Reactive oxygen species drive evolution of pro-biofilm variants in pathogens by modulating cyclic-di-GMP levels.
Chua Song Lin,Ding Yichen,Liu Yang,Cai Zhao,Zhou Jianuan,Swarup Sanjay,Drautz-Moses Daniela I,Schuster Stephan Christoph,Kjelleberg Staffan,Givskov Michael,Yang Liang
The host immune system offers a hostile environment with antimicrobials and reactive oxygen species (ROS) that are detrimental to bacterial pathogens, forcing them to adapt and evolve for survival. However, the contribution of oxidative stress to pathogen evolution remains elusive. Using an experimental evolution strategy, we show that exposure of the opportunistic pathogen Pseudomonas aeruginosa to sub-lethal hydrogen peroxide (HO) levels over 120 generations led to the emergence of pro-biofilm rough small colony variants (RSCVs), which could be abrogated by l-glutathione antioxidants. Comparative genomic analysis of the RSCVs revealed that mutations in the wspF gene, which encodes for a repressor of WspR diguanylate cyclase (DGC), were responsible for increased intracellular cyclic-di-GMP content and production of Psl exopolysaccharide. Psl provides the first line of defence against ROS and macrophages, ensuring the survival fitness of RSCVs over wild-type P. aeruginosa Our study demonstrated that ROS is an essential driving force for the selection of pro-biofilm forming pathogenic variants. Understanding the fundamental mechanism of these genotypic and phenotypic adaptations will improve treatment strategies for combating chronic infections.
Fewer Defects in the Surface Slows the Hydrolysis Rate, Decreases the ROS Generation Potential, and Improves the Non-ROS Antimicrobial Activity of MgO.
Anicˇić Nemanja,Vukomanović Marija,Koklicˇ Tilen,Suvorov Danilo
Small (Weinheim an der Bergstrasse, Germany)
Magnesium oxide (MgO) is recognised as exhibiting a contact-based antibacterial activity. However, a comprehensive study of the impact of atomic-scale surface features on MgO's antibacterial activity is lacking. In this study, the nature and abundance of the native surface defects on different MgO powders are thoroughly investigated. Their impacts on the hydrolysis kinetics, antibacterial activity against Escherichia coli (ATCC 47076), Staphylococcus epidermidis and Pseudomonas aeruginosa and the reactive oxygen species (ROS) generation potential are determined and explained. It is shown that a reduction in the abundance of low-coordinated oxygen atoms on the surface of the MgO improves its resistance to both hydrolysis and antibacterial activity. The ROS generation potential, determined in-situ using a fluorescence microplate assay and electron paramagnetic resonance spectroscopy, is not an inherent property of the studied MgO, rather it is a side product of hydrolysis (only for the most highly defected MgO particles) and/or a consequence of the MgO/bacteria interaction. The evaluation of the mutual correlations of the hydrolysis, the antibacterial activity and the ROS generation, with their origin in the surface defects' peculiarities, led to the conclusion that the acid/base reaction between the MgO surface and the bacterial wall contributes considerably to the MgO's antibacterial activity.
The Effect of Quorum-Sensing and Efflux Pumps Interactions in Against Photooxidative Stress.
Rezaie Parizad,Pourhajibagher Maryam,Chiniforush Nasim,Hosseini Nava,Bahador Abbas
Journal of lasers in medical sciences
Resistant infections essentially cause mortality in a burn unit. Several bacteria contribute to burn infections; among these, majorly contributes to these infections revealing significant drug resistance. Similar to other bacteria, reveals various mechanisms to attain highest pathogenicity and resistance; among these, efflux pumps and quorum sensing are crucial. Quorum sensing enables effective communication between bacteria and synchronizes their gene expression resulting in optimum effect of the secreted proteins; alternatively, efflux pumps increase the bacterial resistance by pumping out the antimicrobial factors as well as the QS signals and precursors. Of recent, increasing episodes of drug resistance led to new findings and approaches for killing pathogenic bacteria without inducing the drug-resistant species. Photodynamic therapy (PDT), considered as an adjuvant and innovative method for conventional antibiotic therapy, is a photochemical reaction that includes visible light, oxygen, and a photosensitizer (PS). In this therapy, after exposure to visible light, the PS generates reactive oxygen species (ROS) that are bacteriostatic or bactericidal. Furthermore, this oxidative stress can disrupt the coordination of gene expression and alter the bacterial behavior. Considering the fact that the adaption and several gene expression patterns of microorganisms within the biofilm make them notably resistant to the recent antimicrobial treatments, this study aimed to emphasize the relationship between the efflux pump and QS under oxidative stress and their role in s reaction to PDT.
Evaluation of the interaction between polymyxin B and Pseudomonas aeruginosa biofilm and planktonic cells: reactive oxygen species induction and zeta potential.
Lima Marlucy Rodrigues,Ferreira Gabriella Freitas,Nunes Neto Wallace Ribeiro,Monteiro Joveliane de Melo,Santos Áquila Rodrigues Costa,Tavares Priscila Batista,Denadai Ângelo Márcio Leite,Bomfim Maria Rosa Quaresma,Dos Santos Vera Lúcia,Marques Sirlei Garcia,de Souza Monteiro Andrea
BACKGROUND:Although the most widely accepted mechanism of action for polymyxins is related to bacterial lysis via disruption, we hypothesized that this antimicrobial drug class could have other effects on Pseudomonas aeruginosa planktonic and sessile cells. Little is known regarding oxidative burst and zeta potential (ZP) data associated with the interaction between polymyxin B and P. aeruginosa cells. The present study evaluated endogenous reactive oxygen species (ROS) production and changes in the net charges of biofilm and planktonic cells in response to polymyxin B. RESULTS:Polymyxin B induced concentration-dependent killing at all concentrations tested in planktonic and sessile cells from P. aeruginosa strains. Sublethal concentrations of polymyxin B induced oxidative burst. ROS production was higher in resistant planktonic cells than in biofilm cells but this was not observed for susceptible cells. Moreover, no net surface charge alterations were observed in planktonic cells from a susceptible strain treated with polymyxin B, but a significant increase of ZP was noted in planktonic cells from a resistant strain. CONCLUSION:Oxidative burst generated by planktonic and sessile cells from P. aeruginosa strains against polymyxin B indicates that ROS may have an important role in the mechanism of action of this drug. ZP data revealed that electrostatic interactions of the cationic peptide with the anionic surface of the cells are strain-dependent. Therefore, we suggested that the intracellular effects of polymyxin B should be further investigated to understand polymyxin B-induced stress in P. aeruginosa.
polysaccharides inhibit cellular apoptosis and autophagy induced by lipopolysaccharide in A549 cells through sirtuin 1 activation.
Shi Xiaolan,Wei Wenfeng,Wang Ning
In the present study, the role of in cellular apoptosis and autophagy induced by lipopolysaccharide (LPS) in human epithelial A549 lung-cancer cells was investigated. Initially, it was demonstrated that LPS attenuated A549 cell viability in a time- and dose-dependent manner. Furthermore, LPS induced apoptotic cell death and autophagy in A549 cells and increased reactive oxygen species (ROS) production in a time-dependent manner. In addition, LPS treatment was demonstrated to markedly suppress sirtuin 1 (SIRT1) protein expression in A549 cells. Notably, it was demonstrated that polysaccharides activate SIRT1, leading to increased p62 expression, decreased p53 acetylation and B-cell lymphoma 2-associated X protein expression, and subsequently attenuate LPS-induced apoptotic cell death and autophagy. The results of the present study demonstrated that polysaccharides activate SIRT1 and inhibit LPS-induced ROS production, apoptosis and autophagy. This may have critical implications for the treatment of infection.
A long-chain flavodoxin protects Pseudomonas aeruginosa from oxidative stress and host bacterial clearance.
