Pyocyanin Biosynthesis Research Articles

1H-1,2,3-triazol-1,4-naphthoquinone Derivatives: Novel Inhibitors Targeting Pyocyanin Biosynthesis for P. Aeruginosa Infection Treatment Advances.

This study investigates the potential of eleven 1H-1,2,3-triazol-1,4-naphthoquinone conjugates as virulence factor inhibitors (like Pyocyanin) and their affinity for PhzM, a crucial enzyme for Pyocyanin biosynthesis in Pseudomonas aeruginosa infections. A straightforward synthetic pathway enabled the production of these compounds, which were characterized and structurally confirmed through spectroscopic analyses. Evaluation of their impact on PhzM thermal stability identified promising candidates for PhzM binders. Concentration-response behavior elucidated their binding affinity, revealing them as the first reported micromolar affinity ligands for PhzM. Structure-activity relationship analysis emphasized the role of specific molecular moieties in binding affinity modulation, paving the way for future advanced inhibitors' development. These findings highlight the potential of naphthoquinone-triazole derivatives as leads for novel therapeutics against P. aeruginosa infections.

Potassium Chloride, Sodium Lactate and Sodium Citrate Impaired the Antimicrobial Resistance and Virulence of Pseudomonas aeruginosa NT06 Isolated from Fish.

Sodium chloride (NaCl) is a commonly used additive in minimally processed fish-based products. The addition of NaCl to fish products and packaging in a modified atmosphere is usually efficient with regard to limiting the occurrence of the aquatic environmental pathogen Pseudomonas aeruginosa. Given the negative effects of excess NaCl in the diet, there is a growing demand to reduce NaCl in food products with safer substituents, but the knowledge of their impact on antibiotic resistant P. aeruginosa is limited. This study aimed to evaluate the physiological and transcriptome characteristics of P. aeruginosa NT06 isolated from fish and to determine the effect of selected concentrations of alternative NaCl compounds (KCl/NaL/NaC) on the P. aeruginosa NT06 virulence phenotype and genotype. In the study, among the isolated microorganisms, P. aeruginosa NT06 showed the highest antibiotic resistance (to ampicillin, ceftriaxone, nalidixic acid, and norfloxacin) and the ability to grow at 4 °C. The Comprehensive Antibiotic Resistance Database (CARD) and the Virulence Factor Database (VFDB) revealed the presence of 24 and 134 gene products assigned to AMR and VF in the P. aeruginosa NT06 transcriptome, respectively. KCl, KCl/NaL and KCl/NaL/NaC inhibited pyocyanin biosynthesis, elastase activity, and protease activity from 40 to 77%. The above virulence phenotypic observations were confirmed via RT-qPCR analyses, which showed that all tested AMR and VF genes were the most downregulated due to KCl/NaL/NaC treatment. In conclusion, this study provides insight into the potential AMR and VF among foodborne P. aeruginosa and the possible impairment of those features by KCl, NaL, and NaC, which exert synergistic effects and can be used in minimally processed fish-based products.

Diallyl trisulfide attenuates Pseudomonas aeruginosa virulence via inhibiting quorum sensing

Diallyl trisulfide (DATS), diallyl disulfide (DADS), and diallyl sulfide (DAS), three sulfur-rich compounds commonly found in garlic, can inhibit Pseudomonas aeruginosa quorum sensing (QS). We previously reported the inhibitory effects of DAS and DADS on the PAO1 QS system. To delineate any differences in QS inhibitory mechanisms among these three compounds and further verify their functional chemical groups, the QS-inhibitory effects and mechanism of DATS on P. aeruginosa PAO1 were investigated based on transcriptomic analysis, qRT-PCR, and physiological and chemical experiments in this study. The results indicated that DATS was mildly toxic to P. aeruginosa PAO1 and inhibited its growth slightly in a dose-dependent manner. Furthermore, DATS downregulated the transcriptional levels of all key genes involved in PAO1 QS systems, including lasI/lasR, rhlI/rhlR, and pqsABCDEH/pqsR, and downregulated the transcriptional levels of some related virulence genes, including the genes encoding exoprotease, elastase, lectin, and rhamnolipids, as well as those involved in PQS, pyocyanin biosynthesis, and biofilm formation. As a result, the production of related virulence factors by the PAO1 strain decreased upon DATS exposure. Therefore, DATS attenuated P. aeruginosa virulence and pathogenicity by inhibiting its QS systems. Through a comparative analysis of the QS-inhibitory effects of DATS, DADS, and DAS on the PAO1 strain, DATS was found to have the strongest QS-inhibitory activity, followed by DADS and DAS. In conclusion, the inhibitory activities of these sulfur compounds against P. aeruginosa QS and virulence were related to the thioether and disulfide groups, which increase with an increase in the number of sulfur atoms in the chemical formula. These findings are of great significance for the control of P. aeruginosa virulence and pathogenicity in the future.

