Levels Of Pyocyanin Research Articles

Exploration of the inhibitory effect of Cassia fistula on quorum sensing mediated virulence factor production and biofilm activity in Pseudomonas aeruginosa: an in vivo study in model organism Caenorhabditis elegans.

Introduction. Resistance to antibiotics is leading to challenges in the treatment of microbial diseases. One amongst the various approaches to control these pathogens is quorum sensing (QS), which is used to rectify resistance issues. Blocking the bacterial QS circuit is the most reliable anti-virulence therapy to control pathogenicity-associated genes. Pseudomonas aeruginosa is a contagious bacterium that proliferates in the host by using signalling molecules like acyl-homoserine lactones; these molecules generate and disseminate toxins and virulence factors for increasing host infection.Hypothesis. The herb Cassia fistula is known to have antimicrobial, antidiabetic, anti-inflammatory, antitumor medicinal properties amongst others. We hypothesize that its crude extracts will inhibit the QS circuit of Pseudomonas aeruginosa (P. aeruginosa).Aim. The research work was aimed at evaluating anti-quorum sensing and anti-biofilm activity of various crude extracts from Cassia fistula against P. aeruginosa.Methodology. Various extraction methods and solvents were availed for maximum separation, and the extracts were screened for anti-quorum sensing activity. The most potent Fruit Ethyl acetate (FEE) extract at non-inhibitory concentrations was found to interrupt both short-chain (RhlI/R) and long-chain (LasI/R) QS circuits and other virulence factors (P<0.05) such as elastase, protease, rhamnolipids and pyocyanin levels in P. aeruginosa. Biofilm inhibitory properties of FEE were demonstrated using atomic force microscopy, scanning electron microscope and confocal laser microscope. Caenorhabditis elegans infection model (Paralytic assay) was developed to determine the protective role of FEE by reducing the pathogenicity of P. aeruginosa.Results. The study results suggest that hot crude FEE extract interfered in the QS circuit, leading to comprehensive debilitation of QS-controlled virulence factors. The extract reduced virulence factor production in P. aeruginosa at 4 mg ml-1 concentration whilst paradoxically promoting biofilm formation. Possibly, higher sugar content in the extract promoted clump formation of biofilm architecture by increasing exopolysaccharide production. Moreover, in vivo analysis of bacterial pathogenesis on Caenorhabditis elegans reveals a drastic increase in survival rates in FEE treated worms compared to untreated control.Conclusions. FEE showed promising QS inhibitory activity against P. aeruginosa. In the future, additional purification of crude FEE is required to remove carbohydrates, and pure isolated phytochemicals from FEE could be used as therapeutic agents to control QS-mediated infections in P. aeruginosa.

Cellulose-based laser-induced graphene devices for electrochemical monitoring of bacterial phenazine production and viability

As an easily disposable substrate with a microporous texture, paper is a well-suited, generic substrate to build analytical devices for studying bacteria. Using a multi-pass lasing process, cellulose-based laser-induced graphene (cLIG) with a sheet resistance of 43.7 ± 2.3 Ω sq-1 is developed and utilized in the fabrication of low-cost and environmentally-friendly paper-based sensor arrays. Two case studies with Pseudomonas aeruginosa and Escherichia coli demonstrate the practicality of the cLIG sensors for the electrochemical analysis of bacteria. The first study measures the time-dependent profile of phenazines, such as pyocyanin, released from both planktonic (up to 60 h) and on-chip-grown (up to 22 h) Pseudomonas aeruginosa cultures. While similarities do exist, marked differences in phenazine production are seen with cells grown directly on cLIG compared to the planktonic culture. Moreover, in planktonic cultures, pyocyanin levels increase early on and plateau around 20 h, while optical density measurements increase monotonically over the duration of testing. The second study monitors the viability and metabolic activity of Escherichia coli using a resazurin-based electrochemical assay. These results demonstrate the utility of cLIG-based sensors as an inexpensive and versatile platform for monitoring bacteria and could enable new opportunities in high-throughput antibiotic susceptibility testing, ecological studies, and biofilm studies.