Moyano Alejandro J,Tobares Romina A,Rizzi Yanina S,Krapp Adriana R,Mondotte Juan A,Bocco José L,Saleh Maria-Carla,Carrillo Néstor,Smania Andrea M
Long-chain flavodoxins, ubiquitous electron shuttles containing flavin mononucleotide (FMN) as prosthetic group, play an important protective role against reactive oxygen species (ROS) in various microorganisms. Pseudomonas aeruginosa is an opportunistic pathogen which frequently has to face ROS toxicity in the environment as well as within the host. We identified a single ORF, hereafter referred to as fldP (for fl avo d oxin from P . aeruginosa), displaying the highest similarity in length, sequence identity and predicted secondary structure with typical long-chain flavodoxins. The gene was cloned and expressed in Escherichia coli. The recombinant product (FldP) could bind FMN and exhibited flavodoxin activity in vitro. Expression of fldP in P. aeruginosa was induced by oxidative stress conditions through an OxyR-independent mechanism, and an fldP-null mutant accumulated higher intracellular ROS levels and exhibited decreased tolerance to H2O2 toxicity compared to wild-type siblings. The mutant phenotype could be complemented by expression of a cyanobacterial flavodoxin. Overexpression of FldP in a mutT-deficient P. aeruginosa strain decreased H2O2-induced cell death and the hypermutability caused by DNA oxidative damage. FldP contributed to the survival of P. aeruginosa within cultured mammalian macrophages and in infected Drosophila melanogaster, which led in turn to accelerated death of the flies. Interestingly, the fldP gene is present in some but not all P. aeruginosa strains, constituting a component of the P. aeruginosa accessory genome. It is located in a genomic island as part of a self-regulated polycistronic operon containing a suite of stress-associated genes. The collected results indicate that the fldP gene encodes a long-chain flavodoxin, which protects the cell from oxidative stress, thereby expanding the capabilities of P. aeruginosa to thrive in hostile environments.
Antioxidant Defense Mechanisms in Pseudomonas aeruginosa: Role of Iron-Cofactored Superoxide Dismutase in Response to UV-C Radiations.
Ghorbal Salma Kloula Ben,Maalej Lobna,Chourabi Kalthoum,Khefacha Sana,Ouzari Hadda-Imene,Chatti Abdelwaheb
The role of SOD gene in response to UV-C radiations was studied in Pseudomonas aeruginosa. Firstly, our results showed that the inactivation of sodM and/or sodB genes decreases the resistance of P. aeruginosa after exposure to UV-C rays. Furthermore, our results showed that SOD activity is dose dependant in all strains. However, significant increase in SOD activity was only shown at UV-C exposure time of 5 min in sodB mutant. At an elevated dose equivalent to 30 min of exposure, significant increase in SOD activity was observed in sodM. Catalase activities showed significant decrease in WT and in sodB mutant after an exposure time of 30 min. CAT enzyme was present at higher levels than SOD, reflecting that alternate enzymes such as POX, is poorly associated with CAT activity, and an increase in POX activity is related to increase in stress tolerance. The overall results showed that sodB gene has an important protective role against UV-C radiations in P. aeruginosa, compared to SodM isoform.
Pseudomonas aeruginosa pyocyanin induces neutrophil death via mitochondrial reactive oxygen species and mitochondrial acid sphingomyelinase.
Managò Antonella,Becker Katrin Anne,Carpinteiro Alexander,Wilker Barbara,Soddemann Matthias,Seitz Aaron P,Edwards Michael J,Grassmé Heike,Szabò Ildiko,Gulbins Erich
Antioxidants & redox signaling
AIMS:Pulmonary infections with Pseudomonas aeruginosa are a serious clinical problem and are often lethal. Because many strains of P. aeruginosa are resistant to antibiotics, therapeutic options are limited. Neutrophils play an important role in the host's early acute defense against pulmonary P. aeruginosa. Therefore, it is important to define the mechanisms by which P. aeruginosa interacts with host cells, particularly neutrophils. RESULTS:Here, we report that pyocyanin, a membrane-permeable pigment and toxin released by P. aeruginosa, induces the death of wild-type neutrophils; its interaction with the mitochondrial respiratory chain results in the release of reactive oxygen species (ROS), the activation of mitochondrial acid sphingomyelinase, the formation of mitochondrial ceramide, and the release of cytochrome c from mitochondria. A genetic deficiency in acid sphingomyelinase prevents both the activation of this pathway and pyocyanin-induced neutrophil death. This reduced death, on the other hand, is associated with an increase in the release of interleukin-8 from pyocyanin-activated acid sphingomyelinase-deficient neutrophils but not from wild-type cells. INNOVATION:These studies identified the mechanisms by which pyocyanin induces the release of mitochondrial ROS and by which ROS induce neutrophil death via mitochondrial acid sphingomyelinase. CONCLUSION:These findings demonstrate a novel mechanism of pyocyanin-induced death of neutrophils and show how this apoptosis balances innate immune reactions.
SIRT1‑mediated regulation of oxidative stress induced by Pseudomonas aeruginosa lipopolysaccharides in human alveolar epithelial cells.
Liu Xiaoli,Yang Tuo,Sun Tieying,Shao Kuiqing
Molecular medicine reports
Sirtuin1 (SIRT1) is an NAD+‑dependent deacetylase that exhibits multiple biological functions, including cell differentiation inhibition, transcription regulation, cell cycle regulation and anti‑apoptosis. Lipopolysaccharides (LPS) are crucial virulence factors produced by Pseudomonas aeruginosa and serve an important role in adjusting the interactions between the host and the pathogen. However, the effect of SIRT1 in the regulation of LPS‑induced A459 human alveolar epithelial cells (AECs) oxidative stress remains unclear. The cellular reactive oxygen species (ROS) production was examined in A549 cells that were supplemented with LPS. Relative cell signaling pathway proteins were further investigated by western blot analysis. It was identified that LPS downregulated SIRT1 expression, however, upregulated ROS generation, which was associated with the increase of nuclear factor (NF)‑κB and acetyl‑NF‑κB. Activation of SIRT1 by resveratrol significantly reversed the effects of LPS on A549 cells. By contrast, inhibition of SIRT1 by nicotinamide had the opposite effects that enhance cell ROS production. Thus, the results indicated that SIRT1 serves an important role in the regulation of oxidative stress induced by LPS in human AECs.
Pseudomonas aeruginosa Quorum Sensing Molecule Alters Skeletal Muscle Protein Homeostasis by Perturbing the Antioxidant Defense System.
Bandyopadhaya Arunava,Tzika A Aria,Rahme Laurence G
Skeletal muscle function is compromised in many illnesses, including chronic infections. The quorum sensing (QS) signal, 2-amino acetophenone (2-AA), is produced during acute and chronic infections and excreted in human tissues, including the lungs of cystic fibrosis patients. We have shown that 2-AA facilitates pathogen persistence, likely via its ability to promote the formation of bacterial persister cells, and that it acts as an interkingdom immunomodulatory signal that epigenetically reprograms innate immune functions. Moreover, 2-AA compromises muscle contractility and impacts the expression of genes involved in reactive oxygen species (ROS) homeostasis in skeletal muscle and in mitochondrial functions. Here, we elucidate the molecular mechanisms of 2-AA's impairment of skeletal muscle function and ROS homeostasis. Murine and differentiated C2C12 myotube cell studies showed that 2-AA promotes ROS generation in skeletal muscle via the modulation of xanthine oxidase (XO) activity, NAD(P)H oxidase2 (NOX2) protein level, and the activity of antioxidant enzymes. ROS accumulation triggers the activity of AMP-activated protein kinase (AMPK), likely upstream of the observed locations of induction of ubiquitin ligases Muscle RING Finger 1 (MuRF1) and Muscle Atrophy F-box (MAFbx), and induces autophagy-related proteins. The protein-level perturbation in skeletal muscle of silent mating type information regulation 2 homolog 1 (SIRT1), peroxisome proliferator-activated receptor gamma coactivator 1 (PGC-1), and uncoupling protein 3 (UCP3) is rescued by the antioxidant N-acetyl-l-cysteine (NAC). Together, these results unveil a novel form of action of a QS bacterial molecule and provide molecular insights into the 2-AA-mediated skeletal muscle dysfunction caused by , a bacterium that is resistant to treatment, causes serious acute, persistent, and relapsing infections in humans. There is increasing evidence that bacterial excreted small molecules play a critical role during infection. We have shown that a quorum sensing (QS)-regulated excreted small molecule, 2-AA, which is abundantly produced by , promotes persistent infections, dampens host inflammation, and triggers mitochondrial dysfunction in skeletal muscle. QS is a cell-to-cell communication system utilized by bacteria to promote collective behaviors. The significance of our study in identifying a mechanism that leads to skeletal muscle dysfunction, via the action of a QS molecule, is that it may open new avenues in the control of muscle loss as a result of infection and sepsis. Given that QS is a common characteristic of prokaryotes, it is possible that 2-AA-like molecules promoting similar effects may exist in other pathogens.
