RESUMO
Various virulence determinants in Pseudomonas aeruginosa are regulated by the quorum sensing (QS) network producing and releasing signalling molecules. Two of these virulence determinants are the pyocyanin and pyoverdine, which interfere with multiple cellular functions during infection. The application of QS-inhibiting agents, such as cyclodextrins (CDs), appears to be a promising approach. Further to method development, this research tested in large-volume test systems the effect of α- and ß-CD (ACD, BCD) at 1, 5, and 10 mM concentrations on the production of pyocyanin in the P. aeruginosa model system. The concentration and time-dependent quorum quenching effect of native CDs and their derivatives on pyoverdine production was tested in a small-volume high-throughput system. In the large-volume system, both ACD and BCD significantly inhibited pyocyanin production, but ACD to a greater extent. 10 mM ACD resulted in 58% inhibition, while BCD only ~40%. Similarly, ACD was more effective in the inhibition of pyoverdine production; nevertheless, the results of RMANOVA demonstrated the significant efficiency of both ACD and BCD, as well as their derivatives. Both the contact time and the cyclodextrin treatments significantly influenced pyoverdine production. In this case, the inhibitory effect of ACD after 48 h at 12.5 mM was 57%, while the inhibitory effect of BCD and its derivatives was lower than 40%. The high-level significant inhibition of both pyocyanin and pyoverdine production by ACD was detectable. Consequently, the potential value of CDs as QS inhibitors and the antivirulence strategy should be considered. KEYPOINTS: ⢠Applicability of a simplified method for quantification of pyocyanin production was demonstrated. ⢠The cyclodextrins significantly affected the pyocyanin and pyoverdine production. ⢠The native ACD exhibited the highest attenuation in pyoverdine production.
Assuntos
Oligopeptídeos , Infecções por Pseudomonas , Percepção de Quorum , Humanos , Pseudomonas aeruginosa , Virulência , Piocianina , Fatores de Virulência , Antibacterianos/farmacologia , BiofilmesRESUMO
The pyoverdine siderophore is produced by Pseudomonas aeruginosa to access iron. Its synthesis involves the complex coordination of four nonribosomal peptide synthetases (NRPSs), which are responsible for assembling the pyoverdine peptide backbone. The precise cellular organization of these NRPSs and their mechanisms of interaction remain unclear. Here, we used a combination of several single-molecule microscopy techniques to elucidate the spatial arrangement of NRPSs within pyoverdine-producing cells. Our findings reveal that PvdL differs from the three other NRPSs in terms of localization and mobility patterns. PvdL is predominantly located in the inner membrane, while the others also explore the cytoplasmic compartment. Leveraging the power of multicolor single-molecule localization, we further reveal co-localization between PvdL and the other NRPSs, suggesting a pivotal role for PvdL in orchestrating the intricate biosynthetic pathway. Our observations strongly indicates that PvdL serves as a central orchestrator in the assembly of NRPSs involved in pyoverdine biosynthesis, assuming a critical regulatory function.
