Disarming the virulence arsenal of by blocking two-component system signaling.
Goswami Manibarsha,Espinasse Adeline,Carlson Erin E
Chemical science
infections have reached a "critical" threat status making novel therapeutic approaches required. Inhibiting key signaling enzymes known as the histidine kinases (HKs), which are heavily involved with its pathogenicity, has been postulated to be an effective new strategy for treatment. Herein, we demonstrate the potential of this approach with benzothiazole-based HK inhibitors that perturb multiple virulence pathways in the burn wound isolate, PA14. Specifically, our compounds significantly reduce the level of toxic metabolites generated by this organism that are involved in quorum-sensing and redox-balancing mechanisms. They also decrease the ability of this organism to swarm and attach to surfaces, likely by influencing their motility appendages. Quantitative transcription analysis of inhibitor-treated cultures showed substantial perturbations to multiple pathways including expression of response regulator GacA, the cognate partner of the "super regulator" of virulence, HK GacS, as well as flagella and pili formation. These promising results establish that blocking of bacterial signaling in has dramatic consequences on virulence behaviours, especially in the context of surface-associated infections.
10.1039/c8sc02496k
Bacterial quorum-sensing signal IQS induces host cell apoptosis by targeting POT1-p53 signalling pathway.
Wang Jianhe,Wang Chao,Yu Hong-Bing,Dela Ahator Stephen,Wu Xiaoyan,Lv Shumei,Zhang Lian-Hui
Cellular microbiology
Pseudomonas aeruginosa, an opportunistic life-threatening human bacterial pathogen, employs quorum-sensing (QS) signal molecules to modulate virulence gene expression. 2-(2-hydroxyphenyl)-thiazole-4-carbaldehyde (IQS) is a recently identified QS signal that integrates the canonical lasR-type QS of P. aeruginosa and host phosphate stress response to fine-tune its virulence production for a successful infection. To address the role of IQS in pathogen-host interaction, we here present that IQS inhibits host cell growth and stimulates apoptosis in a dosage-dependent manner. By downregulating the telomere-protecting protein POT1 in host cells, IQS activates CHK1, CHK2, and p53 in an Ataxia telangiectasia mutated (ATM)/ATM and RAD3-related (ATR)-dependent manner and induces DNA damage response. Overexpression of POT1 in host cells presents a resistance to IQS treatment. These results suggest a pivotal role of IQS in host apoptosis, highlighting the complexity of pathogenesis mechanisms developed by P. aeruginosa during infection.
10.1111/cmi.13076
Revisiting the quorum-sensing hierarchy in Pseudomonas aeruginosa: the transcriptional regulator RhlR regulates LasR-specific factors.
Dekimpe Valérie,Déziel Eric
Microbiology (Reading, England)
Pseudomonas aeruginosa uses the two major quorum-sensing (QS) regulatory systems las and rhl to modulate the expression of many of its virulence factors. The las system is considered to stand at the top of the QS hierarchy. However, some virulence factors such as pyocyanin have been reported to still be produced in lasR mutants under certain conditions. Interestingly, such mutants arise spontaneously under various conditions, including in the airways of cystic fibrosis patients. Using transcriptional lacZ reporters, LC/MS quantification and phenotypic assays, we have investigated the regulation of QS-controlled factors by the las system. Our results show that activity of the rhl system is only delayed in a lasR mutant, thus allowing the expression of multiple virulence determinants such as pyocyanin, rhamnolipids and C(4)-homoserine lactone (HSL) during the late stationary phase. Moreover, at this stage, RhlR is able to overcome the absence of the las system by activating specific LasR-controlled functions, including production of 3-oxo-C(12)-HSL and Pseudomonas quinolone signal (PQS). P. aeruginosa is thus able to circumvent the deficiency of one of its QS systems by allowing the other to take over. This work demonstrates that the QS hierarchy is more complex than the model simply presenting the las system above the rhl system.
10.1099/mic.0.022764-0
The hierarchy quorum sensing network in Pseudomonas aeruginosa.
Lee Jasmine,Zhang Lianhui
Protein & cell
Pseudomonas aeruginosa causes severe and persistent infections in immune compromised individuals and cystic fibrosis sufferers. The infection is hard to eradicate as P. aeruginosa has developed strong resistance to most conventional antibiotics. The problem is further compounded by the ability of the pathogen to form biofilm matrix, which provides bacterial cells a protected environment withstanding various stresses including antibiotics. Quorum sensing (QS), a cell density-based intercellular communication system, which plays a key role in regulation of the bacterial virulence and biofilm formation, could be a promising target for developing new strategies against P. aeruginosa infection. The QS network of P. aeruginosa is organized in a multi-layered hierarchy consisting of at least four interconnected signaling mechanisms. Evidence is accumulating that the QS regulatory network not only responds to bacterial population changes but also could react to environmental stress cues. This plasticity should be taken into consideration during exploration and development of anti-QS therapeutics.
10.1007/s13238-014-0100-x
Electrochemical sensors for identifying pyocyanin production in clinical Pseudomonas aeruginosa isolates.
Sismaet Hunter J,Pinto Ameet J,Goluch Edgar D
Biosensors & bioelectronics
In clinical practice, delays in obtaining culture results impact patient care and the ability to tailor antibiotic therapy. Despite the advancement of rapid molecular diagnostics, the use of plate cultures inoculated from swab samples continues to be the standard practice in clinical care. Because the inoculation culture process can take between 24 and 48h before a positive identification test can be run, there is an unmet need to develop rapid throughput methods for bacterial identification. Previous work has shown that pyocyanin can be used as a rapid, redox-active biomarker for identifying Pseudomonas aeruginosa in clinical infections. However, further validation is needed to confirm pyocyanin production occurs in all clinical strains of P. aeruginosa. Here, we validate this electrochemical detection strategy using clinical isolates obtained from patients with hospital-acquired infections or with cystic fibrosis. Square-wave voltammetric scans of 94 different clinical P. aeruginosa isolates were taken to measure the concentration of pyocyanin. The results showed that all isolates produced measureable concentrations of pyocyanin with production rates correlated with patient symptoms and comorbidity. Further bioinformatics analysis confirmed that 1649 genetically sequenced strains (99.9%) of P. aeruginosa possess the two genes (PhzM and PhzS) necessary to produce pyocyanin, supporting the specificity of this biomarker. Confirming the production of pyocyanin by all clinically-relevant strains of P. aeruginosa is a significant step towards validating this strategy for rapid, point-of-care diagnostics.
10.1016/j.bios.2017.05.042
Enzyme-Mediated Quenching of the Quinolone Signal (PQS) Promotes Biofilm Formation of by Increasing Iron Availability.