Pathogenesis of plant-associated Pseudomonas aeruginosa in Caenorhabditis elegans model

BackgroundPseudomonas aeruginosa is a globally dreaded pathogen that triggers fatality in immuno-compromised individuals. The agricultural ecosystem is a massive reservoir of this bacterium, and several studies have recommended P. aeruginosa to promote plant growth. However, there were limited attempts to evaluate the health risks associated with plant-associated P. aeruginosa. The current study hypothesized that agricultural P. aeruginosa strains exhibit eukaryotic pathogenicity despite their plant-beneficial traits.ResultsWe have demonstrated that feeding with the plant-associated P. aeruginosa strains significantly affects Caenorhabditis elegans health. Out of the 18 P. aeruginosa strain tested, PPA03, PPA08, PPA10, PPA13, PPA14, PPA17, and PPA18 isolated from cucumber, tomato, eggplant, and chili exhibited higher virulence and pathogenicity. Correlation studies indicated that nearly 40% of mortality in C. elegans was triggered by the P. aeruginosa strains with high levels of pyocyanin (> 9 µg/ml) and biofilm to planktonic ratio (> 8).ConclusionThis study demonstrated that plant-associated P. aeruginosa could be a potential threat to human health similar to the clinical strains. Pyocyanin could be a potential biomarker to screen the pathogenic P. aeruginosa strains in the agricultural ecosystem.

Design and Evaluation of New Quinazolin-4(3H)-one Derived PqsR Antagonists as Quorum Sensing Quenchers in Pseudomonas aeruginosa.

P. aeruginosa (PA) continues to pose a threat to global public health due to its high levels of antimicrobial resistance (AMR). The ongoing AMR crisis has led to an alarming shortage of effective treatments for resistant microbes, and hence there is a pressing demand for the development of novel antimicrobial interventions. The potential use of antivirulence therapeutics to tackle bacterial infections has attracted considerable attention over the past decades as they hamper the pathogenicity of target microbes with reduced selective pressure, minimizing the emergence of resistance. One such approach is to interfere with the PA pqs quorum sensing system which upon the interaction of PqsR, a Lys-R type transcriptional regulator, with its cognate signal molecules 4-hydroxy-2-heptylquinoline (HHQ) and 2-heptyl-3-hydroxy-4-quinolone (PQS), governs multiple virulence traits and host-microbe interactions. In this study, we report the hit identification and optimization of PqsR antagonists using virtual screening coupled with whole cell assay validation. The optimized hit compound 61 ((R)-2-(4-(3-(6-chloro-4-oxoquinazolin-3(4H)-yl)-2-hydroxypropoxy)phenyl)acetonitrile) was found to inhibit the expression of the PA PpqsA promoter controlled by PqsR with an IC50 of 1 μM. Using isothermal titration calorimetry, a Kd of 10 nM for the PqsR ligand binding domain (PqsRLBD) was determined for 61. Furthermore, the crystal structure of 61 with PqsRLBD was attained with a resolution of 2.65 Å. Compound 61 significantly reduced levels of pyocyanin, PQS, and HHQ in PAO1-L, PA14 lab strains and PAK6085 clinical isolate. Furthermore, this compound potentiated the effect of ciprofloxacin in early stages of biofilm treatment and in Galleria mellonella infected with PA. Altogether, this data shows 61 as a potent PqsR inhibitor with potential for hit to lead optimization toward the identification of a PA QS inhibitor which can be advanced into preclinical development.