Molecular Investigation of Outer Membrane Channel Genes Among Multidrug Resistance Clinical Pseudomonas Aeruginosa Isolates.

Multidrug resistance Pseudomonas aeruginosa (MDRPA) is most important issue in healthcare setting. It can secrete many virulence effector proteins via its secretion system type (T1SS-T6SS). They are using them as conductor for delivering the effector proteins outside to begins harmful effect on host cell increasing pathogenicity, competition against other microorganism and nutrient acquisition. The study include investigation of 50 isolates of MDRPA for transport secretion system and resistance for antibiotics. Molecular diagnosis using P. aeruginosa specific primer pairs, investigation of AprF, HasF, XcpQ, HxcQ, PscC, CdrB, CupB3, and Hcp using specific primer pairs by PCR were also performed. The results revealed high resistance to beta lactam antibiotics (78% for ceftazidime, 78% for cefepime and 46% for piperacillin) can indicate possessing of isolates for beta lactamases and this confirmed by dropping resistance to piperacillin to 16% when combined with tazobactam. Also, the results shown the ability of MDRPA for pyocyanin biosynthesis using the system of genes. The current study conclude that all isolates of P. aeruginosa were highly virulent due to their possessing of all transport secretion system to deliver different effector proteins with possible harmful effects of these proteins.

Increased production of pyocyanin in recombinant Pseudomonas aeruginosa PS39-phzMS strain harboring the pUCP24-phzMS plasmid

PhzM and PhzS are two “core” enzymes that are necessary for conversion of phenazine-1-carboxylic acid (PCA) into pyocyanin (PYO) phenazine in Gram (-) bacterium Pseudomonas aeruginosa. Apparently, the raise in copy number of their genes could increase the amount of pyocyanin phenazine in the microbe. In previous research, two genes phzM and phzS originated from Pseudomonas aeruginosa PS39 strain had been inserted into a Pseudomonas – Escherichia coli shutlle vector pUCP24 to generate a plasmid pUCP24-phzMS. The obtained plasmid had been transformed into P. aeruginosa PS39 strain to create the recombinant P. aeruginosa PS39-phzMS strain. In this study, pUCP24-phzMS was sequenced to verify the polycistronic expression cassette containing both phzM and phzS genes. The results demonstrated that the recombinant plasmid comprised the ori of Pseudomonas and E. coli, gentamicin resistance-cassette, and polycistronic expression cassette for expression of PhzM and PhzS. In which, both genes will be transcripted together in one mRNA strand by the regulation of Lac promoter and operator. The translation from the mRNA to the corresponding proteins will be started by binding ribosome into RBS located upstream of each gene. The nucleotide sequence of these genes were completely homologous (100%) to the submitted sequences MF673740 (phzM) and MF770713 (phzS) on the NCBI GenBank database. Result on assessing the synthesis of pyocyanin in the recombinant strain with the presence of pUCP24-phzMS plasmid showed that pyocyanin concentration in the recombinant strain increased significantly over 2 times (31.22 mg/mL) more than that in the wild strain (13.47 mg/mL). The absorbance at 360 nm of PCA from the P. aeruginosa PS39-phzMS (OD367 = 0.03) strain decreased significantly compared to the one from wild type strain (OD367 = 0.39). Therefore, the plasmid with phzM and phzS genes was proved to improve the pyocyanin biosynthesis through a better conversion of PCA phenazine into PYO via PhzM and PhzS enzymes.