Tettmann Beatrix,Niewerth Christine,Kirschhöfer Frank,Neidig Anke,Dötsch Andreas,Brenner-Weiss Gerald,Fetzner Susanne,Overhage Joerg
Frontiers in microbiology
The 2-alkyl-3-hydroxy-4(1)-quinolone 2,4-dioxygenase HodC was previously described to cleave the quinolone signal, PQS, which is exclusively used in the complex quorum sensing (QS) system of , an opportunistic pathogen employing QS to regulate virulence and biofilm development. Degradation of PQS by exogenous addition of HodC to planktonic cells of attenuated production of virulence factors, and reduced virulence . However, proteolytic cleavage reduced the efficacy of HodC. Here, we identified the secreted protease LasB of to be responsible for HodC degradation. In static biofilms of the PA14 ::Tn mutant, the catalytic activity of HodC led to an increase in viable biomass in newly formed but also in established biofilms, and reduced the expression of genes involved in iron metabolism and siderophore production, such as , and . This is likely due to an increase in the levels of bioavailable iron by degradation of PQS, which is able to sequester iron from the surrounding environment. Thus, HodC, despite its ability to quench the production of virulence factors, is contraindicated for combating biofilms.
10.3389/fmicb.2016.01978
Chinese medicinal herb extract inhibits PQS-mediated quorum sensing system in Pseudomonas aeruginosa.
Wei Qing,Bhasme Pramod,Wang Zhiguo,Wang Li,Wang Shiwei,Zeng Yunfei,Wang Yi,Ma Luyan Z,Li Yan
Journal of ethnopharmacology
ETHNOPHARMACOLOGICAL RELEVANCE:Chinese medicinal herbs have long been recognized as important resources that can be used for the struggle against diseases and a significant component of health care system for thousands of years. AIM OF THE STUDY:In order to understand their roles in the treatment against bacterial infections, we examined the underlying mechanisms of one of the medicinal herb extracts (MHE) (Artemisiae argyi Folium, the root bark of Cortex dictamni and the root of Solanum melongena) on the human opportunistic pathogen Pseudomonas aeruginosa. MATERIALS AND METHODS:We combined phenotypic assays, transcriptional analysis and chemical investigations to identify the mechanisms underlying MHE inhibition. The standard sample was prepared and transcriptional reporters for quorum sensing systems were constructed. Electrophoretic mobility shift assays were used to clarify the mechanism. GC-MS and molecular docking were used to identify the chemicals in MHE and potential binding agents. RESULTS:We found that co-culturing of MHE with bacterial cells did not change the growth rate but substantially attenuate the production of virulence factors such as phenazine pyocyanin, siderophore pyoverdine and biofilm formation. Transcriptional responses of three major quorum sensing (QS) systems of P. aeruginosa to MHE showed that Pseudomonas quinolone signaling (PQS) system was completely repressed, rhlR/rhlI QS system was moderately inhibited, while lasR/lasI QS system was only slightly affected, suggesting that MHE might selectively target the PQS system to inhibit bacterial virulence. Furthermore, electrophoretic mobility shift assays (EMSA) showed that MHE inhibited the binding of MvfR the corresponding pqsA promoter region, suggesting that MHE serves as a competitive agent to quench the QS functionality in P. aeruginosa. CONCLUSION:We prove that MHE functions as an effective countermeasure against bacterial infections.
10.1016/j.jep.2019.112272
Human single-chain antibodies that neutralize Pseudomonas aeruginosa-exotoxin A-mediated cellular apoptosis.
Scientific reports
Targeting bacterial virulence factors directly provides a new paradigm for the intervention and treatment of bacterial diseases. Pseudomonas aeruginosa produces a myriad of virulence factors to cause fatal diseases in humans. In this study, human single-chain antibodies (HuscFvs) that bound to P. aeruginosa exotoxin A (ETA) were generated by phage display technology using recombinant ETA, ETA-subdomains and the synthetic peptide of the ETA-catalytic site as baits for selecting ETA-bound-phages from the human-scFv phage display library. ETA-bound HuscFvs derived from three phage-transfected E. coli clones neutralized the ETA-induced mammalian cell apoptosis. Computerized simulation demonstrated that these HuscFvs used several residues in their complementarity-determining regions (CDRs) to form contact interfaces with the critical residues in ETA-catalytic domain essential for ADP-ribosylation of eukaryotic elongation factor 2, which should consequently rescue ETA-exposed-cells from apoptosis. The HuscFv-treated ETA-exposed cells also showed decremented apoptosis-related genes, i.e., cas3 and p53. The effective HuscFvs have high potential for future evaluation in animal models and clinical trials as a safe, novel remedy for the amelioration of exotoxin A-mediated pathogenesis. HuscFvs may be used either singly or in combination with the HuscFv cognates that target other P. aeruginosa virulence factors as an alternative therapeutic regime for difficult-to-treat infections.
10.1038/s41598-019-51089-w
Anti-Virulence Strategy against the Multidrug-Resistant Bacterial Pathogen Pseudomonas aeruginosa: Pseudolysin (Elastase B) as a Potential Druggable Target.
Galdino Anna Clara M,de Oliveira Matheus P,Ramalho Teodorico C,de Castro Alexandre A,Branquinha Marta H,Santos André L S
Current protein & peptide science
Pseudomonas aeruginosa is a non-fermentative, gram-negative bacterium that is one of the most common pathogens responsible for hospital-acquired infections worldwide. The management of the infections caused by P. aeruginosa represents a huge challenge in the healthcare settings due to the increased emergence of resistant isolates, some of them resistant to all the currently available antimicrobials, which results in elevated morbimortality rates. Consequently, the development of new therapeutic strategies against multidrug-resistant P. aeruginosa is urgent and needful. P. aeruginosa is wellrecognized for its extreme genetic versatility and its ability to produce a lush variety of virulence factors. In this context, pseudolysin (or elastase B) outstands as a pivotal virulence attribute during the infectious process, playing multifunctional roles in different aspects of the pathogen-host interaction. This protein is a 33-kDa neutral zinc-dependent metallopeptidase that is the most abundant peptidase found in pseudomonal secretions, which contributes to the invasiveness of P. aeruginosa due to its ability to cleave several extracellular matrix proteins and to disrupt the basolateral intercellular junctions present in the host tissues. Moreover, pseudolysin makes P. aeruginosa able to overcome host defenses by the hydrolysis of many immunologically relevant molecules, including antibodies and complement components. The attenuation of this striking peptidase therefore emerges as an alternative and promising antivirulence strategy to combat antibiotic-refractory infections caused by P. aeruginosa. The anti-virulence approach aims to disarm the P. aeruginosa infective arsenal by inhibiting the expression/activity of bacterial virulence factors in order to reduce the invasiveness of P. aeruginosa, avoiding the emergence of resistance since the proliferation is not affected. This review summarizes the most relevant features of pseudolysin and highlights this enzyme as a promising target for the development of new anti-virulence compounds.