Participation of the pvd Gene Cluster in the Mechanism of Quorum Sensing and Virulence of Pseudomonas aeruginosa PAO1

ObjectiveDetermination of the molecular mechanism involved in the virulence modulation of Pseudomonas aeruginosa PAO1 involving the pvdI and pvdL genes.MethodsSupernatants of the P. aeruginosa PAO1 strain and mutants pvdI, pvdJ, and pvdL were obtained from LB liquid medium after 24 h of growth. Evaluation of virulence factors: biofilm was determined by growth of P. aeruginosa strains in 96‐well plates using violet crystal staining. Rhamnolipids production was determined by quantification of rhamnose by the orcinol method. Survival assays were carried out in Caenorhabditis elegans using supernatants of PAO1 strains.AbstractPseudomonas aeruginosa is an opportunistic pathogenic bacterium for humans, animals and plants. This bacterium possesses a system of perception of bacterial quorum (quorum sensing, QS), which modulates several virulence factors. The LasI/LasR QS system modifies the production of the cyclodipeptides (CDPs) cyclo(L‐Pro‐L‐Val), cyclo(L‐Pro‐L‐Phe) and cyclo(L‐Pro‐L‐Tyr). Although CDPs are molecules with bioactive properties, low elements concern in the mechanism of biosynthesis in bacteria and their role in communication with host cells. In addition, it has been described that the CDPs production in P. aeruginosa mainly depends of the multi‐modular non‐ribosomal peptide synthetases (MM‐NRPS), whose products such as the aeruginaldehyde has been involved in the bacterial QS mechanisms. In this study, was found that levels of pyocyanin (a virulence factor in P. aeruginosa), it was increased in mutant strains in the pvdJ gene, which encodes for a NPRS. The pvd gene cluster is directly related to the pioverdin synthesis, which is a fluorescent peptide‐siderophore involved in the iron acquisition, being essential in the bacterial pathogenicity on the host. Therefore, is important to elucidate the mechanism by which the CDPs of P. aeruginosa PAO1 contribute to its pathogenesis and/or virulence. Production of several virulence factors in P. aeruginosa PAO1 with different single and double mutants in pvdI, pvdJ and pvdL genes were compared. Mutants in the pvdI, pvdL and pvdL/pvdI genes decrease 50% the production of biofilm respect to the WT strain. Likewise, when evaluating rhamnolipids amount, the production of rhamnolipids was significantly increased in the single mutants pvdJ and pvdL compared with the WT strain, but not modified in the double mutant pvdJ/pvdL. Survival in the C. elegans worms indicated that the pvdJ mutant increase the death of the nematode 40% than the WT. The results suggest that the pvd gene cluster was involved in the production of CDPs in P. aeruginosa PAO1 and may to regulate the production of virulence factors (biofilm and rhamnolipids), impacting its pathogenicity on the nematode in vivo model.

BIOSYNTHESIS OF GOLD NANOPARTICLES BY BACTERIA FROM HYPERALKALINE SPRING AND EVALUATION OF THEIR INHIBITORY ACTIVITY AGAINST PYOCYANIN PRODUCTION

Nanoparticles are used in wide range of applications given their unique optical, chemical and electronic properties. Microbial biosynthesis of nanoparticles has offered a milder and eco-friendly alternative to physical and chemical methods of synthesis. One potential use of nanoparticles is for the inhibition of quorum sensing-mediated processes by microorganisms during pathogenic colonization and infection. In this study, bacteria from hyperalkaline spring (pH 11) were isolated using various enrichment media. The isolates, phylogenetically related to Lysinibacillus sp. and Pseudomonas stutzeri, were investigated for their ability to biosynthesize gold nanoparticles (AuNPs) via reduction of chloroauric acid (HAuCl4) at pH 9. Extracellular synthesis of gold nanoparticles was confirmed by UV-Vis absorption analysis which showed a peak at 500-600 nm wavelength range corresponding to the surface plasmon resonance of AuNPs. The AuNPs synthesized were of spherical and irregular shapes as revealed by SEM analysis. The presence of elemental gold was further confirmed by EDX analysis. FTIR results showed that various functional groups are possibly involved in the reduction of HAuCl4 and stabilization of synthesized gold nanoparticles. The inhibitory effect of biosynthesized AuNPs was tested on the growth and pyocyanin production of Pseudomonas aeruginosa PA01 during a 72-hour incubation period. Although the biosynthesized AuNPs showed no toxicity to the test organism, an increasing inhibition level of pyocyanin production was observed with increasing volumes of nanoparticles used. The levels of pyocyanin in setups treated with biosynthesized gold nanoparticles at 10, 30 and 50 L were significantly lower compared to the untreated setups (P ≤ 0.05). Our findings demonstrate that bacteria adapted to alkaline conditions can be used for efficient biosynthesis of AuNPs, which exhibited potential biomedical application in inhibiting pyocyanin production.