Assuntos
Oligopeptídeos , Peptídeo Sintases , Pseudomonas aeruginosa , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/enzimologia , Oligopeptídeos/biossíntese , Oligopeptídeos/metabolismo , Peptídeo Sintases/metabolismo , Peptídeo Sintases/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Sideróforos/biossíntese , Sideróforos/metabolismoRESUMO
The bacterial nitrogen-related phosphotransfer (PTSNtr; here, Nitro-PTS) system bears homology to well-known PTS systems that facilitate saccharide import and phosphorylation. The Nitro-PTS comprises an enzyme I (EI), PtsP; an intermediate phosphate carrier, PtsO; and a terminal acceptor, PtsN, which is thought to exert regulatory effects that depend on its phosphostate. For instance, biofilm formation by Pseudomonas aeruginosa can be impacted by the Nitro-PTS, as deletion of either ptsP or ptsO suppresses Pel exopolysaccharide production and additional deletion of ptsN elevates Pel production. However, the phosphorylation state of PtsN in the presence and absence of its upstream phosphotransferases has not been directly assessed, and other targets of PtsN have not been well defined in P. aeruginosa. We show that PtsN phosphorylation via PtsP requires the GAF domain of PtsP and that PtsN is phosphorylated on histidine 68, as in Pseudomonas putida. We also find that FruB, the fructose EI, can substitute for PtsP in PtsN phosphorylation but only in the absence of PtsO, implicating PtsO as a specificity factor. Unphosphorylatable PtsN had a minimal effect on biofilm formation, suggesting that it is necessary but not sufficient for the reduction of Pel in a ptsP deletion. Finally, we use transcriptomics to show that the phosphostate and the presence of PtsN do not appear to alter the transcription of biofilm-related genes but do influence genes involved in type III secretion, potassium transport, and pyoverdine biosynthesis. Thus, the Nitro-PTS influences several P. aeruginosa behaviors, including the production of its signature virulence factors. IMPORTANCE The PtsN protein impacts the physiology of a number of bacterial species, and its control over downstream targets can be altered by its phosphorylation state. Neither its upstream phosphotransferases nor its downstream targets are well understood in Pseudomonas aeruginosa. Here, we examine PtsN phosphorylation and find that the immediate upstream phosphotransferase acts as a gatekeeper, allowing phosphorylation by only one of two potential upstream proteins. We use transcriptomics to discover that PtsN regulates the expression of gene families that are implicated in virulence. One emerging pattern is a repression hierarchy by different forms of PtsN: its phosphorylated state is more repressive than its unphosphorylated state, but the expression of its targets is even higher in its complete absence.
Assuntos
Proteínas de Bactérias , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Virulência , Fosforilação , Fosfotransferases/genética , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/genética , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/metabolismo , Regulação Bacteriana da Expressão GênicaRESUMO
Fluorescent pseudomonads such as Pseudomonas aeruginosa or Pseudomonas fluorescens produce pyoverdine siderophores that ensure iron-supply in iron-limited environments. After its synthesis in the cytoplasm, the nonfluorescent pyoverdine precursor ferribactin is exported into the periplasm, where the enzymes PvdQ, PvdP, PvdO, PvdN, and PtaA are responsible for fluorophore maturation and tailoring steps. While the roles of all these enzymes are clear, little is known about the role of PvdM, a human renal dipeptidase-related protein that is predicted to be periplasmic and that is essential for pyoverdine biogenesis. Here, we reveal the subcellular localization and functional role of PvdM. Using the model organism P. fluorescens, we show that PvdM is anchored to the periplasmic side of the cytoplasmic membrane, where it is indispensable for the activity of the tyrosinase PvdP. While PvdM does not share the metallopeptidase function of renal dipeptidase, it still has the corresponding peptide-binding site. The substrate of PvdP, deacylated ferribactin, is secreted by a ΔpvdM mutant strain, indicating that PvdM prevents loss of this periplasmic biosynthesis intermediate into the medium by ensuring the efficient transfer of ferribactin to PvdP in vivo. We propose that PvdM belongs to a new dipeptidase-related protein subfamily with inactivated Zn2+ coordination sites, members of which are usually genetically linked to TonB-dependent uptake systems and often associated with periplasmic FAD-dependent oxidoreductases related to d-amino acid oxidases. We suggest that these proteins are necessary for selective binding, exposure, or transfer of specific d- and l-amino acid-containing peptides and other periplasmic biomolecules in manifold pathways.
Assuntos
Proteínas de Bactérias/metabolismo , Periplasma , Pseudomonas aeruginosa , Aminoácidos/metabolismo , Humanos , Ferro/metabolismo , Oligopeptídeos , Peptídeos Cíclicos , Periplasma/metabolismo , Pseudomonas aeruginosa/metabolismo , Sideróforos/metabolismoRESUMO
BACKGROUND: Bacterial siderophores are chelating compounds with the potential of application in agriculture, due to their plant growth-promoting (PGP) properties, however, high production and purification costs are limiting factors for their wider application. Cost-efficiency of the production could be increased by omitting purification processes, especially since siderophores accompanying metabolites (SAM) often also possess PGP traits. In this study, the metabolism versatility of Pseudomonas sp. ANT_H12B was used for the optimization of siderophores production and the potential of these metabolites and SAM was characterized in the context of PGP properties. RESULTS: The metabolic diversity of ANT_H12B was examined through genomic analysis and phenotype microarrays. The strain was found to be able to use numerous C, N, P, and S sources, which allowed for the design of novel media suitable for efficient production of siderophores in the form of pyoverdine (223.50-512.60 µM). Moreover, depending on the culture medium, the pH of the siderophores and SAM solutions varied from acidic (pH < 5) to alkaline (pH > 8). In a germination test, siderophores and SAM were shown to have a positive effect on plants, with a significant increase in germination percentage observed in beetroot, pea, and tobacco. The PGP potential of SAM was further elucidated through GC/MS analysis, which revealed other compounds with PGP potential, such as indolic acetic acids, organic acids, fatty acids, sugars and alcohols. These compounds not only improved seed germination but could also potentially be beneficial for plant fitness and soil quality. CONCLUSIONS: Pseudomonas sp. ANT_H12B was presented as an efficient producer of siderophores and SAM which exhibit PGP potential. It was also shown that omitting downstream processes could not only limit the costs of siderophores production but also improve their agricultural potential.
