◽  
Acceptor Molecule ◽  
Wild Type ◽  
Gram Negative ◽  
Content Type ◽  
Persistent Infections ◽  
Structural Identity ◽  
Complement Component C3

Pseudomonas aeruginosais a versatile opportunistic pathogen that can cause devastating persistent infections. Complement is a highly conserved pathway of the innate immune system, and its role in the first line of defense against pathogens is widely appreciated. One of the earliest events in the complement cascade is the conversion of C3 to C3a and C3b, the latter typically binds to one or more acceptor molecules on the pathogen surface. We previously demonstrated that complement C3b binding acceptors exist on theP. aeruginosasurface. In the current study, we utilized either C3 polyclonal or C3b monoclonal antibodies in a far-Western technique followed by mass spectroscopy to identify the C3b acceptor molecule(s) on theP. aeruginosasurface. Our data provide evidence that OprF (an outer membrane porin, highly conserved in thePseudomonadaceae) binds C3b. AnoprF-deficientP. aeruginosastrain exhibits reduced C3 deposition compared to the wild type. We observed reduced internalization ofoprF-deficient bacteria by neutrophils after opsonization compared with wild-typeP. aeruginosa. Heterologous expression of OprF significantly enhanced C3b binding and increased serum-mediated bactericidal effects in complement-susceptibleEscherichia coli. Furthermore, the predicted secondary structure of the C-terminal, surface-exposed region of OprF has high structural identity to the OmpA domain of several other Gram-negative bacteria, one of which is known to bind C3b. Therefore, these findings provide new insights into the biology of complement interactions withP. aeruginosaand other Gram-negative bacteria.

scholarly journals ThePseudomonas aeruginosaPrrF1 and PrrF2 Small Regulatory RNAs Promote 2-Alkyl-4-Quinolone Production through Redundant Regulation of theantRmRNA

2018 ◽  
Vol 200 (10) ◽  
Author(s):  
Louise Djapgne ◽  
Subrata Panja ◽  
Luke K. Brewer ◽  
Jonathan H. Gans ◽  
Maureen A. Kane ◽  
...  
Keyword(s):  
Quorum Sensing ◽  
Iron Homeostasis ◽  
Molecular Mechanisms ◽  
Biological Activities ◽  
Content Type ◽  
Downstream Effects ◽  
Infection Models ◽  
Small Regulatory Rnas ◽  
Regulatory Rnas ◽  
Translational Start

ABSTRACTPseudomonas aeruginosais an opportunistic Gram-negative pathogen that requires iron for growth and virulence. Under low-iron conditions,P. aeruginosatranscribes 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 ofantR, 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 ofantR, demonstrating that the PrrF sRNAs interact with theantR5′ untranslated region (UTR) at sequences overlapping the translational start site of this mRNA. TheP. aeruginosaHfq protein interacted with UA-rich sequences in both PrrF sRNAs (Kd[dissociation constant] = 50 nM and 70 nM). Hfq bound with lower affinity to theantRmRNA (0.3 μM), and PrrF was able to bind toantRmRNA in the absence of Hfq. Nevertheless, Hfq increased the rate of PrrF annealing to theantRUTR by 10-fold. These studies provide a mechanistic description of how the PrrF1 and PrrF2 sRNAs mediate virulence traits, such as AQ production, inP. aeruginosa.IMPORTANCEThe iron-responsive PrrF sRNAs play a central role in regulatingP. aeruginosairon 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 theantRmRNA, 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 inP. aeruginosa.

scholarly journals Functional Analyses of the RsmY and RsmZ Small Noncoding Regulatory RNAs inPseudomonas aeruginosa

2018 ◽  
Vol 200 (11) ◽  
Author(s):  
Kayley H. Janssen ◽  
Manisha R. Diaz ◽  
Matthew Golden ◽  
Justin W. Graham ◽  
Wes Sanders ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Binding Site ◽  
Binding Sites ◽  
Rna Binding ◽  
Opportunistic Pathogen ◽  
Binding Properties ◽  
Content Type ◽  
Preferential Binding ◽  
Regulatory Rnas