10.2174/1389203720666190207100415
Isolation of a putative virulence agent, cytotoxic serine-elastase, from a newly isolated ZuhP13.
Kotb Essam,El-Zawahry Yehia A,Saleh Ghadeer E
Journal of biosciences
A 48 kDa ZuhP13 elastase from P. aeruginosa isolated from a urine sample was successfully purified to 8.8-fold and 39% recovery by DEAE-Sepharose CL-6B and Sephadex G-100 chromatography. Its ideal reaction values were pH 7.5 and 40°C. It showed stability at pH 6-9 for 1 h and up to 60°C for 30 min with midpoint temperature (T) at 61.3°C and isoelectric value (pI) at 5.6+/-0.2. Its K and catalytic efficiency (K/K) for the substrate azocasein were 1.3 mg/mL and 4.62910 Ms, respectively. On contrary to most proteases, Zn, EDTA, 2,2'-bipyridine and o-phenanthroline showed slight inhibition upon its activity, while, the elastase inhibitors (elastatinal and elastase inhibitor II) and the serine protease inhibitors (TLCK, PMSF, SBTI, and aprotinin) markedly decreased the enzymatic activity. Taken together, we suggest that ZuhP13 is a serine elastase-type. Interestingly, the tested enzyme showed both hemolytic and hemorrhagic activities . Furthermore, it induced nuclear lysis yielding hyperchromatism within leaky and malformed hepatocytes, suggesting ZuhP13 elastase as a high molecular weight potential pathological agent.
Attenuation of quorum sensing controlled virulence factors and biofilm formation in Pseudomonas aeruginosa by pentacyclic triterpenes, betulin and betulinic acid.
Rajkumari Jobina,Borkotoky Subhomoi,Murali Ayaluru,Suchiang Kitlangki,Mohanty Saswat Kumar,Busi Siddhardha
Microbial pathogenesis
The production of virulence determinants and biofilm formation in numerous pathogens is regulated by the cell-density-dependent phenomenon, Quorum sensing (QS). The QS system in multidrug resistant opportunistic pathogen, P. aeruginosa constitutes of three main regulatory circuits namely Las, Rhl, and Pqs which are closely linked to its pathogenicity and establishment of chronic infections. In spite intensive antibiotic therapy, P. aeruginosa continue to be an important cause of nosocomial infections and also the major cause of mortality in Cystic Fibrosis patients with 80% of the adults suffering from chronic P. aeruginosa infection. Hence, targeting QS circuit offers an effective intervention to the ever increasing problem of drug resistant pathogens. In the present study, the pentacyclic triterpenes i.e. Betulin (BT) and Betulinic acid (BA) exhibited significant attenuation in production of QS-regulated virulence factors and biofilm formation in P. aeruginosa, at the sub-lethal concentration. The test compound remarkably interfered in initial stages of biofilm development by decreasing the exopolysaccharide production and cell surface hydrophobicity. Based on the in vivo studies, the test compounds notably enhanced the survival of Caenorhabditis elegans infected with P. aeruginosa. Furthermore, molecular docking analysis revealed that BT and BA can act as a strong competitive inhibitor for QS receptors, LasR and RhlR. The findings suggest that BT and BA can serve as potential anti-infectives in the controlling chronic infection of P. aeruginosa.
10.1016/j.micpath.2018.03.012
Modulation of Quorum Sensing by Glutathione.
Zhou Hui,Wang Meizhen,Smalley Nicole E,Kostylev Maxim,Schaefer Amy L,Greenberg E Peter,Dandekar Ajai A,Xu Feng
Journal of bacteriology
uses quorum sensing (QS) to regulate the production of a battery of secreted products. At least some of these products are shared among the population and serve as public goods. When is grown on casein as the sole carbon and energy source, the QS-induced extracellular protease elastase is required for growth. We isolated a variant, which showed increased production of QS-induced factors after repeated transfers in casein broth. This variant, QS*, had a mutation in the glutathione synthesis gene We describe several experiments that show a coding variant and glutathione affect the QS response. The QS transcription factor LasR has a redox-sensitive cysteine (C79). We report that GshA variant cells with a LasR C79S substitution show a similar QS response to that of wild-type Surprisingly, it is not LasR but the QS transcription factor RhlR that is more active in bacteria containing the variant Our results demonstrate that QS integrates information about cell density and the cellular redox state via glutathione levels. and other bacteria coordinate group behaviors using a chemical communication system called quorum sensing (QS). The QS system of is complex, with several regulators and signals. We show that decreased levels of glutathione lead to increased gene activation in , which did not occur in a strain carrying the redox-insensitive variant of a transcription factor. The ability of QS transcription factors to integrate information about cell density and cellular redox state shows these transcription factors can fine-tune levels of the gene products they control in response to at least two types of signals or cues.
10.1128/JB.00685-18
Both toxic and beneficial effects of pyocyanin contribute to the lifecycle of Pseudomonas aeruginosa.
Meirelles Lucas A,Newman Dianne K
Molecular microbiology
Pseudomonas aeruginosa, an opportunistic pathogen, produces redox-active pigments called phenazines. Pyocyanin (PYO, the blue phenazine) plays an important role during biofilm development. Paradoxically, PYO auto-poisoning can stimulate cell death and release of extracellular DNA (eDNA), yet PYO can also promote survival within biofilms when cells are oxidant-limited. Here, we identify the environmental and physiological conditions in planktonic culture that promote PYO-mediated cell death. We demonstrate that PYO auto-poisoning is enhanced when cells are starved for carbon. In the presence of PYO, cells activate a set of genes involved in energy-dependent defenses, including: (i) the oxidative stress response, (ii) RND efflux systems and (iii) iron-sulfur cluster biogenesis factors. P. aeruginosa can avoid PYO poisoning when reduced carbon is available, but blockage of adenosine triphosphate (ATP) synthesis either through carbon limitation or direct inhibition of the F F -ATP synthase triggers death and eDNA release. Finally, even though PYO is toxic to the majority of the population when cells are nutrient limited, a subset of cells is intrinsically PYO resistant. The effect of PYO on the producer population thus appears to be dynamic, playing dramatically different yet predictable roles throughout distinct stages of growth, helping rationalize its multifaceted contributions to biofilm development.
10.1111/mmi.14132
Activation Mechanism and Cellular Localization of Membrane-Anchored Alginate Polymerase in Pseudomonas aeruginosa.