Assuntos
Pseudomonas , Sideróforos , Sideróforos/química , Sideróforos/metabolismo , Pseudomonas/genética , Pseudomonas/metabolismo , Bactérias/metabolismo , Germinação , Plantas , Agricultura , Microbiologia do SoloRESUMO
Pseudomonas aeruginosa is an opportunistic pathogenic bacterium for humans, animals, and plants, through producing different molecular factors such as biofilm, siderophores, and other virulence factors which favor bacterial establishment and infection in the host. In P. aeruginosa PAO1, the production of these factors is regulated by the bacterial quorum sensing (QS) mechanisms. From them, siderophores are involved in iron acquisition, transport, and homeostasis. They are also considered some of the main virulence factors in P. aeruginosa; however, detailed mechanisms to induce bacterial pathogenesis are poorly understood. In this work, through reverse genetics, we evaluated the function of bacterial pathogenesis in the pvd cluster genes, which are required for synthesizing the siderophore pyoverdine (PVD). Single pvdI, pvdJ, pvdL, and double mutant strains were analyzed, and contrary to expected, the pvdL and pvdI mutations increased the concentration of PVD and other phenazines, such as pyocyanin (PYO) and phenazine-1-carboxylic acid (PCA) and also an increased biofilm production and morphology depending on the autoinducer 2-alkyl-4-quinolone (PQS) and the QS molecules acyl-homoserine lactones. Consequently, in the in vivo pathogenicity model of Caenorhabditis elegans, the mutations in pvdI, pvdJ, and pvdL increased the survival of the worms exposed to supernatants or biofilms of the bacterial cultures. However, the double mutant pvdI/pvdJ increased its toxicity in agreeing with the biofilm production, PVD, PYO, and PCA. The findings indicate that the mutations in pvd genes encode non-ribosomal peptide synthetases impacted the biofilm's structure, but suppressively also of the phenazines, confirming that the siderophores contribute to the bacterial establishment and pathogenicity of P. aeruginosa PAO1.
Assuntos
Percepção de Quorum , Sideróforos , Humanos , Animais , Pseudomonas aeruginosa/genética , Piocianina , Biofilmes , Fatores de Virulência/genética , Fenazinas , Proteínas de Bactérias/genéticaRESUMO
Pseudomonas aeruginosa is a Gram-negative bacterium which can cause serious infections among immune-depressed people including cystic fibrosis patients where it can colonize the lungs causing chronic infections. Iron is essential for P. aeruginosa and can be provided via three sources under aerobic conditions: its own siderophores pyochelin (PCH) and pyoverdine (PVD), xenosiderophores, or heme, respectively. Pyoverdine is the high affinity siderophore and its synthesis and uptake involve more than 30 genes organized in different operons. Its synthesis and uptake are triggered by iron scarcity via the Fur regulator and involves two extra cytoplasmic sigma factors (ECF), PvdS for the biosynthesis of PVD and FpvI for the uptake via the TonB-dependent FpvA outer membrane transporter and other periplasmic and inner membrane proteins. It appeared recently that the regulation of PVD biosynthesis and uptake involves other regulators, including other ECF factors, and LysR regulators. This is the case especially for the genes coding for periplasmic and inner membrane proteins involved in the reduction of Fe3+ to Fe2+ and the transport of ferrous iron to the cytoplasm that appears to represent a crucial step in the uptake process.