Pyocyanin biosynthesis protects Pseudomonas aeruginosa from nonthermal plasma inactivation.

Summary Pseudomonas aeruginosa is an important opportunistic human pathogen, which raises a worldwide concern for its increasing resistance. Nonthermal plasma, which is also called cold atmospheric plasma (CAP), is an alternative therapeutic approach for clinical infectious diseases. However, the bacterial factors that affect CAP treatment remain unclear. The sterilization effect of a portable CAP device on different P. aeruginosa strains was investigated in this study. Results revealed that CAP can directly or indirectly kill P. aeruginosa in a time‐dependent manner. Scanning electron microscopy and transmission electron microscope showed negligible surface changes between CAP‐treated and untreated P. aeruginosa cells. However, cell leakage occurred during the CAP process with increased bacterial lactate dehydrogenase release. More importantly, pigmentation of the P. aeruginosa culture was remarkably reduced after CAP treatment. Further mechanical exploration was performed by utilizing mutants with loss of functional genes involved in pyocyanin biosynthesis, including P. aeruginosa PAO1 strain‐derived phzA1::Tn, phzA2::Tn, ΔphzA1/ΔphzA2, phzM::Tn and phzS::Tn, as well as corresponding gene deletion mutants based on clinical PA1 isolate. The results indicated that pyocyanin and its intermediate 5‐methyl phenazine‐1‐carboxylic acid (5‐Me‐PCA) play important roles in P. aeruginosa resistance to CAP treatment. The unique enzymes, such as PhzM in the pyocyanin biosynthetic pathway, could be novel targets for the therapeutic strategy design to control the growing P. aeruginosa infections.

RNA-seq-based transcriptomic analysis of AHL-induced biofilm and pyocyanin inhibition in Pseudomonas aeruginosa by Lactobacillus brevis.

Quorum sensing (QS) regulates hundreds of genes in Pseudomonas aeruginosa, and many of which encode extracellular virulence factors. Lactobacillus as a probiotic has been verified to inhibit pathogenesis in P. aeruginosa via quenching QS. The objective of this study was to investigate mechanism of the QS quenching function of Lactobacillus via analyzing the gene expression by transcriptome. We previously isolated a Lactobacillus brevis strain 3M004 from an aquafeed and identified the strain has the function of degrading QS molecular AHL (OC12-HSL). The result showed that 3M004 cells/lysate inhibited biofilm and pyocyanin production of P. aeruginosa PA002. The biofilm inhibition rates were 16.92% and 33.0% after treatment by 1 and 2mg/mL 3M004 lysate, respectively, and the rates for pyocyanin inhibition were 25.16% and 30.75%, respectively. Transcriptomic analysis showed that down-regulation of genes of LasA and LasB in PA002 was essential in regulating the QS system. The biofilm decrease of PA002 seems not only resulted from gene biosynthesizing of polysaccharides but also from other genes regulating components biosynthesis. Pyocyanin biosynthesis appears to be inhibited by down-regulating the key gene of PhzAB on the nonreversing action from chorismite to pyocyanin.

Lemon Oils Attenuate the Pathogenicity of Pseudomonas aeruginosa by Quorum Sensing Inhibition.

The chemical composition of three Citrus limon oils: lemon essential oil (LEO), lemon terpenes (LT) and lemon essence (LE), and their influence in the virulence factors production and motility (swarming and swimming) of two Pseudomonas aeruginosa strains (ATCC 27853 and a multidrug-resistant HT5) were investigated. The main compound, limonene, was also tested in biological assays. Eighty-four compounds, accounting for a relative peak area of 99.23%, 98.58% and 99.64%, were identified by GC/MS. Limonene (59–60%), γ-terpinene (10–11%) and β-pinene (7–15%) were the main compounds. All lemon oils inhibited specific biofilm production and bacterial metabolic activities into biofilm in a dose-dependent manner (20–65%, in the range of 0.1–4 mg mL−1) of both strains. Besides, all samples inhibited about 50% of the elastase activity at 0.1 mg mL−1. Pyocyanin biosynthesis decreases until 64% (0.1–4 mg mL−1) for both strains. Swarming motility of P. aeruginosa ATCC 27853 was completely inhibited by 2 mg mL−1 of lemon oils. Furthermore, a decrease (29–55%, 0.1–4 mg mL−1) in the synthesis of Quorum sensing (QS) signals was observed. The oils showed higher biological activities than limonene. Hence, their ability to control the biofilm of P. aeruginosa and reduce the production of virulence factors regulated by QS makes lemon oils good candidates to be applied as preservatives in the food processing industry.