Detection of the Bacterial Warfare Toxin, Pyocyanin, Using Transparent Carbon Utlramicroelectrode Arrays

Pyocyanin is a virulence factor exclusively secreted as a secondary metabolite by the opportunistic human pathogen Pseudomonas aeruginosa. Fast and direct detection of pyocyanin is of importance as it could provide fundamental insights regarding P. aeruginosa’s virulence mechanisms. Here, we demonstrate an electrochemical-sensing platform that is able to quantitatively determine the bio-synthesis and release of redox-active pyocyanin in real time using transparent carbon ultramicroelectrode arrays (T-CUAs). We quantified pyocyanin concentrations on various types of T-CUA electrodes using square-wave voltammetry to determine limits of detection (LODs) and linear dynamic ranges (LDRs). LODs and LDRs fall within the micromolar range for a variety of in vitro and in vivo cellular environments and offer promise of the application of T-CUAs as sensing devices for the quantitative study of biotoxins, bacterial group behavior phenotypes, and pathogenesis. We also demonstrate successful use of T-CUAs for electrochemical detection of pyocyanin secreted from P. aeruginosa strains while optically imaging the cells through the transparent electrodes. Secreted pyocyanin levels from two virulent bacterial strains, clinical PA11 and wild-type PA14, were measured. Finally, we present real-time electrochemical monitoring of pyocyanin using T-CUAs while optically and fluorescently analyzing aggregate formation of various clinical and laboratory bacterial strains.

Controlling localization of Escherichia coli populations using a two-part synthetic motility circuit: An accelerator and brake.

Probiotics, whether taken as capsules or consumed in foods, have been regarded as safe for human use by regulatory agencies. Being living cells, they serve as "tunable" factories for the synthesis of a vast array of beneficial molecules. The idea of reprogramming probiotics to act as controllable factories, producing potential therapeutic molecules under user-specified conditions, represents a new and powerful concept in drug synthesis and delivery. Probiotics that serve as drug delivery vehicles pose several challenges, one being targeting (as seen with nanoparticle approaches). Here, we employ synthetic biology to control swimming directionality in a process referred to as "pseudotaxis." Escherichia coli, absent the motility regulator cheZ, swim sporadically, missing the traditional "run" in the run:tumble swimming paradigm. Upon introduction of cheZ in trans and its signal-generated upregulation, engineered bacteria can be "programmed" to swim toward the source of the chemical cue. Here, engineered cells that encounter sufficient levels of the small signal molecule pyocyanin, produce an engineered CheZ and swim with programmed directionality. By incorporating a degradation tag at the C-terminus of CheZ, the cells stop running when they exit spaces containing pyocyanin. That is, the engineered CheZ modified with a C-terminal extension derived from the putative DNA-binding transcriptional regulator YbaQ (RREERAAKKVA) is consumed by the ClpXP protease machine at a rate sufficient to "brake" the cells when pyocyanin levels are too low. Through this process, we demonstrate that over time, these engineered E. coli accumulate in pyocyanin-rich locales. We suggest that such approaches may find utility in engineering probiotics so that their beneficial functions can be focused in areas of principal benefit.

Transparent Carbon Ultramicroelectrode Arrays for the Electrochemical Detection of a Bacterial Warfare Toxin, Pyocyanin.