Assuntos
Proteínas da Membrana Bacteriana Externa , Pseudomonas aeruginosa , Humanos , Pseudomonas aeruginosa/genética , Proteínas da Membrana Bacteriana Externa/genética , Ferro/metabolismo , Transporte Biológico , Sideróforos/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão GênicaRESUMO
Siderophores are iron-chelating molecules produced by bacteria and other microbes. They are involved with virulence in infections and play key roles in bacterial community assembly and as plant protectants due to their pathogen control properties. Although assays exist to screen whether newly isolated bacteria can produce siderophores, the chemical structures of many of these bio-active molecules remain unidentified due to the lack of rapid analytical procedures. An important group of siderophores are pyoverdines. They consist of a structurally diverse group of chromopeptides, whose amino acid sequence is characteristic for the fluorescent Pseudomonas species that secrets them. Although over 60 pyoverdine structures have been described so far, their characterization is cumbersome and several methods (isoelectrofocusing, iron uptake measurement, mass determination) are typically combined as ambiguous results are often achieved by a single method. Those additional experiments consume valuable time and resources and prevent high-throughput analysis. In this work, we present a new pyoverdine characterisation option by recording their collision cross sections (CCS) using trapped ion mobility spectrometry. This can be done simultaneously in combination with UHPLC and high-resolution MS resulting in a rapid identification of pyoverdines. The high specificity of CCS values is presented for 17 pyoverdines secreted by different Pseudomonas strains. The pyoverdine mass determination by full scan MS was supported by fragments obtained from broadband collision induced dissociation (bbCID). As iron contaminations in laboratories are not uncommon, CCS values of ferripyoverdines were also evaluated. Thereby, unusual and highly characteristic ion mobility patterns were obtained that are suitable as an alternative identification marker.
Assuntos
Pseudomonas , Sideróforos , Pseudomonas/metabolismo , Sideróforos/química , Cromatografia Líquida de Alta Pressão , Ferro/metabolismo , CorantesRESUMO
Pseudmonas aeruginosa is a Gram-negative bacterium known to be ubiquitous and recognized as one of the leading causes of infections such as respiratory, urinary tract, burns, cystic fibrosis, and in immunocompromised individuals. Failure of antimicrobial therapy has been documented to be attributable due to the development of various resistance mechanisms, with a proclivity to develop additional resistance mechanisms rapidly. P. aeruginosa virulence attenuation is an alternate technique for disrupting pathogenesis without impacting growth. The iron-scavenging siderophores (pyoverdine and pyochelin) generated by P. aeruginosa have various properties like scavenging iron, biofilm formation, quorum sensing, increasing virulence, and toxicity to the host. As a result, developing an antivirulence strategy, specifically inhibiting the P. aeruginosa siderophore, has been a promising therapeutic option to limit their infection. Several natural, synthetic compounds and nanoparticles have been identified as potent inhibitors of siderophore production/biosynthesis, function, and transport system. The current review discussed pyoverdine and pyochelin's synthesis and transport system in P. aeruginosa. Furthermore, it is also focused on the role of several natural and synthetic compounds in reducing P. aeruginosa virulence by inhibiting siderophore synthesis, function, and transport. The underlying mechanism involved in inhibiting the siderophore by natural and synthetic compounds has also been explained. KEY POINTS: ⢠Pseudomonas aeruginosa is an opportunistic pathogen linked to chronic respiratory, urinary tract, and burns infections, as well as cystic fibrosis and immunocompromised patients. ⢠P. aeruginosa produces two virulent siderophores forms: pyoverdine and pyochelin, which help it to survive in iron-deficient environments. ⢠The inhibition of siderophore production, transport, and activity using natural and synthesized drugs has been described as a potential strategy for controlling P. aeruginosa infection.