ABSTRACTPseudomonas aeruginosais a Gram-negative opportunistic pathogen with distinct acute and chronic virulence phenotypes. Whereas acute virulence is typically associated with expression of a type III secretion system (T3SS), chronic virulence is characterized by biofilm formation. Many of the phenotypes associated with acute and chronic virulence are inversely regulated by RsmA and RsmF. RsmA and RsmF are both members of the CsrA family of RNA-binding proteins and regulate protein synthesis at the posttranscriptional level. RsmA activity is controlled by two small noncoding regulatory RNAs (RsmY and RsmZ). Bioinformatic analyses suggest that RsmY and RsmZ each have 3 or 4 putative RsmA binding sites. Each predicted binding site contains a GGA sequence presented in the loop portion of a stem-loop structure. RsmY and RsmZ regulate RsmA, and possibly RsmF, by sequestering these proteins from target mRNAs. In this study, we used selective 2′-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) chemistry to determine the secondary structures of RsmY and RsmZ and functional assays to characterize the contribution of each GGA site to RsmY/RsmZ activity. Our data indicate that RsmA has two preferential binding sites on RsmY and RsmZ, while RsmF has one preferential binding site on RsmY and two sites on RsmZ. Despite RsmF and RsmA sharing a common consensus site, RsmF binding properties are more restrictive than those of RsmA.IMPORTANCECsrA homologs are present in many bacteria. The opportunistic pathogenPseudomonas aeruginosauses RsmA and RsmF to inversely regulate factors associated with acute and chronic virulence phenotypes. RsmA has an affinity for RsmY and RsmZ higher than that of RsmF. The goal of this study was to understand the differential binding properties of RsmA and RsmF by using the RsmY and RsmZ regulatory small RNAs (sRNAs) as a model. Mutagenesis of the predicted RsmA/RsmF binding sites on RsmY and RsmZ revealed similarities in the sites required to control RsmA and RsmF activityin vivo. Whereas binding by RsmA was relatively tolerant of binding site mutations, RsmF was sensitive to disruption to all but two of the sites, further demonstrating that the requirements for RsmF binding activityin vivoandin vitroare more stringent than those for RsmA.

scholarly journals Facultative Control of Matrix Production Optimizes Competitive Fitness in Pseudomonas aeruginosa PA14 Biofilm Models

2015 ◽  
Vol 81 (24) ◽  
pp. 8414-8426 ◽  
Author(s):  
Jonas S. Madsen ◽  
Yu-Cheng Lin ◽  
Georgia R. Squyres ◽  
Alexa Price-Whelan ◽  
Ana de Santiago Torio ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Competitive Advantage ◽  
Electron Acceptor ◽  
Resource Limitation ◽  
Opportunistic Pathogen ◽  
Wild Type ◽  
Pellicle Formation ◽  
Competitive Fitness ◽  
Content Type ◽  
Matrix Production

ABSTRACTAs biofilms grow, resident cells inevitably face the challenge of resource limitation. In the opportunistic pathogenPseudomonas aeruginosaPA14, electron acceptor availability affects matrix production and, as a result, biofilm morphogenesis. The secreted matrix polysaccharide Pel is required for pellicle formation and for colony wrinkling, two activities that promote access to O2. We examined the exploitability and evolvability of Pel production at the air-liquid interface (during pellicle formation) and on solid surfaces (during colony formation). Although Pel contributes to the developmental response to electron acceptor limitation in both biofilm formation regimes, we found variation in the exploitability of its production and necessity for competitive fitness between the two systems. The wild type showed a competitive advantage against a non-Pel-producing mutant in pellicles but no advantage in colonies. Adaptation to the pellicle environment selected for mutants with a competitive advantage against the wild type in pellicles but also caused a severe disadvantage in colonies, even in wrinkled colony centers. Evolution in the colony center produced divergent phenotypes, while adaptation to the colony edge produced mutants with clear competitive advantages against the wild type in this O2-replete niche. In general, the structurally heterogeneous colony environment promoted more diversification than the more homogeneous pellicle. These results suggest that the role of Pel in community structure formation in response to electron acceptor limitation is unique to specific biofilm models and that the facultative control of Pel production is required for PA14 to maintain optimum benefit in different types of communities.