PA1426 regulates Pseudomonas aeruginosa quorum sensing and virulence: an in vitro study

Abstract Objective: Pseudomonas aeruginosa (P. aeruginosa) contains a hierarchy of quorum sensing (QS) network, consisting of the las, rhl and pqs systems, which play a key role in coordinating the expression of virulence factors. PA2146 was found to be associated with P. aeruginosa pathogenicity in macrophage and host Immune response. The aim of this study was to investigate the effects of PA2146 on the virulence of P. aeruginosa and explore its mechanism. Methods: PA2146 gene knockout strain and complement strain of P. aeruginosa PAO1 were constructed. The biomass of biofilm was detected by crystal violet staining; the virulence factors were measured, including pyocyanin, rhamnolipid, LasA elastase, LasB elastase and hemolytic activity; RNA-seq and label-free relative quantitative proteomics analyses were carried out to test the influence of PA2146 on transcriptomics and proteomics. This study was approved by the Institutional Review Board of the Third Xiangya Hospital, Central South University, China (approval No. 2019-S021). Results: PA2146-deficient strains showed reduced biofilm formation and increased pyocyanin, rhamnolipid, LasA elastase, LasB elastase, and hemolytic activity, as well as increased motility, compared with the wild-type strain. RNA-seq and label-free relative quantitative proteomics analyses revealed that PA2146 repressed the transcription of several genes that are integral to the pqs system and to pyocyanin biosynthesis, and increased the expression of MexEF-OprN efflux pump components at the gene and protein level. Conclusion: PA2146 gene was found to inhibit the pqs system. PA2146 may affect quorum sensing by directly inhibiting the pqs system or by enhancing the expression of MexEF-OprN efflux pump components, thereby promoting efflux of 2-heptyl-4(1H)-quinolone, a Pseudomonas quinolone signal precursor, and thus affecting P. aeruginosa virulence.

Synthesis and biological evaluation of thiazolidinedione derivatives with high ligand efficiency to P. aeruginosa PhzS

The thiazolidinone ring is found in compounds that have widespan biology activity and there is mechanism-based evidence that compounds bearing this moiety inhibit P. aeruginosa PhzS (PaPzhS), a key enzyme in the biosynthesis of the virulence factor named pyocyanin. Ten novel thiazolidinone derivatives were synthesised and screened against PaPhzS, using two orthogonal assays. The biological results provided by these and 28 other compounds, whose synthesis had been described, suggest that the dihydroquinazoline ring, found in the previous hit (A- Kd = 18 µM and LE = 0.20), is not required for PaPzhS inhibition, but unsubstituted nitrogen at the thiazolidinone ring is. The molecular simplification approach, pursued in this work, afforded an optimised lead compound (13- 5-(2,4-dimethoxyphenyl)thiazolidine-2,4-dione) with 10-fold improvement in affinity (Kd= 1.68 µM) and more than 100% increase in LE (0.45), which follows the same inhibition mode as the original hit compound (competitive to NADH). Executive summary PhzS is a key enzyme in the pyocyanin biosynthesis pathway in P. aeruginosa. Orthogonal assays (TSA and FITC) show that fragment-like thiazolidinedione derivatives bind to PaPhzS with one-digit micromolar affinity. Fragment-like thiazolidinedione derivatives bind to the cofactor (NADH) binding site in PaPhzS. The molecular simplification optimised the ligand efficiency and affinity of the lead compound.