Assuntos
Fibrose Cística , Sideróforos , Humanos , Pseudomonas aeruginosa , Virulência , Fibrose Cística/microbiologia , FerroRESUMO
We investigated the function of pyoverdine in the biofilm formation, motility, and spoilage potential of Pseudomonas fluorescens. We targeted and identified two major genes (pvdA and pvdE) that are involved in the biosynthesis of siderophores. We next constructed ΔpvdA and ΔpvdE mutants of P. fluorescens PF08 and found that the deletion of pyoverdine led to a biofilm-to-motivity transition as both ΔpvdA and ΔpvdE mutants displayed enhanced motility, reduced level of exopolysaccharides (EPSs), and attenuated biofilm formation. In addition, the lack of synthesis of pyoverdine promoted the spoilage of fish fillets stored at 4 °C. Based on the effect of pyoverdine deletion on the phenotype; we report that pyoverdine regulates the transcription levels of htpX, phoA, flip, flgA, and RpoS, suggesting that pyoverdine-mediated iron absorption may affect the regulation of flagellum and stress resistance. This study emphasizes the important role of pyoverdine in the formation of biofilm, motility, and spoilage of P. fluorescens PF08.
Assuntos
Pseudomonas fluorescens , Sideróforos , Animais , Pseudomonas fluorescens/genética , BiofilmesRESUMO
The bio-synthesis of pyoverdine (PVD) in Pseudomonas aeruginosa involves multiple enzymatic steps including the action of non-ribosomal peptide synthetases (NRPSs). One hallmark of NRPS is their ability to make usage of non-proteinogenic amino-acids synthesized by co-expressed accessory enzymes. It is generally proposed that different enzymes of a secondary metabolic pathway assemble into large supra-molecular complexes. However, evidence for the assembly of sequential enzymes in the cellular context is sparse. Here, we used in cellulo single-molecule tracking and Förster resonance energy transfer measured by fluorescence lifetime microscopy (FRET-FLIM) to explore the spatial partitioning of the ornithine hydroxylase PvdA and its interactions with NRPS. We found PvdA was mostly diffusing bound to large complexes in the cytoplasm with a small exchangeable trapped fraction. FRET-FLIM clearly showed that PvdA is physically interacting with PvdJ, PvdI, PvdL, and PvdD, the four NRPS involved in the PVD pathway in Pseudomonas aeruginosa PAO1. The binding modes of PvdA were strikingly different according to the NRPS it is interacting with, suggesting that PvdA binding sites have co-evolved with the enzymatic active sites of NRPS. Our data provide evidence for strongly organized multi-enzymatic complexes responsible for the bio-synthesis of PVD and illustrate how binding sites have evolved to finely control the co-localization of sequential enzymes and promote metabolic pathway efficiency.
Assuntos
Oxigenases de Função Mista/metabolismo , Oligopeptídeos/química , Pseudomonas aeruginosa/metabolismo , Imagem Individual de Molécula/métodos , Análise por Conglomerados , Citoplasma/metabolismo , Escherichia coli/metabolismo , Transferência Ressonante de Energia de Fluorescência , Proteínas de Fluorescência Verde/metabolismo , Microscopia de Fluorescência/métodos , Mutação , Oligopeptídeos/metabolismo , Ligação ProteicaRESUMO
Microbial invasions can compromise ecosystem services and spur dysbiosis and disease in hosts. Nevertheless, the mechanisms determining invasion outcomes often remain unclear. Here, we examine the role of iron-scavenging siderophores in driving invasions of Pseudomonas aeruginosa into resident communities of environmental pseudomonads. Siderophores can be 'public goods' by delivering iron to individuals possessing matching receptors; but they can also be 'public bads' by withholding iron from competitors lacking these receptors. Accordingly, siderophores should either promote or impede invasion, depending on their effects on invader and resident growth. Using supernatant feeding and invasion assays, we show that invasion success indeed increased when the invader could use its siderophores to inhibit (public bad) rather than stimulate (public good) resident growth. Conversely, invasion success decreased the more the invader was inhibited by the residents' siderophores. Our findings identify siderophores as a major driver of invasion dynamics in bacterial communities under iron-limited conditions.