Moradali M Fata,Ghods Shirin,Rehm Bernd H A
Applied and environmental microbiology
The exopolysaccharide alginate, produced by the opportunistic human pathogen , confers a survival advantage to the bacterium by contributing to the formation of characteristic biofilms during infection. Membrane-anchored proteins Alg8 (catalytic subunit) and Alg44 (copolymerase) constitute the alginate polymerase that is being activated by the second messenger molecule bis-(3', 5')-cyclic dimeric GMP (c-di-GMP), but the mechanism of activation remains elusive. To shed light on the c-di-GMP-mediated activation of alginate polymerization , an structural model of Alg8 fused to the c-di-GMP binding PilZ domain informed by the structure of cellulose synthase, BcsA, was developed. This structural model was probed by site-specific mutagenesis and different cellular levels of c-di-GMP. Results suggested that c-di-GMP-mediated activation of alginate polymerization involves amino acids residing at two loops, including H323 (loop A) and T457 and E460 (loop B), surrounding the catalytic site in the predicted model. The activities of the respective Alg8 variants suggested that c-di-GMP-mediated control of substrate access to the catalytic site of Alg8 is dissimilar to the known activation mechanism of BcsA. Alg8 variants responded differently to various c-di-GMP levels, while MucR imparted c-di-GMP for activation of alginate polymerase. Furthermore, we showed that Alg44 copolymerase constituted a stable dimer, with its periplasmic domains required for protein localization and alginate polymerization and modification. Superfolder green fluorescent protein (GFP) fusions of Alg8 and Alg44 showed a nonuniform, punctate, and patchy arrangement of both proteins surrounding the cell. Overall, this study provides insights into the c-di-GMP-mediated activation of alginate polymerization while assigning functional roles to Alg8 and Alg44, including their subcellular localization and distribution. The exopolysaccharide alginate is an important biofilm component of the opportunistic human pathogen and the principal cause of the mucoid phenotype that is the hallmark of chronic infections of cystic fibrosis patients. The production of alginate is mediated by interacting membrane proteins Alg8 and Alg44, while their activity is posttranslationally regulated by the second messenger c-di-GMP, a well-known regulator of the synthesis of a range of other exopolysaccharides in bacteria. This study provides new insights into the unknown activation mechanism of alginate polymerization by c-di-GMP. Experimental evidence that the activation of alginate polymerization requires the engagement of specific amino acid residues residing at the catalytic domain of Alg8 glycosyltransferase was obtained, and these residues are proposed to exert an allosteric effect on the PilZ domain upon c-di-GMP binding. This mechanism is dissimilar to the proposed mechanism of the autoinhibition of cellulose polymerization imposed by salt bridge formation between amino acid residues and released upon c-di-GMP binding, leading to activation of polymerization. On the other hand, conserved amino acid residues in the periplasmic domain of Alg44 were found to be involved in alginate polymerization as well as modification events, i.e., acetylation and epimerization. Due to the critical role of c-di-GMP in the regulation of many biological processes, particularly the motility-sessility switch and also the emergence of persisting mucoid phenotypes, these results aid to reach a better understanding of biofilm-associated regulatory networks and c-di-GMP signaling and might assist the development of inhibitory drugs.
10.1128/AEM.03499-16
Pseudomonas aeruginosa AlgU Contributes to Posttranscriptional Activity by Increasing rsmA Expression in a mucA22 Strain.
Stacey Sean D,Pritchett Christopher L
Journal of bacteriology
UNLABELLED:Pseudomonas aeruginosa thrives in multiple environments and is capable of causing life-threatening infections in immunocompromised patients. RsmA is a posttranscriptional regulator that controls virulence factor production and biofilm formation. In this study, we investigated the expression and activity of rsmA and the protein that it encodes, RsmA, in P. aeruginosa mucA mutant strains, which are common in chronic infections. We determined that AlgU regulates a previously unknown rsmA promoter in P. aeruginosa Western blot analysis confirmed that AlgU controls rsmA expression in both a laboratory strain and a clinical isolate. RNase protection assays confirmed the presence of two rsmA transcripts and suggest that RpoS and AlgU regulate rsmA expression. Due to the increased amounts of RsmA in mucA mutant strains, a translational leader fusion of the RsmA target, tssA1, was constructed and tested in mucA, algU, retS, gacA, and rsmA mutant backgrounds to examine posttranscriptional activity. From these studies, we determined that RsmA is active in mucA22 mutants, suggesting a role for RsmA in mucA mutant strains. Taken together, we have demonstrated that AlgU controls rsmA transcription and is responsible for RsmA activity in mucA mutant strains. We propose that RsmA is active in P. aeruginosa mucA mutant strains and that RsmA also plays a role in chronic infections. IMPORTANCE:P. aeruginosa causes severe infections in immunocompromised patients. The posttranscriptional regulator RsmA is known to control virulence and biofilm formation. We identify a new rsmA promoter and determine that AlgU is important in the control of rsmA expression. Mutant mucA strains that are considered mucoid were used to confirm increased rsmA expression from the AlgU promoter. We demonstrate, for the first time, that there is RsmA activity in mucoid P. aeruginosa strains. Our work suggests that RsmA may play a role during chronic infections as well as acute infections.
10.1128/JB.00133-16
Mutations in Gene as a Novel Mechanism of Aminoglycoside Resistance in Clinical Strains of Pseudomonas aeruginosa.
Bolard Arnaud,Plésiat Patrick,Jeannot Katy
Antimicrobial agents and chemotherapy
Resistance of clinical strains of to aminoglycosides can result from production of transferable aminoglycoside-modifying enzymes, of 16S rRNA methylases, and/or mutational derepression of intrinsic multidrug efflux pump MexXY(OprM). We report here the characterization of a new type of mutant that is 4- to 8-fold more resistant to 2-deoxystreptamine derivatives (e.g., gentamicin, amikacin, and tobramycin) than the wild-type strain PAO1. The genetic alterations of three mutants were mapped on and found to result in single amino acid substitutions in domains II, III, and V of elongation factor G (EF-G1A), a key component of translational machinery. Transfer of the mutated alleles into PAO1 reproduced the resistance phenotype. Interestingly, mutants with other amino acid changes in domains G, IV, and V of EF-G1A were identified among clinical strains with decreased susceptibility to aminoglycosides. Allelic-exchange experiments confirmed the relevance of these latter mutations and of three other previously reported alterations located in domains G and IV. Pump MexXY(OprM) partly contributed to the resistance conferred by the mutated EF-G1A variants and had additive effects on aminoglycoside MICs when mutationally upregulated. Altogether, our data demonstrate that cystic fibrosis (CF) and non-CF strains of can acquire a therapeutically significant resistance to important aminoglycosides via a new mechanism involving mutations in elongation factor EF-G1A.
10.1128/AAC.01835-17
Pseudomonas aeruginosa twitching motility: type IV pili in action.