Pyocyanin is a virulence factor produced as a secondary metabolite by the opportunistic human pathogen, Pseudomonas aeruginosa. Fast and direct detection of pyocyanin is of importance as it could provide important insights regarding P. aeruginosa's virulence mechanisms. Here, we present an electrochemical sensing platform of redox-active pyocyanin using transparent carbon ultramicroelectrode arrays (T-CUAs), which were made using a previously reported simple fabrication process ( Duay et al. Anal. Chem. 2015 , 87 , 10109 ). Square-wave voltammetry was used to quantify pyocyanin concentrations on T-CUAs with and without chitosan gold nanoparticles (CS/GNP) and planar transparent macroelectrodes (T-Macro). The response time (RT), limit of detection (LOD), and linear dynamic range (LDR) differ for each electrode type due to subtle influences in how the detectable signal varies in relation to the charging time and resistive and capacitive noise. In general lower LODs can be achieved at the consequence of smaller LDRs. The LOD for T-Macro was 0.75 ± 0.09 μM with a LDR of 0.75-25 μM, and the LOD for the CS/GNP 1.54 T-CUA was determined to be 1.6 ± 0.2 μM with a LDR of 1-100 μM, respectively. The LOD for the 1.54T-CUA with a larger LDR of 1-250 μM was 1.0 ± 0.3 μM. These LODs and LDRs fall within the range of PYO concentrations for a variety of in vitro and in vivo cellular environments and offer promise of the application of T-CUAs for the quantitative study of biotoxins, quorum sensing, and pathogenesis. Finally, we demonstrate the successful use of T-CUAs for the electrochemical detection of pyocyanin secreted from P. aeruginosa strains while optically imaging the cells. The secreted pyocyanin levels from two bacterial strains, PA11 and PA14, were measured to have concentrations of 45 ± 5 and 3 ± 2 μM, respectively.

Reduction of PCN biosynthesis by NO in Pseudomonas aeruginosa

Pyocyanin (PCN), a virulence factor synthesized by Pseudomonas aeruginosa, plays an important role during clinical infections. There is no study of the effect of nitric oxide (NO) on PCN biosynthesis. Here, the effect of NO on PCN levels in Pseudomonas aeruginosa strain PAO1, a common reference strain, was tested. The results showed that the NO donor sodium nitroprusside (SNP) can significantly reduce PCN levels (82.5% reduction at 60μM SNP). Furthermore, the effect of endogenous NO on PCN was tested by constructing PAO1 nor (NO reductase gene) knockout mutants. Compared to the wild-type strain, the Δnor strain had a lower PCN (86% reduction in Δnor). To examine whether the results were universal with other P. aeruginosa strains, we collected 4 clinical strains from a hospital, tested their PCN levels after SNP treatment, and obtained similar results, i.e., PCN biosynthesis was inhibited by NO. These results suggest that NO treatment may be a new strategy to inhibit PCN biosynthesis and could provide novel insights into eliminating P. aeruginosa virulence as a clinical goal.

Differential Potentiation of the Virulence of the Pseudomonas aeruginosa Cystic Fibrosis Liverpool Epidemic Strain by Oral Commensal Streptococci

The Pseudomonas aeruginosa Liverpool epidemic strain (LES) is an important cystic fibrosis (CF) pathogen and is associated with increased morbidity and a worsened prognosis, compared with other CF-associated strains. However, interactions of common LES phenotypic variants with other members of the polymicrobial biofilms associated with chronic CF respiratory disease, such as oral commensal streptococci, have not been investigated. Biofilm population dynamics, virulence factor production, and pathogenicity in Galleria mellonella larvae of common LES phenotypes (ie, low production, intermediate production, and overproduction of pyocyanin) in the presence or absence of anginosus group streptococci (AGS) were compared. AGS populations isolated from biofilm cocultures were P. aeruginosa phenotypic variant dependent, with higher AGS cell densities than those in monoculture frequently observed. Coexistence of AGS with a producer of low or intermediate levels of pyocyanin was found to result in enhancement of virulence factor production. In addition, the LES formed pathogenic partnerships with AGS in the G. mellonella infection model, with killing dependent on LES phenotype and AGS species. The pathogenic potential of LES phenotypic variants can be enhanced by the presence of oral commensal streptococci. As adaptive mutations leading to reduced virulence factor production are commonplace, the observations made are relevant in the general context of the biology of P. aeruginosa infection during CF.