Assuntos
Ecossistema , Sideróforos , Humanos , Ferro , Oligopeptídeos , Pseudomonas aeruginosaRESUMO
BACKGROUND: Iron is essential for almost all bacterial pathogens and consequently it is actively withheld by their hosts. However, the production of extracellular siderophores enables iron sequestration by pathogens, increasing their virulence. Another function of siderophores is extracellular detoxification of non-ferrous metals. Here, we experimentally link the detoxification and virulence roles of siderophores by testing whether the opportunistic pathogen Pseudomonas aeruginosa displays greater virulence after exposure to copper. To do this, we incubated P. aeruginosa under different environmentally relevant copper regimes for either two or twelve days. Subsequent growth in a copper-free environment removed phenotypic effects, before we quantified pyoverdine production (the primary siderophore produced by P. aeruginosa), and virulence using the Galleria mellonella infection model. RESULTS: Copper selected for increased pyoverdine production, which was positively correlated with virulence. This effect increased with time, such that populations incubated with high copper for twelve days were the most virulent. Replication of the experiment with a non-pyoverdine producing strain of P. aeruginosa demonstrated that pyoverdine production was largely responsible for the change in virulence. CONCLUSIONS: We here show a direct link between metal stress and bacterial virulence, highlighting another dimension of the detrimental effects of metal pollution on human health.
Assuntos
Pseudomonas aeruginosa , Sideróforos , Humanos , Virulência , FerroRESUMO
Pseudomonas aeruginosa is an opportunistic pathogen and is the major cause of corneal infections in India and worldwide. The increase in antimicrobial resistance among Pseudomonas has prompted rise in significant research to develop alternative therapeutics. Antimicrobial peptides (AMPs) are considered as potent alternatives to combat bacterial infections. In this study, we investigated the role of S100A12, a host defense peptide, against PAO1 and an ocular clinical isolate. Increased expression of S100A12 was observed in corneal tissues obtained from Pseudomonas keratitis patients by immunohistochemistry. S100A12 significantly inhibited growth of Pseudomonas in vitro as determined from colony forming units. Furthermore, recombinant S100A12 reduced the corneal opacity and the bacterial load in a mouse model of Pseudomonas keratitis. Transcriptome changes in PAO1 in response to S100A12 was investigated using RNA sequencing. The pathway analysis of transcriptome data revealed that S100A12 inhibits expression of genes involved in pyoverdine synthesis and biofilm formation. It also impedes several important pathways like redox, pyocyanin synthesis and type 6 secretion system (T6SS). The transcriptome data was further validated by checking the expression of several affected genes by quantitative PCR. Our study sheds light on how S100A12 impacts Pseudomonas and that it might have the potential to be used as therapeutic intervention in addition to antibiotics to combat infection in future.
Assuntos
Ceratite , Infecções por Pseudomonas , Sistemas de Secreção Tipo VI , Animais , Peptídeos Antimicrobianos , Biofilmes , Ceratite/microbiologia , Complexo Antígeno L1 Leucocitário/metabolismo , Camundongos , Oligopeptídeos , Pseudomonas/metabolismo , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/genética , Proteína S100A12/metabolismo , Sistemas de Secreção Tipo VI/genéticaRESUMO
Microbial secondary metabolites represent a rich source for drug discovery, plant protective agents, and biotechnologically relevant compounds. Among them are siderophores, iron-chelating molecules, that show a great influence on bacterial community assembly and the potential to control pathogen invasions. One of such a siderophore is pyoverdine that is produced by fluorescent Pseudomonas members and consists of different peptide chains specific to each bacterial species. The identification and structural elucidation of such suites of siderophores remain widely underexplored as general high-throughput analytical protocols are missing. Therefore, a dedicated method was established allowing a rapid localization and structural elucidation of pyoverdines. Liquid bacterial culture samples were purified by an easy small-scale solid-phase extraction (SPE). Ultra-high-performance liquid chromatography high-resolution tandem mass spectrometry (UHPLC-HR-MS/MS) separated highly polar pyoverdines and their derivatives. All ion fragmentation (AIF) generated mass spectra containing the characteristic fragments of the biological precursor of pyoverdine, ferribactin. This led to the revelation of the mass of secreted pyoverdines. Targeted MS/MS experiments at multiple collision energies accomplished the full structure elucidation of the pyoverdine peptide chain. A mass calculator and a fragmentation predictor facilitated greatly the interpretation of MS/MS spectra by providing accurate masses for a straightforward comparison of measured and theoretical values. The method was successfully validated using four well-known pyoverdines with various peptide chains. Finally, the applicability was proven by the analysis of 13 unknown pyoverdines secreted by sampled bacterial cultures. Among these, 4 novel pyoverdine peptide chains were discovered and are herein reported for the first time.