Pseudomonas aeruginosa promotes autophagy to suppress macrophage-mediated bacterial eradication.
Wu Yongjian,Li Dandan,Wang Yi,Chen Kang,Yang Kun,Huang Xi,Zhang Yuanqing,Wu Minhao
OBJECTIVES:To explore the role of autophagy on macrophage-mediated phagocytosis and intracellular killing of Pseudomonas aeruginosa (PA), a common extracellular bacterium which often causes various opportunistic infections. METHODS:Macrophages were infected with PA or stimulated with zymosan bioparticles. Autophagy was tested by fluorescent microscopy and Western blot for LC3. Phagocytosis and killing efficiency were assessed by plate count assay, flow cytometry or immunofluorescent staining. Phagocytic receptor expression, ROS generation and NO production were examined by PCR, flow cytometry and Griess reaction, respectively. RESULTS:PA infection induced autophagy activation in both mouse and human macrophages. Induction of autophagy by rapamycin or starvation significantly inhibited PA internalization by downregulating phagocytosis receptor expression, and suppressed intracellular killing of PA via reducing ROS and NO production in macrophages. While knockdown of autophagy molecules ATG7 or Beclin1 enhanced macrophage-mediated phagocytosis and intracellular killing of PA. Additionally, confocal microscopy data showed that induction of autophagy reduced the number of phagosomes and phagolysosomes in macrophages after stimulation with zymosan bioparticles. CONCLUSIONS:Our study suggested that PA promotes autophagy to suppress macrophage-mediated bacterial phagocytosis and intracellular killing. These insights demonstrated a novel immune evasion mechanism employed by PA, which may provide potential therapeutic strategies of PA infectious diseases.
Antioxidant enzymes expression in Pseudomonas aeruginosa exposed to UV-C radiation.
Salma Kloula Ben Ghorbal,Lobna Maalej,Sana Khefacha,Kalthoum Chourabi,Imene Ouzari,Abdelwaheb Chatti
Journal of basic microbiology
It was well known that, UV-C irradiation increase considerably the reactive oxygen species (ROS) levels in eukaryotic and prokaryotic organisms. In the enzymatic ROS-scavenging pathways, superoxide dismutase (SOD), Catalase (CAT), and peroxidase (POX) were developed to deal with oxidative stress. In this study, we investigated the effects of UV-C radiations on antioxidant enzymes (catalase, superoxide dismutase, and peroxidases) expression in Pseudomonas aeruginosa. Catalase, superoxide dismutase, and peroxidases activities were determined spectrophotometrically. Isozymes of superoxide dismutase were revealed by native gel activity staining method. Lipid peroxidation was determined by measuring malondialdehyde formation. Our results showed that superoxide dismutase, catalase and peroxidase activities exhibited a gradual increase during the exposure time (30 min). However, the superoxide dismutase activity was maximized at 15 min. Native gel activity staining assays showed the presence of three superoxide dismutase isozymes. The iron-cofactored isoform activity was altered after exposure to UV-C stress. These finding suggest that catalase and peroxidase enzymes have the same importance toward UV-C rays at shorter and longer exposure times and this may confer additional protection to superoxide dismutase from damage caused by lipid peroxidation. Moreover, our data demonstrate the significant role of the antioxidant system in the resistance of this important human pathogen.
Exposure to low UVA doses increases KatA and KatB catalase activities, and confers cross-protection against subsequent oxidative injuries in Pseudomonas aeruginosa.
Pezzoni Magdalena,Tribelli Paula M,Pizarro Ramón A,López Nancy I,Costa Cristina S
Microbiology (Reading, England)
Solar UVA radiation is one of the main environmental stress factors for Pseudomonas aeruginosa. Exposure to high UVA doses produces lethal effects by the action of the reactive oxygen species (ROS) it generates. P. aeruginosa has several enzymes, including KatA and KatB catalases, which provide detoxification of ROS. We have previously demonstrated that KatA is essential in defending P. aeruginosa against high UVA doses. In order to analyse the mechanisms involved in the adaptation of this micro-organism to UVA, we investigated the effect of exposure to low UVA doses on KatA and KatB activities, and the physiological consequences. Exposure to UVA induced total catalase activity; assays with non-denaturing polyacrylamide gels showed that both KatA and KatB activities were increased by radiation. This regulation occurred at the transcriptional level and depended, at least partly, on the increase in H2O2 levels. We demonstrated that exposure to low UVA produced a protective effect against subsequent lethal doses of UVA, sodium hypochlorite and H2O2. Protection against lethal UVA depends on katA, whilst protection against sodium hypochlorite depends on katB, demonstrating that different mechanisms are involved in the defence against these oxidative agents, although both genes can be involved in the global cellular response. Conversely, protection against lethal doses of H2O2 could depend on induction of both genes and/or (an)other defensive factor(s). A better understanding of the adaptive response of P. aeruginosa to UVA is relevant from an ecological standpoint and for improving disinfection strategies that employ UVA or solar irradiation.
Phytol has antibacterial property by inducing oxidative stress response in Pseudomonas aeruginosa.
Lee Wonjong,Woo Eun-Rhan,Lee Dong Gun
Free radical research
Phytol, isolated from Aster yomena, is widely distributed as a constituent of chlorophyll. In the present study, we confirmed the antibacterial activity of phytol and its mechanism inducing oxidative cell death in Pseudomonas aeruginosa. In phytol-treated cells, elevated level of intracellular reactive oxygen species (ROS) and transient NADH depletion were observed. These results demonstrated that phytol induced ROS accumulation and that the electron transport chain was involved in increase of ROS. Due to this ROS generation, the imbalance developed between intracellular ROS and the antioxidant defense system, leading to decrease of reduced glutathione (GSH). Moreover, severe DNA damage was shown after treatment with phytol. DNA electrophoresis and a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay were conducted with pretreatment with the antioxidant N-acetylcysteine (NAC) to evaluate the cause of DNA damage. In NAC-pretreated cells, alleviated damage was confirmed and it supports that phytol induces oxidative stress-mediated DNA damage. In conclusion, phytol exerts the antibacterial property via inducing oxidative stress response in P. aeruginosa.
Expression analysis of the Pseudomonas aeruginosa AlgZR two-component regulatory system.
Pritchett Christopher L,Little Alexander S,Okkotsu Yuta,Frisk Anders,Cody William L,Covey Christopher R,Schurr Michael J
Journal of bacteriology
Pseudomonas aeruginosa virulence components are subject to complex regulatory control primarily through two-component regulatory systems that allow for sensing and responding to environmental stimuli. In this study, the expression and regulation of the P. aeruginosa AlgZR two-component regulatory system were examined. Primer extension and S1 nuclease protection assays were used to identify two transcriptional initiation sites for algR within the algZ coding region, and two additional start sites were identified upstream of the algZ coding region. The two algR transcriptional start sites, RT1 and RT2, are directly regulated by AlgU, consistent with previous reports of increased algR expression in mucoid backgrounds, and RpoS additionally plays a role in algR transcription. The expression of the first algZ promoter, ZT1, is entirely dependent upon Vfr for expression, whereas Vfr, RpoS, or AlgU does not regulate the second algZ promoter, ZT2. Western blot, real-time quantitative PCR (RT-qPCR), and transcriptional fusion analyses show that algZR expression is Vfr dependent. The algZ and algR genes also are cotranscribed in both nonmucoid and mucoid backgrounds. Furthermore, algZR was found to be cotranscribed with hemCD by RT-PCR. RT-qPCR confirmed that hemC transcription in the PAO1 ΔalgZ mutant was 40% of the level of the wild-type strain. Taken together, these results indicate that algZR transcription involves multiple factors at multiple start sites that control individual gene expression as well as coexpression of this two-component system with heme biosynthetic genes.
Co-expressional conservation in virulence and stress related genes of three Gammaproteobacterial species: Escherichia coli, Salmonella enterica and Pseudomonas aeruginosa.