Diallyl sulfide from garlic suppresses quorum‐sensing systems of Pseudomonas aeruginosa and enhances biosynthesis of three B vitamins through its thioether group

Diallyl sulfide (DAS) and diallyl disulfide (DADS), two constituents of garlic, can inhibit quorum sensing (QS) systems of Pseudomonas aeruginosa. However, the differences in the mechanism of QS inhibition between DAS and DADS, and the functional chemical groups of these sulfides that contribute in QS inhibition have not been elucidated yet. We assumed that the sulfide group might play a key role in QS inhibition. To prove this hypothesis and to clarify these unsolved problems, in this study, we synthesized diallyl ether (DAE), and compared and investigated the effects of DAS and DAE on the growth and production of virulence factors, including Pseudomonas quinolone signal (PQS), elastase and pyocyanin, of P. aeruginosa PAO1. Transcriptome analysis and qRT-PCR were used to compare and analyse the differentially expressed genes between the different treatment groups (DAS, DAE and control). The results indicated that DAS did not affect the growth dynamics of P. aeruginosa PAO1; however, DAS inhibited transcription of most of the QS system genes, including lasR, rhlI/rhlR and pqsABCDE/pqsR; thus, biosynthesis of the signal molecules C4 -HSL (encoded by rhlI) and PQS (encoded by pqsABCDE) was inhibited. Furthermore, DAS inhibited the transcription of virulence genes regulated by the QS systems, including rhlABC, lasA, lasB, lecA and phzAB, phzDEFG, phzM and phzS that encode for rhamnolipid, exoprotease, elastase, lectin and pyocyanin biosynthesis respectively. DAS also enhanced the expression of the key genes involved in the biosynthesis of three B vitamins: folate, thiamine and riboflavin. In conclusion, DAS suppressed the production of some virulence factors toxic to the host and enhanced the production of some nutrition factors beneficial to the host. These actions of DAS may be due to its thioether group. These findings would be significant for development of an effective drug to control the virulence and pathogenesis of the opportunistic pathogen P. aeruginosa.

Pip serves as an intermediate in RpoS-modulated phz2 expression and pyocyanin production in Pseudomonas aeruginosa

Pyocyanin, a main virulence factor that is produced by Pseudomonas aeruginosa, plays an important role in pathogen-host interaction during infection. Two copies of phenazine-biosynthetic operons on genome, phz1 (phzA1B1C1D1E1F1G1) and phz2 (phzA2B2C2D2E2F2G2), contribute to phenazine biosynthesis. In our previous study, we found that RpoS positively regulates expression of the phz2 operon and pyocyanin biosynthesis in P. aeruginosa PAO1. In this work, when a TetR-family regulator gene, pip, was knocked out, we found that pyocyanin production was dramatically reduced, indicating that Pip positively regulates pyocyanin biosynthesis. With further phenazines quantification and β-galactosidase assay, we confirmed that Pip positively regulates phz2 expression, but does not regulate phz1 expression. In addition, while the rpoS gene was deleted, expression of pip was down-regulated. Expression of rpoS in the wild-type PAO1 strain, however, was similar to that in the Pip-deficient mutant PAΔpip, suggesting that expression of pip could positively be regulated by RpoS, whereas rpoS could not be regulated by Pip. Taken together, we drew a conclusion that Pip might serve as an intermediate in RpoS-modulated expression of the phz2 operon and pyocyanin biosynthesis in P. aeruginosa.

Biosíntesis de piocianina por Pseudomonas aeruginosa usando un subproducto del biodiesel

Biosíntesis de piocianina por Pseudomonas aeruginosa usando un subproducto del biodiesel

Mechanism of pyocyanin abolishment caused by mvaT mvaU double knockout in Pseudomonas aeruginosa PAO1