Assuntos
Pseudomonas , Espectrometria de Massas em Tandem , Cromatografia Líquida de Alta Pressão , Oligopeptídeos , Pseudomonas/metabolismo , SideróforosRESUMO
Pseudomonas aeruginosa causes a wide array of life-threatening acute and chronic infections in humans. This opportunistic pathogen is metabolically highly versatile and harbors multiple virulence factors that allow infection of essentially any organ of the human body. The high capacity of this bacterium to acquire iron facilitates its versatility and is considered one of the P. aeruginosa virulence hallmarks. Iron functions as a redox cofactor of enzymes required for vital biological processes and is thus essential for all living organisms. However, in aerobic environments, iron is mainly present in its ferric form, which is insoluble and poorly bioavailable. This problem increases in the human body because, as a reaction to the infection, the host induces a "nutritional immunity" response aiming to reduce the amount of iron available for invading microorganisms. P. aeruginosa contains several mechanisms for iron acquisition including (1) production of siderophores pyoverdine and pyochelin; (2) use of xenosiderophores produced by other microorganisms; (3) direct transport of ferrous ions; and (4) utilization of host iron carriers (e.g., heme). However, although essential, iron results toxic when present in excess because it facilitates the production of reactive oxygen species (ROS) that damage bacterial cells. P. aeruginosa contains ferritins and efflux systems for iron withdrawal to avoid excess of this metal. Production of iron acquisition and removal systems is highly regulated to ensure sufficient iron for metabolic needs while preventing its toxicity. This chapter covers the different mechanisms used by P. aeruginosa to maintain iron homeostasis, which is vital for this pathogen to grow and proliferate in the host. We also highlight current strategies to block P. aeruginosa infections by disrupting iron homeostasis.
Assuntos
Anti-Infecciosos , Pseudomonas aeruginosa , Humanos , Pseudomonas aeruginosa/fisiologia , Sideróforos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ferro/metabolismo , Homeostase , Fatores de Virulência/metabolismo , Heme , Antibacterianos , Ferritinas/metabolismo , Íons , Proteínas de Bactérias/metabolismoRESUMO
Pyoverdines (PVDs) are a class of siderophores produced mostly by members of the genus Pseudomonas. Their primary function is to accumulate, mobilize, and transport iron necessary for cell metabolism. Moreover, PVDs also play a crucial role in microbes' survival by mediating biofilm formation and virulence. In this review, we reorganize the information produced in recent years regarding PVDs biosynthesis and pathogenic mechanisms, since PVDs are extremely valuable compounds. Additionally, we summarize the therapeutic applications deriving from the PVDs' use and focus on their role as therapeutic target themselves. We assess the current biotechnological applications of different sectors and evaluate the state-of-the-art technology relating to the use of synthetic biology tools for pathway engineering. Finally, we review the most recent methods and techniques capable of identifying such molecules in complex matrices for drug-discovery purposes.
Assuntos
Oligopeptídeos , Sideróforos , Ferro/metabolismo , Oligopeptídeos/metabolismo , Pseudomonas/metabolismo , Pseudomonas aeruginosa/metabolismo , Sideróforos/metabolismoRESUMO
Multidrug efflux pumps are critical elements in both intrinsic and acquired antibiotic resistance of bacterial populations. Consequently, most studies regarding these protein machineries focus on this specific phenotype. Nevertheless, different works show that efflux pumps participate in other aspects of bacterial physiology too. Herein, we study the Pseudomonas aeruginosa multidrug efflux pump MexJK. Previous studies, using model strains lacking MexAB-OprM and MexCD-OprJ efflux pumps, support that MexJK can extrude erythromycin, tetracycline, and triclosan. However, the results here reported indicate that this potential increased extrusion, in a mutant overexpressing mexJK, does not alter the antibiotics susceptibility in a wild-type genetic background where all intrinsic multidrug efflux pumps remain functional. Nevertheless, a clear impact on the quorum sensing (QS) response, mainly in the Pqs-dependent QS regulation network and in the expression of Pqs-regulated virulence factors, was observed linked to mexJK overexpression. The production of the siderophore pyoverdine strongly depended on the level of mexJK expression, suggesting that MexJK might participate in P. aeruginosa pyoverdine-dependent iron homeostasis. All in all, the results presented in the current article support that the functions of multidrug efflux pumps, as MexJK, go beyond antibiotic resistance and can modulate other relevant aspects of bacterial physiology.