Hosseinkhan Nazanin,Zarrineh Peyman,Rokni-Zadeh Hassan,Ashouri Mohammad Reza,Masoudi-Nejad Ali
Gene co-expression analysis is one of the main aspects of systems biology that uses high-throughput gene expression data. In the present study we applied cross-species co-expressional analysis on a module of biofilm and stress response associated genes. We addressed different kinds of stresses in three most intensively studied members of Gammaproteobacteria including Escherichia coli K12, Pseudomonas aeruginosa PAO1 and Salmonella enterica for which large sets of gene expression data are available. Our aim was to evaluate the presence of common stress response strategies adopted by these microorganisms that may be assigned to the other members of Gammaproteobacteria. Results of functional annotation analysis revealed distinct categories among co-expressed genes, most of which concerned biological processes associated with virulence and stress response. Transcriptional regulatory analysis of genes present in co-expressed modules showed that the global stress sigma factor, RpoS, besides several local transcription factors accounts for the observed co-expressional response, and that several cases of feed-forward loops exist between global regulators, local transcription factors and their targets. Our results lend partial support to our underlying assumption of the conservation of core biological processes and regulatory interactions among these related Gammaproteobacteria members. This has led to the implementation of transferring gene function annotations from well-studied Gammaproteobacterial species to less-characterized members. These findings can shed light on the discovery of new drug targets capable of controlling severe infections caused by these groups of bacteria.
Carbon Starvation Induces the Expression of PprB-Regulated Genes in Pseudomonas aeruginosa.
Wang Congcong,Chen Wenhui,Xia Aiguo,Zhang Rongrong,Huang Yajia,Yang Shuai,Ni Lei,Jin Fan
Applied and environmental microbiology
can cause severe infections in humans. This bacterium often adopts a biofilm lifestyle that is hard to treat. In several previous studies, the PprA-PprB two-component system (TCS), which controls the expression of type IVb pili, BapA adhesin, and CupE fimbriae, was shown to be involved in biofilm formation (M. Romero, H. Silistre, L. Lovelock, V. J. Wright, K.-G. Chan, et al., Nucleic Acids Res 46:6823-6840, 2018, https://doi.org/10.1093/nar/gky324; S. de Bentzmann, C. Giraud, C. S. Bernard, V. Calderon, F. Ewald F, et al., PLoS Pathog 8:e1003052, 2012, https://doi.org/10.1371/journal.ppat.1003052). However, signals or environmental conditions that can trigger the PprA-PprB TCS are still unknown, and the molecular mechanisms of PprB-mediated biofilm formation are poorly characterized. Here, we report that carbon starvation stress (CSS) can induce the expression of and genes in the PprB regulon. CSS-induced transcription is mediated by the stress response sigma factor RpoS rather than the two-component sensor PprA. We also observed a strong negative regulation of PprB on the transcription of itself. Further experiments showed that PprB overexpression greatly enhanced cell-cell adhesion (CCA) and cell-surface adhesion (CSA) in Specifically, under the background of PprB overexpression, both the BapA adhesin and CupE fimbriae displayed positive effects on CCA and CSA, while the type IVb pili showed an unexpected negative effect on CCA and no effect on CSA. In addition, expression of the PprB regulon genes were significantly increased in 3-day colony biofilms, indicating a possible carbon limitation state. The CSS-RpoS-PprB-Bap/Flp/CupE pathway identified in this study provides a new perspective on the process of biofilm formation in carbon-limited environments. Typically, the determination of the external signals that can trigger a regulatory system is crucial to understand the regulatory logic and inward function of that system. The PprA-PprB two-component system was reported to be involved in biofilm formation in , but the signals triggering this system are unknown. In this study, we found that carbon starvation stress (CSS) induces transcription of and genes in the PprB regulon through an RpoS-dependent pathway. Increased PprB expression leads to enhanced cell-cell adhesion (CCA) and cell-surface adhesion (CSA) in Both CCA and CSA are largely dependent on the Bap secretion system and are moderately dependent on the CupE fimbriae. Our findings suggest that PprB reinforces the structure of biofilms under carbon-limited conditions, and the Bap secretion system and CupE fimbriae are two potential targets for biofilm treatment.
Expression stability of 13 housekeeping genes during carbon starvation of Pseudomonas aeruginosa.
Alqarni Budoor,Colley Brendan,Klebensberger Janosch,McDougald Diane,Rice Scott A
Journal of microbiological methods
Quantitative real-time polymerase chain reaction (qRT-PCR) is a reliable technique for quantifying mRNA levels when normalised by a stable reference gene/s. Many putative reference genes are known to be affected by physiological stresses, such as nutrient limitation and hence may not be suitable for normalisation. In this study of Pseudomonas aeruginosa, the expression of 13 commonly used reference genes, rpoS, proC, recA, rpsL, rho, oprL, anr, tipA, nadB, fabD, ampC, algD and gyrA, were analysed for changes in expression under carbon starvation and nutrient replete conditions. The results showed that rpoS was the only stably expressed housekeeping gene during carbon starvation. In contrast, other commonly used housekeeping genes were shown to vary by as much as 10-100 fold under starvation conditions. This study has identified a suitable reference gene for qRT-PCR in P. aeruginosa during carbon starvation. The results presented here highlight the need to validate housekeeping genes under the chosen experimental conditions.
Contribution of stress responses to antibiotic tolerance in Pseudomonas aeruginosa biofilms.
Stewart Philip S,Franklin Michael J,Williamson Kerry S,Folsom James P,Boegli Laura,James Garth A
Antimicrobial agents and chemotherapy
Enhanced tolerance of biofilm-associated bacteria to antibiotic treatments is likely due to a combination of factors, including changes in cell physiology as bacteria adapt to biofilm growth and the inherent physiological heterogeneity of biofilm bacteria. In this study, a transcriptomics approach was used to identify genes differentially expressed during biofilm growth of Pseudomonas aeruginosa. These genes were tested for statistically significant overlap, with independently compiled gene lists corresponding to stress responses and other putative antibiotic-protective mechanisms. Among the gene groups tested were those associated with biofilm response to tobramycin or ciprofloxacin, drug efflux pumps, acyl homoserine lactone quorum sensing, osmotic shock, heat shock, hypoxia stress, and stationary-phase growth. Regulons associated with Anr-mediated hypoxia stress, RpoS-regulated stationary-phase growth, and osmotic stress were significantly enriched in the set of genes induced in the biofilm. Mutant strains deficient in rpoS, relA and spoT, or anr were cultured in biofilms and challenged with ciprofloxacin and tobramycin. When challenged with ciprofloxacin, the mutant strain biofilms had 2.4- to 2.9-log reductions in viable cells compared to a 0.9-log reduction of the wild-type strain. Interestingly, none of the mutants exhibited a statistically significant alteration in tobramycin susceptibility compared to that with the wild-type biofilm. These results are consistent with a model in which multiple genes controlled by overlapping starvation or stress responses contribute to the protection of a P. aeruginosa biofilm from ciprofloxacin. A distinct and as yet undiscovered mechanism protects the biofilm bacteria from tobramycin.
Effects of Chlorine Stress on Pseudomonas aeruginosa Biofilm and Analysis of Related Gene Expressions.
Kekeç Özge,Gökalsın Barış,Karaltı İskender,Kayhan Figen Esin,Sesal Nüzhet Cenk
Chlorine is deployed worldwide to clean waters and prevent water-originated illnesses. However, chlorine has a limited disinfection capacity against biofilms. Microorganisms form biofilms to protect themselves from biological threats such as disinfectant chemicals. Pseudomonas aeruginosa is an opportunistic pathogen and its biofilm form attaches to surfaces, living buried into exopolysaccharides, can be present in all watery environments including tap water and drinking water. This research aimed to study the biofilm trigger mechanism of the opportunistic pathogen P. aeruginosa PAO1 strain, which is known to form biofilm in water supply systems and human body, under chlorine stress levels. In addition to biofilm staining, certain genes that are relevant to the stress condition were selected for gene expression analysis. The bacteria cultures were grown under chlorine stress with concentrations of 0.5, 0.7 and 1 mg/l. Six gene regions were determined related to biofilm and stress response: rpoS, bifA, migA, katB, soxR, and algC. Biofilm formation was analyzed by basic fuchsin staining, and gene expressions were quantified by quantitative real-time PCR. According to the results, highest biofilm production was observed in P. aeruginosa PAO1 wild strain under no stress conditions. Higher biofilm amounts were observed for bacteria under 0.5 and 0.7 mg/l chlorine stress compared to 1 mg/l chlorine stress.