Proteomics of Pseudomonas aeruginosa Australian Epidemic Strain 1 (AES-1) Cultured under Conditions Mimicking the Cystic Fibrosis Lung Reveals Increased Iron Acquisition via the Siderophore Pyochelin

Pseudomonas aeruginosa is an opportunistic pathogen that is the major cause of morbidity and mortality in patients with cystic fibrosis (CF). While most CF patients are thought to acquire P. aeruginosa from the environment, person-to-person transmissible strains have been identified in CF clinics worldwide, and the molecular basis for transmissibility remains poorly understood. We undertook a complementary proteomics approach to characterize protein profiles from a transmissible, acute isolate of the Australian epidemic strain 1 (AES-1R), the virulent burns/wound isolate PA14, and the poorly virulent, laboratory-associated strain PAO1 when grown in an artificial medium that mimics the CF lung environment compared to growth in standard laboratory medium. Proteins elevated in abundance in AES-1R included those involved in methionine and S-adenosylmethionine biosynthesis and in the synthesis of phenazines. Proteomic data were validated by measuring culture supernatant levels of the virulence factor pyocyanin, which is the final product of the phenazine pathway. AES-1R and PAO1 released higher extracellular levels of pyocyanin compared to PA14 when grown in conditions that mimic the CF lung. Proteins associated with biosynthesis of the iron-scavenging siderophore pyochelin (PchDEFGH and FptA) were also present at elevated abundance in AES-1R and at much higher levels than in PAO1, whereas they were reduced in PA14. These protein changes resulted phenotypically in increased extracellular iron acquisition potential and, specifically, elevated pyochelin levels in AES-1R culture supernatants as detected by chrome azurol-S assay and fluorometry, respectively. Transcript analysis of pyochelin genes (pchDFG and fptA) showed they were highly expressed during the early stage of growth in artificial sputum medium (18 h) but returned to basal levels following the establishment of microcolony growth (72 h) consistent with that observed in the CF lung. This provides further evidence that iron acquisition by pyochelin may play a role in the early stages of transmissible CF infection associated with AES-1R.

155 Fluctuating levels of pyocyanin in cystic fibrosis patient sputum from stable and acute samples

155 Fluctuating levels of pyocyanin in cystic fibrosis patient sputum from stable and acute samples

The Pseudomonas aeruginosa oxidative stress regulator OxyR influences production of pyocyanin and rhamnolipids: protective role of pyocyanin

The LysR-type transcriptional regulator (LTTR) OxyR orchestrates the defence of the opportunistic pathogen Pseudomonas aeruginosa against reactive oxygen species. In previous work we also demonstrated that OxyR is needed for the utilization of the ferrisiderophore pyoverdine, stressing the importance of this regulator. Here, we show that an oxyR mutant is unable to swarm on agar plates, probably as a consequence of absence of production of rhamnolipid surfactant molecules. Another obvious phenotypic change was the increased production of the phenazine redox-active molecule pyocyanin in the oxyR mutant. As already described, the oxyR mutant could not grow in LB medium, unless high numbers of cells (>10( 8) ml(-1)) were inoculated. However, its growth in Pseudomonas P agar (King's A), a medium inducing pyocyanin production, was like that of the wild-type, suggesting a protective action of this redox-active phenazine compound. This was confirmed by the restoration of the capacity to grow in LB medium upon addition of pure pyocyanin. Although both rhamnolipid and pyocyanin production are controlled by quorum sensing, no obvious changes were observed in the production of N-acylhomoserine lactones or the Pseudomonas quinolone signal (PQS). Complementation of rhamnolipid production and motility, and restoration of normal pyocyanin levels, was only possible when the oxyR gene was in single copy, while pyocyanin levels were increased when oxyR was present in a multicopy vector. Conversely, plating efficiency was increased only when the oxyR gene was present in multicopy, but not when in single copy in the chromosome, due to lower expression of oxyR compared with the wild-type, suggesting that some phenotypes are differently affected in function to the levels of OxyR molecules in the cell. Analysis of transcripts of oxidative stress-response enzymes showed a strong decrease of katB, ahpC and ahpB expression in the oxyR mutant grown in LB, but this was not the case when the mutant was grown on P agar, suggesting that the OxyR dependency for the transcription of these genes is not total.