Burrows Lori L
Annual review of microbiology
Type IV pili (T4P) are one of the most common forms of bacterial and archaeal surface structures, involved in adherence, motility, competence for DNA uptake, and pathogenesis. Pseudomonas aeruginosa has emerged as one of the key model systems for the investigation of T4P structure and function. Although its reputation as a serious nosocomial and opportunistic pathogen is well deserved, its facile growth requirements and the ready availability of molecular tools have allowed for rapid advances in our understanding of how T4P are assembled; their contributions to motility, biofilm formation and virulence; and their complex regulation. This review covers recent findings concerning the three different types of T4P found in P. aeruginosa (type IVa, type IVb, and Tad) and provides details about the modes of translocation mediated by T4aP, the architecture and function of the T4aP assembly system, and the complex regulation of T4aP biogenesis and function.
10.1146/annurev-micro-092611-150055
Effect of pyocyanin and 1-hydroxyphenazine on in vivo tracheal mucus velocity.
Munro N C,Barker A,Rutman A,Taylor G,Watson D,McDonald-Gibson W J,Towart R,Taylor W A,Wilson R,Cole P J
Journal of applied physiology (Bethesda, Md. : 1985)
Products of the bacterium Pseudomonas aeruginosa have been shown to slow the beating of human respiratory tract cilia in vitro. We have tested the effects of two of these compounds, pyocyanin and 1-hydroxyphenazine (given as a bolus dose dissolved in 2 microliters Ringer solution), on tracheal mucus velocity of radiolabeled erythrocytes in anesthetized guinea pigs. 1-Hydroxyphenazine (200 ng) caused a rapid slowing of tracheal mucus velocity (maximum fall 47% at 20 min) with recovery by 1 h. The effect of pyocyanin was slower in onset, 600 ng causing 60% reduction in tracheal mucus velocity at 3 h, and no recovery occurred. A combination of pyocyanin and 1-hydroxyphenazine produced an initial rapid slowing equivalent to the same dose of 1-hydroxyphenazine given alone, but the later slowing attributed to pyocyanin was greater than the same dose administered alone. This study demonstrates one mechanism by which products of P. aeruginosa may facilitate its colonization of the respiratory tract.
10.1152/jappl.1989.67.1.316
TesG is a type I secretion effector of Pseudomonas aeruginosa that suppresses the host immune response during chronic infection.
Zhao Kelei,Li Wujiao,Li Jing,Ma Teng,Wang Kailun,Yuan Yang,Li Jing Shirley,Xie Rou,Huang Ting,Zhang Yige,Zhou Yingshun,Huang Nongyu,Wu Wenling,Wang Zhen,Zhang Jun,Yue Bisong,Zhou Zongguang,Li Jiong,Wei Yu-Quan,Zhang Xiuyue,Zhou Xikun
Nature microbiology
Pseudomonas aeruginosa is a versatile Gram-negative pathogen with intricate intracellular regulatory networks that enable it to adapt to and flourish in a variety of biotic and abiotic habitats. However, the mechanism permitting the persistent survival of P. aeruginosa within host tissues and causing chronic symptoms still remains largely elusive. By using in situ RNA sequencing, here we show that P. aeruginosa adopts different metabolic pathways and virulence repertoires to dominate the progression of acute and chronic lung infections. Notably, a virulence factor named TesG, which is controlled by the vital quorum-sensing system and secreted by the downstream type I secretion system, can suppress the host inflammatory response and facilitate the development of chronic lung infection. Mechanically, TesG can enter the intracellular compartment of macrophages through clathrin-mediated endocytosis, competitively inhibit the activity of eukaryotic small GTPase and thus suppress subsequent neutrophil influx, cell cytoskeletal rearrangement of macrophages and the secretion of cytokines and chemokines. Therefore, the identification of TesG in this study reveals a type I secretion apparatus of P. aeruginosa that functions during the host-pathogen interaction, and may open an avenue for the further mechanistic study of chronic respiratory diseases and the development of antibacterial therapy.
10.1038/s41564-018-0322-4
Role of ppGpp in Pseudomonas aeruginosa acute pulmonary infection and virulence regulation.
Xu Xiaohui,Yu Hua,Zhang Di,Xiong Junzhi,Qiu Jing,Xin Rong,He Xiaomei,Sheng Halei,Cai Wenqiang,Jiang Lu,Zhang Kebin,Hu Xiaomei
Microbiological research
During infection, bacteria might generate adaptive responses to facilitate their survival and colonization in the host environment. The alarmone guanosine 5'-triphosphate-3'-diphosphate (ppGpp), the levels of which are regulated by the RelA and SpoT enzymes, plays a critical role in mediating bacterial adaptive responses and virulence. However, the mechanism by which ppGpp regulates virulence-associated traits in Pseudomonas aeruginosa is poorly understood. To investigate the regulatory role of ppGpp, the ppGpp-deficient strain ΔRS (relA and spoT gene double mutant) and the complemented strain ΔRS(++) (complemented with relA and spoT genes) were constructed. Herein, we reported that the ΔRS strain showed decreased cytotoxicity towards A549 human alveolar adenocarcinoma cell lines and led to reduced mortality, lung edema and inflammatory cell infiltration in a mouse model of acute pneumonia compared to wild-type PAO1 and the complemented strain ΔRS(++). Subsequent analyses demonstrated that the ΔRS strain displayed reduced T3SS expression, decreased levels of elastase activity, pyocyanin, pyoverdin and alginate, and inhibited swarming and biofilm formation compared to PAO1 and the complemented strain ΔRS(++). In addition, the results demonstrate that ppGpp-mediated regulation of T3SS, virulence factor production, and swarming occurs in a quinolone quorum-sensing system-dependent manner. Taken together, these results suggest that ppGpp is required for virulence regulation in P. aeruginosa, providing new clues for the development of interference strategies against bacterial infection.
10.1016/j.micres.2016.06.005
Acquisition of 16S rRNA methylase gene in Pseudomonas aeruginosa.
Yokoyama Keiko,Doi Yohei,Yamane Kunikazu,Kurokawa Hiroshi,Shibata Naohiro,Shibayama Keigo,Yagi Tetsuya,Kato Haru,Arakawa Yoshichika
Lancet (London, England)
BACKGROUND:Bacteria develop resistance to aminoglycosides by producing aminoglycoside-modifying enzymes such as acetyltransferase, phosphorylase, and adenyltransferase. These enzymes, however, cannot confer consistent resistance to various aminoglycosides because of their substrate specificity. Notwithstanding, a Pseudomonas aeruginosa strain AR-2 showing high-level resistance (minimum inhibitory concentration >1024 mg/L) to various aminoglycosides was isolated clinically. We aimed to clone and characterise the genetic determinant of this resistance. METHODS:We used conventional methods for DNA manipulation, susceptibility testing, and gene analyses to clone and characterise the genetic determinant of the resistance seen. PCR detection of the gene was also done on a stock of P aeruginosa strains that were isolated clinically since 1997. FINDINGS:An aminoglycoside-resistance gene, designated rmtA, was identified in P aeruginosa AR-2. The Escherichia coli transformant and transconjugant harbouring the rmtA gene showed very high-level resistance to various aminoglycosides, including amikacin, tobramycin, isepamicin, arbekacin, kanamycin, and gentamicin. The 756-bp nucleotide rmtA gene encoded a protein, RmtA. This protein showed considerable similarity to the 16S rRNA methylases of aminoglycoside-producing actinomycetes, which protect bacterial 16S rRNA from intrinsic aminoglycosides by methylation. Incorporation of radiolabelled methyl groups into the 30S ribosome was detected in the presence of RmtA. Of 1113 clinically isolated P aeruginosa strains, nine carried the rmtA gene, as shown by PCR analyses. INTERPRETATION:Our findings strongly suggest intergeneric lateral gene transfer of 16S rRNA methylase gene from some aminoglycoside-producing microorganisms to P aeruginosa. Further dissemination of the rmtA gene in nosocomial bacteria could be a matter of concern in the future.