Marker genes for the metabolic adaptation of Pseudomonas aeruginosa to the hypoxic cystic fibrosis lung environment.
Eichner Anja,Günther Nicole,Arnold Martin,Schobert Max,Heesemann Jürgen,Hogardt Michael
International journal of medical microbiology : IJMM
Pseudomonas aeruginosa is the leading pathogen of chronic cystic fibrosis (CF) lung infection. Life-long persistence in the inflamed and ever fluctuating CF lungs results in the selection of a variety of changes in P. aeruginosa physiology. Accumulating evidence suggests that especially metabolic changes support the survival and growth of P. aeruginosa within the hypoxic and nutritious CF mucus. To investigate if metabolic adaptations we described for hypermutable P. aeruginosa from late CF lung disease (Hoboth et al., 2009. J. Infect. Dis., pp. 118-130) may represent specific changes in response to the selective conditions within the oxygen-restricted CF mucus, we determined the expression of a set of genes during aerobic and hypoxic growth in LB and the artificial sputum medium ASM. We further focused on the regulation of the two isocitrate dehydrogenases Icd and Idh. Interestingly, both isoenzymes may replace each other under aerobic and hypoxic conditions. The NADPH- and RpoS-dependent Icd seems to be the leading isoenzyme under prolonged oxygen limitation and stationary growth phase. LacZ reporter analysis revealed that oxygen-restriction increased the expression levels of azu, cbb3-1, cbb3-2, ccpR, icd, idh and oprF gene, whereas himD and nuoA are increasingly expressed only during hypoxic growth in ASM. Overexpression of the anaerobic regulator Anr improved the expression of azu, ccpR, cbb3-2 and icd. In summary, expression of azu, cbb3-1, cbb3-2, ccpR, icd, idh, oprF, himD, and nuoA appeared to be beneficial for the growth of P. aeruginosa under hypoxic conditions indicating these genes may represent marker genes for the metabolic adaptation to the CF lung environment.
RpoN Modulates Carbapenem Tolerance in Pseudomonas aeruginosa through Pseudomonas Quinolone Signal and PqsE.
Viducic Darija,Murakami Keiji,Amoh Takashi,Ono Tsuneko,Miyake Yoichiro
Antimicrobial agents and chemotherapy
The ability of Pseudomonas aeruginosa to rapidly modulate its response to antibiotic stress and persist in the presence of antibiotics is closely associated with the process of cell-to-cell signaling. The alternative sigma factor RpoN (σ(54)) is involved in the regulation of quorum sensing (QS) and plays an important role in the survival of stationary-phase cells in the presence of carbapenems. Here, we demonstrate that a ΔrpoN mutant grown in nutrient-rich medium has increased expression of pqsA, pqsH, and pqsR throughout growth, resulting in the increased production of the Pseudomonas quinolone signal (PQS). The link between pqsA and its role in carbapenem tolerance was studied using a ΔrpoN ΔpqsA mutant, in which the carbapenem-tolerant phenotype of the ΔrpoN mutant was abolished. In addition, we demonstrate that another mechanism leading to carbapenem tolerance in the ΔrpoN mutant is mediated through pqsE Exogenously supplied PQS abolished the biapenem-sensitive phenotype of the ΔrpoN ΔpqsA mutant, and overexpression of pqsE failed to alter the susceptibility of the ΔrpoN ΔpqsA mutant to biapenem. The mutations in the ΔrpoN ΔrhlR mutant and the ΔrpoN ΔpqsH mutant led to susceptibility to biapenem. Comparison of the changes in the expression of the genes involved in QS in wild-type PAO1 with their expression in the ΔrpoN mutant and the ΔrpoN mutant-derived strains demonstrated the regulatory effect of RpoN on the transcript levels of rhlR, vqsR, and rpoS The findings of this study demonstrate that RpoN negatively regulates the expression of PQS in nutrient-rich medium and provide evidence that RpoN interacts with pqsA, pqsE, pqsH, and rhlR in response to antibiotic stress.
Connected partner-switches control the life style of Pseudomonas aeruginosa through RpoS regulation.
Bouillet Sophie,Ba Moly,Houot Laetitia,Iobbi-Nivol Chantal,Bordi Christophe
Biofilm formation is a complex process resulting from the action of imbricated pathways in response to environmental cues. In this study, we showed that biofilm biogenesis in the opportunistic pathogen Pseudomonas aeruginosa depends on the availability of RpoS, the sigma factor regulating the general stress response in bacteria. Moreover, it was demonstrated that RpoS is post-translationally regulated by the HsbR-HsbA partner switching system as has been demonstrated for its CrsR-CrsA homolog in Shewanella oneidensis. Finally, it was established that HsbA, the anti-sigma factor antagonist, has a pivotal role depending on its phosphorylation state since it binds HsbR, the response regulator, when phosphorylated and FlgM, the anti-sigma factor of FliA, when non-phosphorylated. The phosphorylation state of HsbA thus drives the switch between the sessile and planktonic way of life of P. aeruginosa by driving the release or the sequestration of one or the other of these two sigma factors.
[Regulation of pyocyanin biosynthesis by transcriptional factor sigma38 in Pseudomonas aeruginosa PAO1].
Miao Jing,Chi Xiaoyan,Wang Yanhua,Feng Zhibin,Xue Wenwen,Huang Run,Zhang Haoyi,Tian Lingqian,Zhang Hongqian,Zhai Junjie,Ge Yihe
Wei sheng wu xue bao = Acta microbiologica Sinica
Pyocyanin, an important virulence factor, is synthesized and secreted by Pseudomonas aeruginosa PAO1and plays a critical role in pathogen-host interaction during infection. Sigma38 (σ38, σS) is a central regulator for many virulence production in pathogens. Objective:Our aim is to identify expression and regulation of two phenazine-producing operons mediated by the sigma38 factor in Pseudomonas aeruginosa PAO1. Methods:We first cloned the flanking fragments of rpoS from the chromosomal DNA of P. aeruginosa PAO1 and constructed the deletion mutant ΔrpoS with the insertion of gentamycin resistance cassette (aacC1). Complementation of rpoS was then carried out after construction and introduction of pME10S (containing the whole rpoS region). Finally, we created the mutant ΔrpoSphz1 and ΔrpoSphz2, and measured pyocyanin production by these mutants in GA medium, using the parental strain Δphz1 and Δphz2 as controls. Results:In GA medium, pyocyanin production by mutant ΔrpoS increased dramatically in comparison with the wild-type strain PAO1. Production of pyocyanin, however, was decreased to the level of the wild-type strain with complementation of the derivative ΔrpoS harboring pME10S. Mutant ΔrpoSphz2 produced much more pyocyanin than mutant Δphz2. Mutant ΔrpoSphz1, however, produced much less pyocyanin than mutant Δphz1. Conclusion:By positively regulating the expression of phz2 and negatively regulating the phz1, sigma38 factor exerts negative modulation on pyocyanin biosynthesis in P. aeruginosa PAO1.
The small RNA ReaL: a novel regulatory element embedded in the Pseudomonas aeruginosa quorum sensing networks.
Carloni Sara,Macchi Raffaella,Sattin Sara,Ferrara Silvia,Bertoni Giovanni
The small RNA ReaL of the opportunistic pathogen Pseudomonas aeruginosa has been characterized. Our results indicate that ReaL contributes to P. aeruginosa virulence. In the Galleria mellonella infection model, reaL gene deletion resulted in decreased virulence, while ReaL overexpression resulted in a hyper-virulent phenotype. We also demonstrate that ReaL is embedded in the P. aeruginosa quorum sensing (QS) with the role of linking las to pqs systems. We show that ReaL is negatively regulated by the las regulator LasR and impacts positively the synthesis of the pqs quinolone signal PQS by a positive post-transcriptional effect on the pqsC gene. Perturbations of ReaL levels affect pyocyanin synthesis, biofilm formation and swarming motility, processes that are known to be influenced by PQS synthesis. In addition to being regulated by LasR, ReaL is also responding to infection relevant cues that P. aeruginosa can experience in mammalian hosts such as temperature and oxygen availability. Furthermore, ReaL shows a growth phase-dependent pattern of expression, being up-regulated in stationary phase, due to the activity of the alternative σ factor RpoS. Together, these regulations of ReaL expression are expected to contribute to the fine co-modulation of PQS synthesis and, ultimately, virulence.