Pseudomonas pyocyanin inhibits wound repair by inducing premature cellular senescence: Role for p38 mitogen-activated protein kinase

Pseudomonas aeruginosa is an important nosocomial pathogen of burn wounds. Pyocyanin, a virulence factor produced by the bacterium, induces persistent intracellular oxidative stress and premature senescence in mammalian cells. Our aims were to evaluate pyocyanin levels in infected wound dressings and the potential of the toxin to influence wound repair. Surgical dressings from infected burn patients were examined for pyocyanin and normal primary human diploid fibroblasts (HDFs) were treated with comparable concentrations and their replicative ability examined. Pyocyanin was detected in the exudate of infected wound dressings in amounts up to 5.3 μg/g (mean: 2.0 ± 2.3 μg/g). HDFs exposed to pyocyanin (1–50 μM; 0.2–10.5 μg/ml) underwent growth arrest at all concentrations and developed morphological characteristics associated with cellular senescence, including expression of senescence-associated β-galactosidase. Using an in vitro wound repair model, a single exposure to pyocyanin inhibited wound repair in a concentration-dependent manner. Prior treatment with a specific p38 MAPK inhibitor allowed cells to maintain their replicative ability and pre-senescent morphology indicating pyocyanin operates through the Erk/p38 MAPK senescence pathway. These data support the hypothesis that bacterial virulence factors capable of inducing persistent low-level oxidative stress play a pivotal role in modulating the tissue repair response to infection by inducing premature cellular senescence.

Expression of the phytochrome operon in Pseudomonas aeruginosa is dependent on the alternative sigma factor RpoS

Phytochromes are red/far-red light photoreceptors found in plants, cyanobacteria and heterotrophic bacteria. Biochemical analyses have established that the genes bphO and bphP (PA4116 and PA4117) of Pseudomonas aeruginosa encode both phytochrome components: BphO, a heme oxygenase that produces the linear tetrapyrrole chromophore biliverdin IXalpha, and BphP, the apo-phytochrome. Reverse transcription-PCR established that both genes form a bicistronic operon. Expression of the bphOP operon was induced in the stationary phase, indicating an involvement of the P. aeruginosa quorum-sensing system and/or the stationary-phase alternative sigma factor RpoS. Bioinformatic analyses of the promoter region revealed a potential binding site for the quorum sensing regulators LasR and/or RhlR. While a direct involvement of the quorum-sensing system could be ruled out, the dependence of bphOP expression on RpoS was clearly demonstrated. Chromosomal knock-out mutants showed identical growth behavior as a wild type under various conditions but increased levels of pyocyanin were detected in the DeltabphO strain. Additionally, this strain showed decreased heat tolerance in the stationary phase, indicating a potential protective role of the BphO reaction product biliverdin. Therefore, BphO might have an additional function besides providing the chromophore for BphP and both proteins are likely to fulfill a task in the stationary phase.