Assuntos
Pseudomonas aeruginosa , Percepção de Quorum , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana Múltipla/genética , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Percepção de Quorum/genéticaRESUMO
Pseudomonas chlororaphis YL-1 has extensive antimicrobial activities against phytopathogens, and its genome harbors a pyoverdine (PVD) biosynthesis gene cluster. The alternative sigma factor PvdS in Pseudomonas aeruginosa PAO1 acts as a critical regulator in response to iron starvation. The assembly of the PVD backbone starts with peptide synthetase enzyme PvdL. PvdF catalyzes formylation of l-OH-Orn to produce l-N5-hydroxyornithine. Here, we describe the characterization of PVD production in YL-1 and its antimicrobial activity in comparison with that of its PVD-deficient ΔpvdS, ΔpvdF, and ΔpvdL mutants, which were obtained using a sacB-based site-specific mutagenesis strategy. Using in vitro methods, we examined the effect of exogenous iron under low-iron conditions and an iron-chelating agent under iron-sufficient conditions on PVD production, antibacterial activity, and the relative expression of the PVD transcription factor gene pvdS in YL-1. We found that strain YL-1, the ΔpvdF mutant, and the ΔpvdS(pUCP26-pvdS) complemented strain produced visible PVDs and demonstrated a wide range of inhibitory effects against Gram-negative and Gram-positive bacteria in vitro under low-iron conditions and that with the increase of iron, its PVD production and antibacterial activity were reduced. The antibacterial compounds produced by strain YL-1 under low-iron conditions were PVDs based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Moreover, the antibacterial activity observed in vitro was correlated with in vivo control efficacies of strain YL-1 against rice bacterial leaf blight (BLB) disease caused by Xanthomonas oryzae pv. oryzae. Collectively, PVDs are responsible for the antibacterial activities of strain YL-1 under both natural and induced low-iron conditions.IMPORTANCE The results demonstrated that PVDs are essential for the broad-spectrum antibacterial activities of strain YL-1 against both Gram-positive and Gram-negative bacteria under low-iron conditions. Our findings also highlight the effect of exogenous iron on the production of PVD and the importance of this bacterial product in bacterial interactions. As a biocontrol agent, PVDs can directly inhibit the proliferation of the tested bacteria in addition to participating in iron competition.
Assuntos
Antibacterianos/farmacologia , Ferro/metabolismo , Oligopeptídeos/farmacologia , Pseudomonas chlororaphis/metabolismo , Antibacterianos/química , Cromatografia Líquida , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Oligopeptídeos/química , Pseudomonas chlororaphis/química , Pseudomonas chlororaphis/genética , Espectrometria de Massas em TandemRESUMO
Bacteria often cooperate by secreting molecules that can be shared as public goods between cells. Because the production of public goods is subject to cheating by mutants that exploit the good without contributing to it, there has been great interest in elucidating the evolutionary forces that maintain cooperation. However, little is known about how bacterial cooperation evolves under conditions where cheating is unlikely to be of importance. Here we use experimental evolution to follow changes in the production of a model public good, the iron-scavenging siderophore pyoverdine, of the bacterium Pseudomonas aeruginosa. After 1200 generations of evolution in nine different environments, we observed that cheaters only reached high frequency in liquid medium with low iron availability. Conversely, when adding iron to reduce the cost of producing pyoverdine, we observed selection for pyoverdine hyperproducers. Similarly, hyperproducers also spread in populations evolved in highly viscous media, where relatedness between interacting individuals is increased. Whole-genome sequencing of evolved clones revealed that hyperproduction is associated with mutations involving genes encoding quorum-sensing communication systems, while cheater clones had mutations in the iron-starvation sigma factor or in pyoverdine biosynthesis genes. Our findings demonstrate that bacterial social traits can evolve rapidly in divergent directions, with particularly strong selection for increased levels of cooperation occurring in environments where individual dispersal is reduced, as predicted by social evolution theory. Moreover, we establish a regulatory link between pyoverdine production and quorum-sensing, showing that increased cooperation with respect to one trait (pyoverdine) can be associated with the loss (quorum-sensing) of another social trait.