ABSTRACT MvaT and MvaU are global transcriptional regulators belonging to the H-NS family, and pyocyanin is an important virulence factor produced by Pseudomonas aeruginosa. mvaT mvaU double knockout mutant of P. aeruginosa PAO1 demonstrated pyocyanin abolishment in the previous study. Here, we further explored the mechanism. Two main directions were studied: pyocyanin biosynthesis pathway and QS system. The effect on the expression of the pyocyanin biosynthesis genes was evaluated by promoter strength determination and Real-Time PCR assay, and significant changes leading to low pyocyanin production were found. The effect on the QS system was studied by signal molecule quantification using LC-MS/MS and related gene expression measurements using Real-Time PCR. In mvaT mvaU double knockout, the production of 3-oxo-C12-HSL obviously increased, while those of C4-HSL and PQS obviously decreased, and the changes can be recovered by mvaT or mvaU complementation. The expressions of transcriptional activator genes binding with QS system signal molecules were all decreased, resulting in decreased formation of signal-transcriptional activator complexes. And the decreased expression of rhlR and pqsE also led to the lower expression of phzA1 and phzA2. Further exploration found that QS system downregulation may be related to QsrO, a QS system repressor, which was highly upregulated with mvaT mvaU double knockout. Hence, the synthesis of pyocyanin was suffocated and the biofilm formation ability was decreased. These results were also confirmed by transcriptome analysis, which demonstrated similar gene expression changes of the aforementioned genes together with decreased expression of other virulence factor genes regulated by QS system.

Structure-based Druggability Assessment of Anti-virulence Targets from Pseudomonas aeruginosa.

Antimicrobial Resistance (AMR) represents a serious threat to health and the global economy. However, interest in antibacterial drug development has decreased substantially in recent decades. Meanwhile, anti-virulence drug development has emerged as an attractive alternative to fight AMR. Although several macromolecular targets have been explored for this goal, their druggability is a vital piece of information that has been overlooked. This review explores this subject by showing how structure- based freely available in silico tools, such as PockDrug and FTMap, might be useful for designing novel inhibitors of the pyocyanin biosynthesis pathway and improving the potency/selectivity of compounds that target the Pseudomonas aeruginosa quorum sensing mechanism. The information provided by hotspot analysis, along with binding site features, reveals novel druggable targets (PhzA and PhzS) that remain largely unexplored. However, it also highlights that in silico druggability prediction tools have several limitations that might be overcome in the near future. Meanwhile, anti-virulence drug targets should be assessed by complementary methods, such as the combined use of FTMap/PockDrug, once the consensus druggability classification reduces the risk of wasting resources on undruggable proteins.

Visual detection of Hg2+ by manipulation of pyocyanin biosynthesis through the Hg2+-dependent transcriptional activator MerR in microbial cells

Mercury pollution has always been a huge threat to human health due to its significant toxicity. Thus, it's the continuing goal to obtain new mercury detection techniques that are cost-effective, operational stable, performance efficient, and applicable to the environmental and biological milieus. In this research, the soluble pigment pyocyanin with anti-bacterial and anti-fungal activities, the biosynthesis pathway of which was engineered under the regulation of Hg2+-dependent transcriptional activator MerR, was firstly used as the visual detection signal in the whole-cell biosensor. The engineered biosensor displayed optical sensing window and a good linearity for Hg2+ in the range of 25-1000nM, and the detection limit could reach as low as 10nM. It permitted on-site detection of bioavailable Hg2+ with extraordinary selectivity and could resist the interferences of extra metal ions. What's more, the developed biosensor performed function well in a wide pH range (pH 4-10) as well as the environmental water. By fully imitating and utilizing the biosystems from nature, the engineered colorimetric biosensor has great economic and performance advantages over most chemosensors as well as whole-cell biosensors in the practical application of detecting Hg2+ in the contaminated aquatic systems.

LasR Might Act as an Intermediate in Overproduction of Phenazines in the Absence of RpoS in Pseudomonas aeruginosa.