Role of the interplay between quorum sensing regulator VqsR and the Pseudomonas quinolone signal in mediating carbapenem tolerance in Pseudomonas aeruginosa.
Viducic Darija,Murakami Keiji,Amoh Takashi,Ono Tsuneko,Miyake Yoichiro
Research in microbiology
Pseudomonas aeruginosa coordinates its response to environmental conditions through activation of a quorum sensing (QS) system. In this study, we investigated the regulatory interaction between the QS transcriptional regulator VqsR and the Pseudomonas quinolone signal (PQS) through integration of sigma factor RpoS, and we addressed whether one of the pathways controlling carbapenem tolerance can be attributed to VqsR. We demonstrate that vqsR expression at the transcriptional level is regulated by pqsA, pqsR, and pqsE. Assessment of the transcriptional expression of vqsR, lasI, rhlI, and qscR in ΔpqsA and ΔpqsAΔrpoS mutants provided insight into pqsA- and rpoS-dependent regulation of vqsR and vqsR-controlled genes. Exogenously supplemented PQS reversed expression of vqsR and vqsR-controlled genes in the ΔpqsA mutant to wild-type levels, but failed to increase expression levels of lasI and qscR in the ΔpqsAΔrpoS mutant to levels observed in wild-type PAO1. The ΔvqsR mutant showed reduced survival when challenged with carbapenems compared to wild-type PAO1. Introduction of a pqsA mutation into the ΔvqsR mutant completely abolished its carbapenem-sensitive phenotype. We conclude that a regulatory link between PQS and vqsR exists, and that RpoS is important in their interaction. We also demonstrate that VqsR affects carbapenem tolerance.
RpoS-dependent sRNA RgsA regulates Fis and AcpP in Pseudomonas aeruginosa.
Lu Pei,Wang Yifei,Zhang Yong,Hu Yangbo,Thompson Karl M,Chen Shiyun
RgsA is a phylogenetically conserved small regulatory RNA (sRNA) in Pseudomonas species. This sRNA has been shown to be directly controlled by the major stationary phase and stress sigma factor σ (RpoS), and also indirectly regulated by the GacS/GacA two-component system. However, the role and the regulatory targets of this sRNA remain unclear. Here, two direct regulatory targets of RgsA, the mRNAs coding for the global transcriptional regulator Fis and the acyl carrier protein AcpP, were identified in P. aeruginosa. RgsA downregulates the synthesis of Fis and AcpP by base-pairing, and this regulation requires the RNA chaperone protein Hfq. Alignment of RgsA homologs in Pseudomonas revealed a conserved core region, which is strictly required for RgsA target recognition. Specifically, RgsA inhibits fis expression via an 11 + 11 bp RNA duplex, whereas this interaction region is not sufficient for repression and the 35 nt downstream region is also required. Interestingly, two functional start codons initiate fis mRNA translation and both are repressed by RgsA. Furthermore, deletion of rgsA significantly increased swarming motility in P. aeruginosa. Together, this study suggests a novel regulatory role of sRNA in which the versatile transcriptional regulator Fis and the stress regulator RpoS are connected by RgsA.
In silico analysis and molecular modeling of RNA polymerase, sigma S (RpoS) protein in Pseudomonas aeruginosa PAO1.
Sedighi Mansour,Moghoofei Mohsen,Kouhsari Ebrahim,Pournajaf Abazar,Emadi Behzad,Tohidfar Masoud,Gholami Mehrdad
Reports of biochemistry & molecular biology
BACKGROUND:Sigma factors are proteins that regulate transcription in bacteria. Sigma factors can be activated in response to different environmental conditions. The rpoS (RNA polymerase, sigma S) gene encodes sigma-38 (σ38, or RpoS), a 37.8 kDa protein in Pseudomonas aeruginosa (P. aeruginosa) strains. RpoS is a central regulator of the general stress response and operates in both retroactive and proactive manners; not only does it allow the cell to survive environmental challenges; it also prepares the cell for subsequent stresses (cross-protection). METHODS:The significance of RpoS for stress resistance and protein expression in stationary-phase P. aeruginosa cells was assessed. The goal of the current study was to characterize RpoS of P. aeruginosa PAO1 using bioinformatics tools. RESULTS:The results showed that RpoS is an unstable protein that belongs to the sigma-70 factor family. Secondary structure analysis predicted that random coil is the predominant structure followed by extended alpha helix. The three-dimensional (3D) structure was modeled using SWISS-MODEL Workspace. CONCLUSION:Determination of sequence, function, structure, and predicted epitopes of RpoS is important for modeling of inhibitors that will help in the design of new drugs to combat multi-drug-resistant (MDR) strains. Such information may aid in the development of new diagnostic tools, drugs, and vaccines for treatment in endemic regions.
Negative Control of RpoS Synthesis by the sRNA ReaL in .
Thi Bach Nguyen Hue,Romero A David,Amman Fabian,Sorger-Domenigg Theresa,Tata Muralidhar,Sonnleitner Elisabeth,Bläsi Udo
Frontiers in microbiology
(Pae) is an opportunistic human pathogen, able to resist host defense mechanisms and antibiotic treatment. In Pae, the master regulator of stress responses RpoS (σ) is involved in the regulation of quorum sensing and several virulence genes. Here, we report that the sRNA ReaL translationally silences mRNA, which results in a decrease of the RpoS levels. Our studies indicated that ReaL base-pairs with the Shine-Dalgarno region of mRNA. These studies are underlined by a highly similar transcription profile of a deletion mutant and a over-expressing strain.
LasR Might Act as an Intermediate in Overproduction of Phenazines in the Absence of RpoS in .
He Qiuning,Feng Zhibin,Wang Yanhua,Wang Kewen,Zhang Kailu,Kai Le,Hao Xiuying,Yu Zhifen,Chen Lijuan,Ge Yihe
Journal of microbiology and biotechnology
As an opportunistic bacterial pathogen, PAO1 contains two phenazineproducing gene operons, () and (), 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 operons and regulates pyocyanin biosynthesis in detail. In this study, with deletion of the gene in the PAΔ mutant and the PAΔ mutant respectively, we demonstrated that RpoS exerted opposite regulatory roles on the expression of the and operons. We also confirmed that the operon played a critical role and especially biosynthesized much more phenazines than the operon when the gene was knocked out in . By constructing the translational reporter fusion vector - and the chromosomal fusion mutant PAΔ::, we verified that RpoS deficiency caused increased expression of , a transcription regulator gene in a first quorum sensing system () that activates overexpression of the operon, suggesting that in the absence of RpoS, LasR might act as an intermediate in overproduction of phenazine biosynthesis mediated by the operon in .
RpoS differentially affects the general stress response and biofilm formation in the endophytic Serratia plymuthica G3.
Liu Xiaoguang,Wu Yan,Chen Yuanyuan,Xu Fang,Halliday Nigel,Gao Kexiang,Chan Kok Gan,Cámara Miguel
Research in microbiology
The σ(S) subunit RpoS of RNA polymerase functions as a master regulator of the general stress response in Escherichia coli and related bacteria. RpoS has been reported to modulate biocontrol properties in the rhizobacterium Serratia plymuthica IC1270. However, the role of RpoS in the stress response and biofilm formation in S. plymuthica remains largely unknown. Here we studied the role of RpoS from an endophytic S. plymuthica G3 in regulating these phenotypes. Mutational analysis demonstrated that RpoS positively regulates the global stress response to acid or alkaline stresses, oxidative stress, hyperosmolarity, heat shock and carbon starvation, in addition to proteolytic and chitinolytic activities. Interestingly, rpoS mutations resulted in significantly enhanced swimming motility, biofilm formation and production of the plant auxin indole-3-acetic acid (IAA), which may contribute to competitive colonization and environmental fitness for survival. These findings provide further insight into the strain-specific role of RpoS in the endophytic strain G3 of S. plymuthica, where it confers resistance to general stresses encountered within the plant environment. The heterogeneous functionality of RpoS in rhizosphere and endophytic S. plymuthica populations may provide a selective advantage for better adaptation to various physiological and environmental stresses.
Role of RpoS in stress resistance, quorum sensing and spoilage potential of Pseudomonas fluorescens.