10.1016/S0140-6736(03)14959-8
Extended-spectrum beta-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat.
Bradford P A
Clinical microbiology reviews
Beta-lactamases continue to be the leading cause of resistance to beta-lactam antibiotics among gram-negative bacteria. In recent years there has been an increased incidence and prevalence of extended-spectrum beta-lactamases (ESBLs), enzymes that hydrolyze and cause resistance to oxyimino-cephalosporins and aztreonam. The majority of ESBLs are derived from the widespread broad-spectrum beta-lactamases TEM-1 and SHV-1. There are also new families of ESBLs, including the CTX-M and OXA-type enzymes as well as novel, unrelated beta-lactamases. Several different methods for the detection of ESBLs in clinical isolates have been suggested. While each of the tests has merit, none of the tests is able to detect all of the ESBLs encountered. ESBLs have become widespread throughout the world and are now found in a significant percentage of Escherichia coli and Klebsiella pneumoniae strains in certain countries. They have also been found in other Enterobacteriaceae strains and Pseudomonas aeruginosa. Strains expressing these beta-lactamases will present a host of therapeutic challenges as we head into the 21st century.
10.1128/CMR.14.4.933-951.2001
Muropeptides in Pseudomonas aeruginosa and their Role as Elicitors of β-Lactam-Antibiotic Resistance.
Lee Mijoon,Dhar Supurna,De Benedetti Stefania,Hesek Dusan,Boggess Bill,Blázquez Blas,Mathee Kalai,Mobashery Shahriar
Angewandte Chemie (International ed. in English)
Muropeptides are a group of bacterial natural products generated from the cell wall in the course of its turnover. These compounds are cell-wall recycling intermediates and are also involved in signaling within the bacterium. However, the identity of these signaling molecules remains elusive. The identification and characterization of 20 muropeptides from Pseudomonas aeruginosa is described. The least abundant of these metabolites is present at 100 and the most abundant at 55,000 molecules per bacterium. Analysis of these muropeptides under conditions of induction of resistance to a β-lactam antibiotic identified two signaling muropeptides (N-acetylglucosamine-1,6-anhydro-N-acetylmuramyl pentapeptide and 1,6-anhydro-N-acetylmuramyl pentapeptide). Authentic synthetic samples of these metabolites were shown to activate expression of β-lactamase in the absence of any β-lactam antibiotic, thus indicating that they serve as chemical signals in this complex biochemical pathway.
10.1002/anie.201601693
Rhamnolipids know-how: Looking for strategies for its industrial dissemination.
Lovaglio R B,Silva V L,Ferreira H,Hausmann R,Contiero J
Biotechnology advances
Despite the numerous advantages of biosurfactants, such as low toxicity, biodegradability and high stability, these compounds are not widely used because of the high cost of production. Details about genetics, regulation and biosynthesis of rhamnolipids by Pseudomonas aeruginosa, are extremely important to the development of bioprocesses involving the synthesis of these compounds. The holding of such knowledge associated with the use of metabolic engineering tools allow modification of producing strains and the development of synthetic routes, with the purpose of increasing the production of rhamnolipids. Considering the need to obtain this know-how, this review provides information on the rhamnolipids, covering genetics, biosynthesis of hydrophobic and hydrophilic portions, and regulation, plus some future strategies that would contribute to the expansion of the production of this green surfactant.
10.1016/j.biotechadv.2015.09.002
Multicomponent biosurfactants--A "Green Toolbox" extension.
Jirku Vladimir,Cejkova Alena,Schreiberova Olga,Jezdik Richard,Masak Jan
Biotechnology advances
The unflagging interest in the surfactants of biological origin, representing ecological alternatives to their synthetic counterparts, has enhanced R&D effort both to produce their new types and to resolve the bottlenecks of their commercialization. In this context, the rhamnolipids, offering a relatively large scale of potential applications, variety of congeners, low toxicity as well as stability towards the extremes of environment, logically attract attention. In this connection, the current state of knowledge concerning these compound exploitation, biosynthesis control and non-genetic factors affecting both production yield and final rhamnolipid product is surveyed.
10.1016/j.biotechadv.2015.03.005
Small molecule disruption of quorum sensing cross-regulation in pseudomonas aeruginosa causes major and unexpected alterations to virulence phenotypes.
Welsh Michael A,Eibergen Nora R,Moore Joseph D,Blackwell Helen E
Journal of the American Chemical Society
The opportunistic pathogen Pseudomonas aeruginosa uses three interwoven quorum-sensing (QS) circuits-Las, Rhl, and Pqs-to regulate the global expression of myriad virulence-associated genes. Interception of these signaling networks with small molecules represents an emerging strategy for the development of anti-infective agents against this bacterium. In the current study, we applied a chemical approach to investigate how the Las-Rhl-Pqs QS hierarchy coordinates key virulence phenotypes in wild-type P. aeruginosa. We screened a focused library of synthetic, non-native N-acyl l-homoserine lactones and identified compounds that can drastically alter production of two important virulence factors: pyocyanin and rhamnolipid. We demonstrate that these molecules act by targeting RhlR in P. aeruginosa, a QS receptor that has seen far less scrutiny to date relative to other circuitry. Unexpectedly, modulation of RhlR activity by a single compound induces inverse regulation of pyocyanin and rhamnolipid, a result that was not predicted using genetic approaches to interrogate QS in P. aeruginosa. Further, we show that certain RhlR agonists strongly repress Pqs signaling, revealing disruption of Rhl-Pqs cross-regulation as a novel mechanism for QS inhibition. These compounds significantly expand the known repertoire of chemical probes available to study RhlR in P. aeruginosa. Moreover, our results suggest that designing chemical agents to disrupt Rhl-Pqs crosstalk could be an effective antivirulence strategy to fight this common pathogen.
10.1021/ja5110798
The role of bacterial proteases in the pathogenesis of cystic fibrosis.