As an opportunistic bacterial pathogen, Pseudomonas aeruginosa PAO1 contains two phenazineproducing gene operons, phzA1B1C1D1E1F1G1 (phz1) and phzA2B2C2D2E2F2G2 (phz2), each of which is independently capable of encoding all enzymes for biosynthesizing phenazines, including phenazine-1-carboxylic acid and its derivatives. Other previous study reported that the RpoS-deficient mutant SS24 overproduced pyocyanin, a derivative of phenazine-1- carboxylic acid. However, it is not known how RpoS mediates the expression of two phz operons and regulates pyocyanin biosynthesis in detail. In this study, with deletion of the rpoS gene in the PAΔphz1 mutant and the PAΔphz2 mutant respectively, we demonstrated that RpoS exerted opposite regulatory roles on the expression of the phz1and phz2 operons. We also confirmed that the phz1 operon played a critical role and especially biosynthesized much more phenazines than the phz2 operon when the rpoS gene was knocked out in P. aeruginosa. By constructing the translational reporter fusion vector lasR'-'lacZ and the chromosomal fusion mutant PAΔlasR::lacZ, we verified that RpoS deficiency caused increased expression of lasR, a transcription regulator gene in a first quorum sensing system (las) that activates overexpression of the phz1 operon, suggesting that in the absence of RpoS, LasR might act as an intermediate in overproduction of phenazine biosynthesis mediated by the phz1 operon in P. aeruginosa.

Diallyl Disulfide From Garlic Oil Inhibits Pseudomonas aeruginosa Quorum Sensing Systems and Corresponding Virulence Factors.

Previously, we determined that diallyl disulfide (DADS) from garlic oil can inhibit Pseudomonas aeruginosa PAO1 pathogenic factors by inactivating the transcription of key genes from three quorum sensing (QS) systems (las, rhl, and pqs) based on the effects of DADS on growth, virulence factor production (elastase, pyocyanin, biofilm, and swarming motility), and RNA transcription (real-time q-PCR). To further investigate the mechanisms underlying the inhibition of the three P. aeruginosa QS systems by DADS, high-throughput RNA and proteome sequencing techniques were used to study differences in the transcriptional and proteome expression of P. aeruginosa PAO1 following treatment with DADS. The RNA-seq and proteomic data are available via NCBI Gene Expression Omnibus database with accession number GSE118801 and ProteomeXchange with identifier PXD011144, respectively. The experimental results indicated that all key genes of the three QS systems (las, rhl, and pqs) of P. aeruginosa PAO1 as well as the virulence factors (including exoprotease LasA, elastase LasB, lectin LecA and LecB, pyocyanin biosynthesis, and biofilm formation) regulated by these three QS systems were inhibited. This is consistent with our previous studies on the physiology, biochemistry, and RNA expression of P. aeruginosa treated with DADS. Additionally, our results also indicated that bacterial motility, chemotaxis, and two-component systems were inhibited by DADS treatment. All these changes abolish the sensitivity of P. aeruginosa PAO1 to environmental stimuli and cause the cells to be in a state of passivation. Further research is needed to determine how QS systems regulate these functions. Our findings could potentially contribute to the treatment and control of P. aeruginosa infection, virulence, and pathogenicity.

Insight into Pseudomonas aeruginosa pyocyanin production under low-shear modeled microgravity.

Long-term space flight impairs the immune system of astronauts, rendering them vulnerable to opportunistic infections. Pseudomonas aeruginosa causes opportunistic infections, particularly in individuals with a compromised immune system; it can be a major health hazard for astronauts during space flight missions. Hence, the production of the most abundant redox active virulence factor, pyocyanin by P. aeruginosa, was assessed under low-shear modeled microgravity (LSMMG) conditions, simulated using a high aspect ratio vessel. Moreover, we evaluated changes in the expression of genes involved in pyocyanin biosynthesis and genes involved in the MexGHI-OpmD operon quorum sensing. Extracellular DNA and H2O2 production were measured, and their correlation with pyocyanin production was examined. Interestingly, the pyocyanin quantity was 2.58-fold lower in the LSMMG conditions compared to the normal gravity. LSMMG caused downregulation of the genes associated with pyocyanin biosynthesis. Interestingly, extracellular DNA and H2O2 release were significantly high in the normal gravity environment. Scanning electron microscopy revealed aggregation and elongated cells under LSMMG. Taken together, these findings suggest that LSMMG did not induce pyocyanin secretion in P. aeruginosa.

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.