scholarly journals Intraspecies Signaling between Common Variants of Pseudomonas aeruginosa Increases Production of Quorum-Sensing-Controlled Virulence Factors

mBio ◽  
2020 ◽  
Vol 11 (4) ◽  
Author(s):  
Dallas L. Mould ◽  
Nico J. Botelho ◽  
Deborah A. Hogan
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Opportunistic Pathogen ◽  
Loss Of Function ◽  
Wild Type ◽  
Chronic Infections ◽  
Content Type ◽  
Complex Interactions ◽  
Natural Settings ◽  
Citrate Release

ABSTRACT The opportunistic pathogen Pseudomonas aeruginosa damages hosts through the production of diverse secreted products, many of which are regulated by quorum sensing (QS). The lasR gene, which encodes a central QS regulator, is frequently mutated in clinical isolates from chronic infections, and loss of LasR function (LasR−) generally impairs the activity of downstream QS regulators RhlR and PqsR. We found that in cocultures containing LasR+ and LasR− strains, LasR− strains hyperproduce the RhlR/RhlI-regulated antagonistic factors pyocyanin and rhamnolipids in diverse models and media and in different strain backgrounds. Diffusible QS autoinducers produced by the wild type were not required for this effect. Using transcriptomics, genetics, and biochemical approaches, we uncovered a reciprocal interaction between wild-type and lasR mutant pairs wherein the iron-scavenging siderophore pyochelin produced by the lasR mutant induced citrate release and cross-feeding from the wild type. Citrate, a metabolite often secreted in low iron environments, stimulated RhlR signaling and RhlI levels in LasR−but not in LasR+ strains. These studies reveal the potential for complex interactions between recently diverged, genetically distinct isolates within populations from single chronic infections. IMPORTANCE Coculture interactions between lasR loss-of-function and LasR+ Pseudomonas aeruginosa strains may explain the worse outcomes associated with the presence of LasR− strains. More broadly, this report illustrates how interactions within a genotypically diverse population, similar to those that frequently develop in natural settings, can promote unpredictably high virulence factor production.

scholarly journals Comparative Systems Biology Analysis To Study the Mode of Action of the Isothiocyanate Compound Iberin on Pseudomonas aeruginosa

2014 ◽  
Vol 58 (11) ◽  
pp. 6648-6659 ◽  
Author(s):  
Sean Yang-Yi Tan ◽  
Yang Liu ◽  
Song Lin Chua ◽  
Rebecca Munk Vejborg ◽  
Tim Holm Jakobsen ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Systems Biology ◽  
Rna Sequencing ◽  
Virulence Factor ◽  
Content Type ◽  
Factor Expression ◽  
Small Regulatory Rnas ◽  
Regulatory Rnas ◽  
The Impact ◽  
Comparative Systems

ABSTRACTFood is now recognized as a natural resource of novel antimicrobial agents, including those that target the virulence mechanisms of bacterial pathogens. Iberin, an isothiocyanate compound from horseradish, was recently identified as a quorum-sensing inhibitor (QSI) of the bacterial pathogenPseudomonas aeruginosa. In this study, we used a comparative systems biology approach to unravel the molecular mechanisms of the effects of iberin on QS and virulence factor expression ofP. aeruginosa. Our study shows that the two systems biology methods used (i.e., RNA sequencing and proteomics) complement each other and provide a thorough overview of the impact of iberin onP. aeruginosa. RNA sequencing-based transcriptomics showed that iberin inhibits the expression of the GacA-dependent small regulatory RNAs RsmY and RsmZ; this was verified by usinggfp-based transcriptional reporter fusions with thersmYorrsmZpromoter regions. Isobaric tags for relative and absolute quantitation (iTRAQ) proteomics showed that iberin reduces the abundance of the LadS protein, an activator of GacS. Taken together, the findings suggest that the mode of QS inhibition in iberin is through downregulation of the Gac/Rsm QS network, which in turn leads to the repression of QS-regulated virulence factors, such as pyoverdine, chitinase, and protease IV. Lastly, as expected from the observed repression of small regulatory RNA synthesis, we also show that iberin effectively reduces biofilm formation. This suggests that small regulatory RNAs might serve as potential targets in the future development of therapies against pathogens that use QS for controlling virulence factor expression and assume the biofilm mode of growth in the process of causing disease.

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