Suter S
American journal of respiratory and critical care medicine
Among the roles of mediators damaging the respiratory epithelium in patients with cystic fibrosis (CF) during the course of chronic, purulent bronchitis, that of neutrophil proteases is well established. The role of bacterial proteases is less well known. Among all pathogens colonizing the airways in CF, Pseudomonas aeruginosa is quantitatively the dominant pathogen; Staphylococcus aureus and Haemophilus influenzae are present in lower numbers. Anaerobic bacteria may be detected in numbers exceeding those of Staphylococcus aureus and Haemophilus influenzae. Among all enzymes secreted by these bacterial strains, Pseudomonas elastase and alkaline protease were shown to be secreted in vivo over prolonged periods in the airways. These enzymes, mainly elastase, have proteolytic activity on many proteins involved in host defense mechanisms, often the same as those hydrolyzed by neutrophil proteases. Pseudomonas elastase has damaging effects on the respiratory epithelium; it has recently also been shown to augment the permeability of the respiratory epithelium cultured in vitro by proteolytic attack of tight junctions. The potential role of proteases and other enzymes secreted by anaerobic bacteria has not been studied in this disease. In conclusion, bacterial proteases secreted in vivo may play a role in the pathogenesis of the airway disease in CF; their relative importance to the role of host proteases is, however, often difficult to determine.
10.1164/ajrccm/150.6_Pt_2.S118
Pseudomonas aeruginosa exotoxin A induces pulmonary endothelial cytotoxicity: protection by dibutyryl-cAMP.
The European respiratory journal
In pseudomonal septicaemia, serum levels of antibody to exotoxin A have been demonstrated to be an important independent predictor of survival. Previously, we have demonstrated that exotoxin A directly injures pulmonary endothelial cells, and that dibutyryl-cyclic adenosine monophosphate (Db-cAMP) can attenuate this injury. The object of this study was to examine the mechanisms of this pulmonary endothelial cell injury and the mechanism of Db-cAMP protection. The effects of differing duration of exposure to exotoxin A and a reduction in temperature on endothelial cell injury were examined. In addition, the effect of post-treatment with Db-cAMP on exotoxin A-induced endothelial cell injury was studied. A brief, 5 min, exposure to exotoxin resulted in maximum injury comparable to that produced by 18 h exposure. This injury did not occur at low temperatures, which would inhibit receptor-mediated endocytosis. Db-cAMP protected endothelial cells, even when added up to one hour after exotoxin exposure. These results suggest that, in this model, exotoxin A-induced injury of endothelial cells is receptor-mediated. Furthermore, this injury may be attenuated even after exotoxin A internalization has taken place.
10.1183/09031936.94.07101754
Host translational inhibition by Pseudomonas aeruginosa Exotoxin A Triggers an immune response in Caenorhabditis elegans.
Cell host & microbe
Intestinal epithelial cells are exposed to both innocuous and pathogenic microbes, which need to be distinguished to mount an effective immune response. To understand the mechanisms underlying pathogen recognition, we investigated how Pseudomonas aeruginosa triggers intestinal innate immunity in Caenorhabditis elegans, a process independent of Toll-like pattern recognition receptors. We show that the P. aeruginosa translational inhibitor Exotoxin A (ToxA), which ribosylates elongation factor 2 (EF2), upregulates a significant subset of genes normally induced by P. aeruginosa. Moreover, immune pathways involving the ATF-7 and ZIP-2 transcription factors, which protect C. elegans from P. aeruginosa, are required for preventing ToxA-mediated lethality. ToxA-responsive genes are not induced by enzymatically inactive ToxA protein but can be upregulated independently of ToxA by disruption of host protein translation. Thus, C. elegans has a surveillance mechanism to recognize ToxA through its effect on protein translation rather than by direct recognition of either ToxA or ribosylated EF2.
10.1016/j.chom.2012.02.007
Exotoxin A-eEF2 complex structure indicates ADP ribosylation by ribosome mimicry.
Nature
The bacteria causing diphtheria, whooping cough, cholera and other diseases secrete mono-ADP-ribosylating toxins that modify intracellular proteins. Here, we describe four structures of a catalytically active complex between a fragment of Pseudomonas aeruginosa exotoxin A (ETA) and its protein substrate, translation elongation factor 2 (eEF2). The target residue in eEF2, diphthamide (a modified histidine), spans across a cleft and faces the two phosphates and a ribose of the non-hydrolysable NAD+ analogue, betaTAD. This suggests that the diphthamide is involved in triggering NAD+ cleavage and interacting with the proposed oxacarbenium intermediate during the nucleophilic substitution reaction, explaining the requirement of diphthamide for ADP ribosylation. Diphtheria toxin may recognize eEF2 in a manner similar to ETA. Notably, the toxin-bound betaTAD phosphates mimic the phosphate backbone of two nucleotides in a conformational switch of 18S rRNA, thereby achieving universal recognition of eEF2 by ETA.
10.1038/nature03871
Alginate synthesis by Pseudomonas aeruginosa: a key pathogenic factor in chronic pulmonary infections of cystic fibrosis patients.
May T B,Shinabarger D,Maharaj R,Kato J,Chu L,DeVault J D,Roychoudhury S,Zielinski N A,Berry A,Rothmel R K
Clinical microbiology reviews
Pulmonary infection by mucoid, alginate-producing Pseudomonas aeruginosa is the leading cause of mortality among patients suffering from cystic fibrosis. Alginate-producing P. aeruginosa is uniquely associated with the environment of the cystic fibrosis-affected lung, where alginate is believed to increase resistance to both the host immune system and antibiotic therapy. Recent evidence indicates that P. aeruginosa is most resistant to antibiotics when the infecting cells are present as a biofilm, as they appear to be in the lungs of cystic fibrosis patients. Inhibition of the protective alginate barrier with nontoxic compounds targeted against alginate biosynthetic and regulatory proteins may prove useful in eradicating P. aeruginosa from this environment. Our research has dealt with elucidating the biosynthetic pathway and regulatory mechanism(s) responsible for alginate synthesis by P. aeruginosa. This review summarizes reports on the role of alginate in cystic fibrosis-associated pulmonary infections caused by P. aeruginosa and provides details about the biosynthesis and regulation of this exopolysaccharide.
10.1128/CMR.4.2.191
The multiple signaling systems regulating virulence in Pseudomonas aeruginosa.