Pseudomonas aeruginosaAmpR Is a Global Transcriptional Factor That Regulates Expression of AmpC and PoxB β-Lactamases, Proteases, Quorum Sensing, and Other Virulence Factors

In members of the family Enterobacteriaceae, ampC, which encodes a beta-lactamase, is regulated by an upstream, divergently transcribed gene, ampR. However, in Pseudomonas aeruginosa, the regulation of ampC is not understood. In this study, we compared the characteristics of a P. aeruginosa ampR mutant, PAOampR, with that of an isogenic ampR+ parent. The ampR mutation greatly altered AmpC production. In the absence of antibiotic, PAOampR expressed increased basal beta-lactamase levels. However, this increase was not followed by a concomitant increase in the P(ampC) promoter activity. The discrepancy in protein and transcription analyses led us to discover the presence of another chromosomal AmpR-regulated beta-lactamase, PoxB. We found that the expression of P. aeruginosa ampR greatly altered the beta-lactamase production from ampC and poxB in Escherichia coli: it up-regulated AmpC but down-regulated PoxB activities. In addition, the constitutive P(ampR) promoter activity in PAOampR indicated that AmpR did not autoregulate in the absence or presence of inducers. We further demonstrated that AmpR is a global regulator because the strain carrying the ampR mutation produced higher levels of pyocyanin and LasA protease and lower levels of LasB elastase than the wild-type strain. The increase in LasA levels was positively correlated with the P(lasA), P(lasI), and P(lasR) expression. The reduction in the LasB activity was positively correlated with the P(rhlR) expression. Thus, AmpR plays a dual role, positively regulating the ampC, lasB, and rhlR expression levels and negatively regulating the poxB, lasA, lasI, and lasR expression levels.

Differential roles of the Pseudomonas aeruginosa PA14 rpoN gene in pathogenicity in plants, nematodes, insects, and mice.

We cloned the rpoN (ntrA, glnF) gene encoding the alternate sigma factor sigma(54) from the opportunistic multihost pathogen Pseudomonas aeruginosa strain PA14. A marker exchange protocol was used to construct the PA14 rpoN insertional mutation rpoN::Gen(r). PA14 rpoN::Gen(r) synthesized reduced levels of pyocyanin and displayed a variety of phenotypes typical of rpoN mutants, including a lack of motility and the failure to grow on nitrate, glutamate, or histidine as the sole nitrogen source. Compared to wild-type PA14, rpoN::Gen(r) was ca. 100-fold less virulent in a mouse thermal injury model and was significantly impaired in its ability to kill the nematode Caenorhabditis elegans. In an Arabidopsis thaliana leaf infectivity assay, although rpoN::Gen(r) exhibited significantly reduced attachment to trichomes, stomata, and the epidermal cell surface, did not attach perpendicularly to or perforate mesophyll cell walls, and proliferated less rapidly in Arabidopsis leaves, it nevertheless elicited similar disease symptoms to wild-type P. aeruginosa PA14 at later stages of infection. rpoN::Gen(r) was not impaired in virulence in a Galleria mellonella (greater wax moth) pathogenicity model. These data indicate that rpoN does not regulate the expression of any genes that encode virulence factors universally required for P. aeruginosa pathogenicity in diverse hosts.

The regulation of pyocyanin production in Pseudomonas aeruginosa

Pyocyanin was produced only after the exponential phase of growth on all media examined. Pyocyanin was also found to be produced in response to some nutrient limitation (for example, carbon or oxygen). Furthermore, by controlling the growth rate at less than approximately 0.1 h−1 the repression of pyocyanin production could be overcome to a large degree. An inverse relationship existed at low growth rates between growth rate and pyocyanin production, with a decrease in growth rate resulting in increased pyocyanin levels. Therefore, pyocyanin production appeared to be regulated by the energy status of the cell which would be lowered under conditions of low nutrient concentration, resulting in a decrease in growth rate and an increase in the level of pyocyanin produced. Under conditions of readily available nutrients the energy generating capacity of the cell was increased resulting in an increased growth rate and repression of pyocyanin. The ability of uncouplers of oxidative phosphorylation (e.g. CCCP and FCCP) to induce pyocyanin production, and of inhibitors of the membrane-bound ATPase (e.g. DCCD and sodium azide) to repress pyocyanin production, confirmed the existance of an energy mediated regulatory mechanism. Indeed, the evidence presented here along with the reported regulatory role of inorganic phosphate in pyocyanin production, suggests that production of this antibiotic may be regulated by intracellular ATP levels.