Liu Xiaoxiang,Ji Lei,Wang Xu,Li Jianrong,Zhu Junli,Sun Aihua
International journal of food microbiology
Microorganism activities are considered the main cause of most food spoilage, leading to great economic losses. Pseudomonas fluorescens is a Gram-negative bacterium that is widely found in food and has high spoilage activity. RpoS is considered an important global regulator involved in stress survival and virulence in many pathogens. Thus, it is very possible that RpoS plays an important role in spoilage regulation in P. fluorescens. In this study an in-frame deletion mutation of rpoS was constructed to explore its function in P. fluorescens. The results showed that RpoS positively regulated the resistance of P. fluorescens to HO, heat, ethanol and crystal violet, negatively regulated the resistance to acetic acid, and had no effect on the resistance to NaCl. Further studies indicated that acylated homoserine lactone (AHL) production and the transcription levels of five AHL-related genes were significantly decreased in the rpoS mutant compared with those in the wild-type strain. Interestingly, the two homologous genes coding for AHL synthases contained RpoS-dependent -10 elements, suggesting that AHL quorum sensing is directly regulated by RpoS. RpoS also contributed to the spoilage activities of P. fluorescens by regulating extracellular protease and total volatile basic nitrogen (TVB-N) production in sterilized salmon juice. Our results reveal that RpoS was a key regulatory factor involved in stress resistance, the AHL quorum sensing system, and spoilage potential of P. fluorescens. Our study may benefit food safety control and food preservation.
Pseudomonas aeruginosa Effector ExoS Inhibits ROS Production in Human Neutrophils.
Vareechon Chairut,Zmina Stephanie Elizabeth,Karmakar Mausita,Pearlman Eric,Rietsch Arne
Cell host & microbe
Neutrophils are the first line of defense against bacterial infections, and the generation of reactive oxygen species is a key part of their arsenal. Pathogens use detoxification systems to avoid the bactericidal effects of reactive oxygen species. Here we demonstrate that the Gram-negative pathogen Pseudomonas aeruginosa is susceptible to reactive oxygen species but actively blocks the reactive oxygen species burst using two type III secreted effector proteins, ExoS and ExoT. ExoS ADP-ribosylates Ras and prevents it from interacting with and activating phosphoinositol-3-kinase (PI3K), which is required to stimulate the phagocytic NADPH-oxidase that generates reactive oxygen species. ExoT also affects PI3K signaling via its ADP-ribosyltransferase activity but does not act directly on Ras. A non-ribosylatable version of Ras restores reactive oxygen species production and results in increased bacterial killing. These findings demonstrate that subversion of the host innate immune response requires ExoS-mediated ADP-ribosylation of Ras in neutrophils.
phz1 contributes much more to phenazine-1-carboxylic acid biosynthesis than phz2 in Pseudomonas aeruginosa rpoS mutant.
Sun Longshuo,Chi Xiaoyan,Feng Zhibin,Wang Kewen,Kai Le,Zhang Kailu,Cheng Shiwei,Hao Xiuying,Xie Weihai,Ge Yihe
Journal of basic microbiology
Pseudomonas aeruginosa PAO1, a common opportunistic bacterial pathogen, contains two phenazine-biosynthetic operons, phz1 (phzA B C D E F G ) and phz2 (phzA B C D E F G ). Each of two operons can independently encode a set of enzymes involving in the biosynthesis of phenazine-1-carboxylic acid. As a global transcriptional regulator, RpoS mediates a lot of genes involving secondary metabolites biosynthesis in many bacteria. In an other previous study, it was reported that RpoS deficiency caused overproduction of pyocyanin, a derivative of phenazine-1-carboxylic acid in P. aeruginosa PAO1. But it is not known how RpoS mediates the expression of each of two phz operons and modulates phenazine-1-carboxylic acid biosynthesis in detail. In this study, by deleting the rpoS gene in the mutant PNΔphz1 and the mutant PNΔphz2, we found that the phz1 operon contributes much more to phenazine-1-carboxylic acid biosynthesis than the phz2 operon in the absence of RpoS. With the construction of the translational and transcriptional fusion vectors with the truncated lacZ reporter gene, we demonstrated that RpoS negatively regulates the expression of phz1 and positively controls the expression of phz2, and the regulation of phenazine-1-carboxylic acid biosynthesis mediated by RopS occurs at the posttranscriptional level, not at the transcriptional level. Obviously, two copies of phz operons and their differential expression mediated by RpoS might help P. aeruginosa adapt to its diverse environments and establish infection in its hosts.
Novel RpoS-Dependent Mechanisms Strengthen the Envelope Permeability Barrier during Stationary Phase.
Mitchell Angela M,Wang Wei,Silhavy Thomas J
Journal of bacteriology
Gram-negative bacteria have effective methods of excluding toxic compounds, including a largely impermeable outer membrane (OM) and a range of efflux pumps. Furthermore, when cells become nutrient limited, RpoS enacts a global expression change providing cross-protection against many stresses. Here, we utilized sensitivity to an anionic detergent (sodium dodecyl sulfate [SDS]) to probe changes occurring to the cell's permeability barrier during nutrient limitation. Escherichia coli is resistant to SDS whether cells are actively growing, carbon limited, or nitrogen limited. In actively growing cells, this resistance depends on the AcrAB-TolC efflux pump; however, this pump is not necessary for protection under either carbon-limiting or nitrogen-limiting conditions, suggesting an alternative mechanism(s) of SDS resistance. In carbon-limited cells, RpoS-dependent pathways lessen the permeability of the OM, preventing the necessity for efflux. In nitrogen-limited but not carbon-limited cells, the loss of rpoS can be completely compensated for by the AcrAB-TolC efflux pump. We suggest that this difference simply reflects the fact that nitrogen-limited cells have access to a metabolizable energy (carbon) source that can efficiently power the efflux pump. Using a transposon mutant pool sequencing (Tn-Seq) approach, we identified three genes, sanA, dacA, and yhdP, that are necessary for RpoS-dependent SDS resistance in carbon-limited stationary phase. Using genetic analysis, we determined that these genes are involved in two different envelope-strengthening pathways. These genes have not previously been implicated in stationary-phase stress responses. A third novel RpoS-dependent pathway appears to strengthen the cell's permeability barrier in nitrogen-limited cells. Thus, though cells remain phenotypically SDS resistant, SDS resistance mechanisms differ significantly between growth states. IMPORTANCE:Gram-negative bacteria are intrinsically resistant to detergents and many antibiotics due to synergistic activities of a strong outer membrane (OM) permeability barrier and efflux pumps that capture and expel toxic molecules eluding the barrier. When the bacteria are depleted of an essential nutrient, a program of gene expression providing cross-protection against many stresses is induced. Whether this program alters the OM to further strengthen the barrier is unknown. Here, we identify novel pathways dependent on the master regulator of stationary phase that further strengthen the OM permeability barrier during nutrient limitation, circumventing the need for efflux pumps. Decreased permeability of nutrient-limited cells to toxic compounds has important implications for designing new antibiotics capable of targeting Gram-negative bacteria that may be in a growth-limited state.
RgsA, an RpoS-dependent sRNA, negatively regulates rpoS expression in Pseudomonas aeruginosa.