Jimenez Pol Nadal,Koch Gudrun,Thompson Jessica A,Xavier Karina B,Cool Robbert H,Quax Wim J
Microbiology and molecular biology reviews : MMBR
Cell-to-cell communication is a major process that allows bacteria to sense and coordinately react to the fluctuating conditions of the surrounding environment. In several pathogens, this process triggers the production of virulence factors and/or a switch in bacterial lifestyle that is a major determining factor in the outcome and severity of the infection. Understanding how bacteria control these signaling systems is crucial to the development of novel antimicrobial agents capable of reducing virulence while allowing the immune system of the host to clear bacterial infection, an approach likely to reduce the selective pressures for development of resistance. We provide here an up-to-date overview of the molecular basis and physiological implications of cell-to-cell signaling systems in Gram-negative bacteria, focusing on the well-studied bacterium Pseudomonas aeruginosa. All of the known cell-to-cell signaling systems in this bacterium are described, from the most-studied systems, i.e., N-acyl homoserine lactones (AHLs), the 4-quinolones, the global activator of antibiotic and cyanide synthesis (GAC), the cyclic di-GMP (c-di-GMP) and cyclic AMP (cAMP) systems, and the alarmones guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), to less-well-studied signaling molecules, including diketopiperazines, fatty acids (diffusible signal factor [DSF]-like factors), pyoverdine, and pyocyanin. This overview clearly illustrates that bacterial communication is far more complex than initially thought and delivers a clear distinction between signals that are quorum sensing dependent and those relying on alternative factors for their production.
10.1128/MMBR.05007-11
BrlR from Pseudomonas aeruginosa is a receptor for both cyclic di-GMP and pyocyanin.
Wang Feng,He Qing,Yin Jia,Xu Sujuan,Hu Wei,Gu Lichuan
Nature communications
The virulence factor pyocyanin and the intracellular second messenger cyclic diguanylate monophosphate (c-di-GMP) play key roles in regulating biofilm formation and multi-drug efflux pump expression in Pseudomonas aeruginosa. However, the crosstalk between these two signaling pathways remains unclear. Here we show that BrlR (PA4878), previously identified as a c-di-GMP responsive transcriptional regulator, acts also as a receptor for pyocyanin. Crystal structures of free BrlR and c-di-GMP-bound BrlR reveal that the DNA-binding domain of BrlR contains two separate c-di-GMP binding sites, both of which are involved in promoting brlR expression. In addition, we identify a pyocyanin-binding site on the C-terminal multidrug-binding domain based on the structure of the BrlR-C domain in complex with a pyocyanin analog. Biochemical analysis indicates that pyocyanin enhances BrlR-DNA binding and brlR expression in a concentration-dependent manner.
10.1038/s41467-018-05004-y
Oxylipins produced by Pseudomonas aeruginosa promote biofilm formation and virulence.
Nature communications
The oxygenation of unsaturated fatty acids by dioxygenases occurs in all kingdoms of life and produces physiologically important lipids called oxylipins. The biological roles of oxylipins have been extensively studied in animals, plants, algae and fungi, but remain largely unidentified in prokaryotes. The bacterium Pseudomonas aeruginosa displays a diol synthase activity that transforms several monounsaturated fatty acids into mono- and di-hydroxylated derivatives. Here we show that oxylipins derived from this activity inhibit flagellum-driven motility and upregulate type IV pilus-dependent twitching motility of P. aeruginosa. Consequently, these oxylipins promote bacterial organization in microcolonies, increasing the ability of P. aeruginosa to form biofilms in vitro and in vivo (in Drosophila flies). We also demonstrate that oxylipins produced by P. aeruginosa promote virulence in Drosophila flies and lettuce. Our study thus uncovers a role for prokaryotic oxylipins in the physiology and pathogenicity of bacteria.
10.1038/ncomms13823
Multiple communication mechanisms between sensor kinases are crucial for virulence in Pseudomonas aeruginosa.
Francis Vanessa I,Waters Elaine M,Finton-James Sutharsan E,Gori Andrea,Kadioglu Aras,Brown Alan R,Porter Steven L
Nature communications
Bacteria and many non-metazoan Eukaryotes respond to stresses and threats using two-component systems (TCSs) comprising sensor kinases (SKs) and response regulators (RRs). Multikinase networks, where multiple SKs work together, detect and integrate different signals to control important lifestyle decisions such as sporulation and virulence. Here, we study interactions between two SKs from Pseudomonas aeruginosa, GacS and RetS, which control the switch between acute and chronic virulence. We demonstrate three mechanisms by which RetS attenuates GacS signalling: RetS takes phosphoryl groups from GacS-P; RetS has transmitter phosphatase activity against the receiver domain of GacS-P; and RetS inhibits GacS autophosphorylation. These mechanisms play important roles in vivo and during infection, and exemplify an unprecedented degree of signal processing by SKs that may be exploited in other multikinase networks.
10.1038/s41467-018-04640-8
A Surface-Induced Asymmetric Program Promotes Tissue Colonization by Pseudomonas aeruginosa.
Laventie Benoît-Joseph,Sangermani Matteo,Estermann Fabienne,Manfredi Pablo,Planes Rémi,Hug Isabelle,Jaeger Tina,Meunier Etienne,Broz Petr,Jenal Urs
Cell host & microbe
The opportunistic human pathogen Pseudomonas aeruginosa effectively colonizes host epithelia using pili as primary adhesins. Here we uncover a surface-specific asymmetric virulence program that enhances P. aeruginosa host colonization. We show that when P. aeruginosa encounters surfaces, the concentration of the second messenger c-di-GMP increases within a few seconds. This leads to surface adherence and virulence induction by stimulating pili assembly through activation of the c-di-GMP receptor FimW. Surface-attached bacteria divide asymmetrically to generate a piliated, surface-committed progeny (striker) and a flagellated, motile offspring that leaves the surface to colonize distant sites (spreader). Cell differentiation is driven by a phosphodiesterase that asymmetrically positions to the flagellated pole, thereby maintaining c-di-GMP levels low in the motile offspring. Infection experiments demonstrate that cellular asymmetry strongly boosts infection spread and tissue damage. Thus, P. aeruginosa promotes surface colonization and infection transmission through a cooperative virulence program that we termed Touch-Seed-and-Go.
10.1016/j.chom.2018.11.008
An integrated genomic regulatory network of virulence-related transcriptional factors in Pseudomonas aeruginosa.
Huang Hao,Shao Xiaolong,Xie Yingpeng,Wang Tingting,Zhang Yingchao,Wang Xin,Deng Xin
Nature communications
The virulence of Pseudomonas aeruginosa, a Gram-negative opportunistic pathogen, is regulated by many transcriptional factors (TFs) that control the expression of quorum sensing and protein secretion systems. Here, we report a genome-wide, network-based approach to dissect the crosstalk between 20 key virulence-related TFs. Using chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq), as well as RNA-seq, we identify 1200 TF-bound genes and 4775 differentially expressed genes. We experimentally validate 347 of these genes as functional target genes, and describe the regulatory relationships of the 20 TFs with their targets in a network that we call 'Pseudomonas aeruginosa genomic regulatory network' (PAGnet). Analysis of the network led to the identification of novel functions for two TFs (ExsA and GacA) in quorum sensing and nitrogen metabolism. Furthermore, we present an online platform and R package based on PAGnet to facilitate updating and user-customised analyses.
10.1038/s41467-019-10778-w