Lu Pei,Wang Yifei,Hu Yangbo,Chen Shiyun
Microbiology (Reading, England)
As a master regulator, the alternative sigma factor RpoS coordinates the transcription of genes associated with protection against environmental stresses in bacteria. In Pseudomonas aeruginosa, RpoS is also involved in quorum sensing and virulence. The cellular RpoS level is regulated at multiple levels, whereas the post-transcriptional regulation of rpoS in P. aeruginosa remains unclear. To identify and characterize small regulatory RNAs (sRNAs) regulating RpoS in P. aeruginosa, an sRNA library expressing a total of 263 sRNAs was constructed to examine their regulatory roles on rpoS expression. Our results demonstrate that rpoS expression is repressed by the RpoS-dependent sRNA RgsA at the post-transcriptional level. Unlike OxyS, an sRNA previously known to repress rpoS expression under oxidative stress in Escherichia coli, RgsA represses rpoS expression during the exponential phase. This repression requires the RNA chaperone Hfq. Furthermore, the 71-77 conserved region of RgsA is necessary for full repression of rpoS expression, and the -25 to +27 region of rpoS mRNA is sufficient for RgsA-mediated rpoS repression. Together, our results not only add RgsA to the RpoS regulatory circuits but also highlight the complexity of interplay between sRNAs and transcriptional regulators in bacteria.
phoU inactivation in Pseudomonas aeruginosa enhances accumulation of ppGpp and polyphosphate.
de Almeida Luiz Gustavo,Ortiz Julia Helena,Schneider René P,Spira Beny
Applied and environmental microbiology
Inorganic polyphosphate (polyP) is a linear polymer composed of several molecules of orthophosphate (Pi) linked by energy-rich phosphoanhydride bonds. In Pseudomonas aeruginosa, Pi is taken up by the ABC transporter Pst, encoded by an operon consisting of five genes. The first four genes encode proteins involved in the transport of Pi and the last gene of the operon, phoU, codes for a protein which exact function is unknown. We show here that the inactivation of phoU in P. aeruginosa enhanced Pi removal from the medium and polyP accumulation. The phoU mutant also accumulated high levels of the alarmone guanosine tetraphosphate (ppGpp), which in turn increased the buildup of polyP. In addition, phoU inactivation had several pleiotropic effects, such as reduced growth rate and yield and increased sensitivity to antibiotics and stresses. However, biofilm formation was not affected by the phoU mutation.
Superoxide dismutase activity confers (p)ppGpp-mediated antibiotic tolerance to stationary-phase .
Martins Dorival,McKay Geoffrey,Sampathkumar Gowthami,Khakimova Malika,English Ann M,Nguyen Dao
Proceedings of the National Academy of Sciences of the United States of America
Metabolically quiescent bacteria represent a large proportion of those in natural and host environments, and they are often refractory to antibiotic treatment. Such drug tolerance is also observed in the laboratory during stationary phase, when bacteria face stress and starvation-induced growth arrest. Tolerance requires (p)ppGpp signaling, which mediates the stress and starvation stringent response (SR), but the downstream effectors that confer tolerance are unclear. We previously demonstrated that the SR is linked to increased antioxidant defenses in We now demonstrate that superoxide dismutase (SOD) activity is a key factor in SR-mediated multidrug tolerance in stationary-phase Inactivation of the SR leads to loss of SOD activity and decreased multidrug tolerance during stationary phase. Genetic or chemical complementation of SOD activity of the mutant (ΔSR) is sufficient to restore antibiotic tolerance to WT levels. Remarkably, we observe high membrane permeability and increased drug internalization upon ablation of SOD activity. Combined, our results highlight an unprecedented mode of SR-mediated multidrug tolerance in stationary-phase and suggest that inhibition of SOD activity may potentiate current antibiotics.
Mechanisms of Bacterial Tolerance and Persistence in the Gastrointestinal and Respiratory Environments.
Trastoy R,Manso T,Fernández-García L,Blasco L,Ambroa A,Pérez Del Molino M L,Bou G,García-Contreras R,Wood T K,Tomás M
Clinical microbiology reviews
Pathogens that infect the gastrointestinal and respiratory tracts are subjected to intense pressure due to the environmental conditions of the surroundings. This pressure has led to the development of mechanisms of bacterial tolerance or persistence which enable microorganisms to survive in these locations. In this review, we analyze the general stress response (RpoS mediated), reactive oxygen species (ROS) tolerance, energy metabolism, drug efflux pumps, SOS response, quorum sensing (QS) bacterial communication, (p)ppGpp signaling, and toxin-antitoxin (TA) systems of pathogens, such as , spp., spp., spp., , spp., spp., spp., and , all of which inhabit the gastrointestinal tract. The following respiratory tract pathogens are also considered: , , , , and Knowledge of the molecular mechanisms regulating the bacterial tolerance and persistence phenotypes is essential in the fight against multiresistant pathogens, as it will enable the identification of new targets for developing innovative anti-infective treatments.
A cell-cell communication signal integrates quorum sensing and stress response.
Lee Jasmine,Wu Jien,Deng Yinyue,Wang Jing,Wang Chao,Wang Jianhe,Chang Changqing,Dong Yihu,Williams Paul,Zhang Lian-Hui
Nature chemical biology
Pseudomonas aeruginosa uses a hierarchical quorum sensing (QS) network consisting of las, pqs and rhl regulatory elements to coordinate the expression of bacterial virulence genes. However, clinical isolates frequently contain loss-of-function mutations in the central las system. This motivated us to search for a mechanism that may functionally substitute las. Here we report identification of a new QS signal, IQS. Disruption of IQS biosynthesis paralyzes the pqs and rhl QS systems and attenuates bacterial virulence. Production of IQS is tightly controlled by las under normal culture conditions but is also activated by phosphate limitation, a common stressor that bacteria encounter during infections. Thus, these results have established an integrated QS system that connects the central las system and phosphate-stress response mechanism to the downstream pqs and rhl regulatory systems. Our discovery highlights the complexity of QS signaling systems and extends the gamut of QS and stress-response mechanisms.
Anti-biofilm effect of novel thiazole acid analogs against Pseudomonas aeruginosa through IQS pathways.
Li Shengrong,Chen Siyu,Fan Jilin,Cao Zhen,Ouyang Weihao,Tong Ning,Hu Xin,Hu Jie,Li Peishan,Feng Zifeng,Huang Xi,Li Yuying,Xie Mingshan,He Ruikun,Jian Jingyi,Wu Biyuan,Xu Chen,Wu Weijian,Guo Jialiang,Lin Jing,Sun Pinghua
European journal of medicinal chemistry
IQS has been proven to be a new quorum sensing (QS) system against bacterial biofilm formation, which is activated in the common phosphate-limiting environment of infected tissues taking over the central las system. Up to now, numerous biofilm inhibitors which function by affecting traditional QS system have been reported. However, no compound has been reported to exert anti-biofilm activity through IQS system. Herein, various novel IQS derivatives were synthesized by the reaction of thiazole-4-carboxylic acid with different linear alcohols (R-OH) or amines (R-NH). IQS derivatives with four carbon chain length of R group were found to present the best biofilm inhibition activity. Compound B-11 as the model molecule was observed to inhibit biofilm formation only under phosphate-limiting condition, and increase in B-11 concentration significantly reduced the expression of rhlA-gfp and pqsA-gfp, but lasB-gfp. Moreover, B-11 reduced production of virulence factors of rhamnolipid and pyocyanin under phosphate limitation. These observations indicated that the synthesized compounds possessed the anti-biofilm activity through IQS pathways rather than traditional QS pathways, which pave a path for future molecular design against bacterial biofilm formation.
TrmB, a tRNA m7G46 methyltransferase, plays a role in hydrogen peroxide resistance and positively modulates the translation of katA and katB mRNAs in Pseudomonas aeruginosa.
Thongdee Narumon,Jaroensuk Juthamas,Atichartpongkul Sopapan,Chittrakanwong Jurairat,Chooyoung Kamonchanok,Srimahaeak Thanyaporn,Chaiyen Pimchai,Vattanaviboon Paiboon,Mongkolsuk Skorn,Fuangthong Mayuree
Nucleic acids research
Cellular response to oxidative stress is a crucial mechanism that promotes the survival of Pseudomonas aeruginosa during infection. However, the translational regulation of oxidative stress response remains largely unknown. Here, we reveal a tRNA modification-mediated translational response to H2O2 in P. aeruginosa. We demonstrated that the P. aeruginosa trmB gene encodes a tRNA guanine (46)-N7-methyltransferase that catalyzes the formation of m7G46 in the tRNA variable loop. Twenty-three tRNA substrates of TrmB with a guanosine residue at position 46 were identified, including 11 novel tRNA substrates. We showed that loss of trmB had a strong negative effect on the translation of Phe- and Asp-enriched mRNAs. The trmB-mediated m7G modification modulated the expression of the catalase genes katA and katB, which are enriched with Phe/Asp codons at the translational level. In response to H2O2 exposure, the level of m7G modification increased, consistent with the increased translation efficiency of Phe- and Asp-enriched mRNAs. Inactivation of trmB led to decreased KatA and KatB protein abundance and decreased catalase activity, resulting in H2O2-sensitive phenotype. Taken together, our observations reveal a novel role of m7G46 tRNA modification in oxidative stress response through translational regulation of Phe- and Asp-enriched genes, such as katA and katB.