Search

Interactions between tumoral cells and tumor‐associated bacteria within the tumor microenvironment play a significant role in tumor survival and progression, potentially impacting cancer treatment outcomes. In lung cancer patients, the Gram‐negative pathogen Pseudomonas aeruginosa raises questions about its role in tumor survival. Here, a microfluidic‐based 3D‐human lung tumor spheroid‐P. aeruginosa model is developed to study the bacteria's impact on tumor survival. P. aeruginosa forms a tumor‐associated biofilm by producing Psl exopolysaccharide and secreting iron‐scavenging pyoverdine, which is critical for establishing a bacterial community in tumors. Consequently, pyoverdine promotes cancer progression by reducing susceptibility to iron‐induced death (ferroptosis), enhancing cell viability, and facilitating several cancer hallmarks, including epithelial–mesenchymal transition and metastasis. A promising combinatorial therapy approach using antimicrobial tobramycin, ferroptosis‐inducing thiostrepton, and anti‐cancer doxorubicin could eradicate biofilms and tumors. This work unveils a novel phenomenon of cross‐kingdom cooperation, where bacteria protect tumors from death, and it paves the way for future research in developing antibiofilm cancer therapies. Understanding these interactions offers potential new strategies for combatting cancer and enhancing treatment efficacy. [ABSTRACT FROM AUTHOR]

Aspergillus fumigatus (Af) and Pseudomonas aeruginosa (Pa) are pathogens inhabiting the lungs of persons with cystic fibrosis (CF), or immune-compromised patients, causing or aggravating disease. We previously investigated their microbial interaction as well as susceptibility to anti-fungal drugs using RPMI medium (contains undetectable iron concentrations), as is standard for susceptibility testing. Here we investigated microbial interaction in synthetic sputum medium (SSPM), a complex mixture designed to mimic the milieu in CF lungs. SSPM contains Fe2+. Pa laboratory strain PA14 or PA14 siderophore mutant planktonic culture filtrate, prepared in RPMI or SSPM, were compared for inhibition of Af biofilm formation. SSPM enhanced bacterial and fungal growth and the production of the Pa molecules pyoverdine, phenazines, and rhamnolipids. Af was more susceptible to these molecules in SSPM (with the exception of pyoverdine). SSPM interfered with fungal susceptibility to pyoverdine. Studies with the mutant helped to reveal that the reduced anti-fungal activity of pyoverdine in SSPM appears to be compensated by higher production of other anti-fungal molecules, e.g., rhamnolipids, phenazines, and PQS, and higher Af sensitivity to these molecules. In summary, SSPM better defines Pa–Af intermicrobial competition in the milieu of CF lungs. [ABSTRACT FROM AUTHOR]

Pyoverdines are high affinity siderophores produced by most Pseudomonas with a wide role in microbial interspecies interactions. They are primarily composed of a conserved chromophore moiety, an acyl side chain and a peptide backbone which may be highly variable among strains. Upon ferric iron sequestration, pyoverdines are internalized through specialized receptors. The peptide precursor of pyoverdine, termed ferribactin, is synthesized by a set of non-ribosomal peptide synthetase (NRPS) enzymes and further modified by tailoring enzymes. While PvdL, the NRPS responsible for the synthesis of the peptide moiety that derives into the chromophore is conserved, the NRPSs for the peptide backbone are different across fluorescent Pseudomonas. Although the variation of pyoverdine is a widely recognized characteristic within the genus, the evolutionary events associated with the diversity and distribution of this trait remain mostly unknown. This study analyzed the NRPSs clusters for the biosynthesis of the peptide backbone of ferribactin in the genomes of a representative subset of strains of the Pseudomonas fluorescens complex. Bioinformatic analysis of the specificity of adenylation domains of the NRPSs allowed the prediction of 30 different pyoverdine variants. Phylogenetic reconstruction and mapping of the NRPS clusters pinpointed two different general levels of modifications. In the first level, a complete replacement of the set of NRPRs by horizontal transfer occurs. In the second level, the original set of NRPSs is modified through different mechanisms, including partial substitution of the NRPS genes by horizontal transfer, adenylation domain specificity change or NRPS accessory domain gain/loss. [ABSTRACT FROM AUTHOR]

Pyoverdines are iron-chelating siderophores employed by various pseudomonads to promote their growth in iron-limited environments, facilitating both beneficial and detrimental interactions with co-inhabiting microbes or hosts, including plants and animals. The fluorescent pseudomonads produce fluorescent pyoverdines comprised of a conserved central chromophore and a unique strain-specific peptidic side chain produced by non-ribosomal peptide synthetases. Pyoverdine Pf5 (PVD-Pf5) is produced by Pseudomonas protegens Pf-5, a species known for supporting plant growth and its involvement in plant pathogen control. To develop a means of exploring the dynamics of P. protegens activity in soil and in the rhizosphere, we selected DNA aptamers that specifically recognize PVD-Pf5 with high affinities. Two selected aptamers with only 16% identity in sequence were examined for structure and function. We found evidence that both aptamers form structures in their apo-forms and one aptamer has structural features suggesting the presence of a G-quadruplex. Although their tertiary structures are predicted to be different, both aptamers bind the target PVD-Pf5 with similar affinities and do not bind other siderophores, including the related pyoverdine, pseudobactin, produced by Pseudomonas sp. B10. One aptamer binds the pyoverdine peptide component and may also interact with the chromophore. This aptamer was integrated into a nanoporous aluminum oxide biosensor and demonstrated to successfully detect PVD-Pf5 and not to detect other siderophores that do not bind to the aptamer when evaluated in solution. This sensor provides a future opportunity to track the locations of P. protegens around plant roots and to monitor PVD-Pf5 production and movement through the soil. [ABSTRACT FROM AUTHOR]

ABSTRACT Bacteria assume two distinct lifestyles: the planktonic and biofilm modes of growth. Additionally, dispersion has emerged as a third phenotype, accompanied by the distinct phenotypes and the unique expression of >600 genes. Here, we asked whether the distinct phenotype of dispersed cells is already apparent within minutes of egressing from the biofilm. We used RNA-seq to show that the physiology of freshly dispersed cells from Pseudomonas aeruginosa biofilms is highly different from those of planktonic and biofilm cells, apparent by dispersed cells uniquely expressing 194 genes. Unique and differentially expressed genes relative to planktonic or biofilm cells include genes associated with type IV pili, pyoverdine, type III and type VI secretion systems, and antibiotic resistance that are downregulated in dispersed cells, whereas the transcript abundance of genes involved in swimming motility, Hxc type II secretion system and various other virulence factors, and metabolic and energy-generating pathways are increased, indicative of dispersion coinciding with an awakening and re-energizing of dispersed cells, and a switch in virulence, further apparent by freshly dispersed cells significantly subverting engulfment by macrophages. The findings suggest that dispersed cells display a distinct phenotype within minutes of egressing from the biofilm, with freshly dispersed cells already capable of efficiently evading phagocytosis.IMPORTANCEDispersion is considered a transitionary phenotype, enabling bacteria to switch between the communal, biofilm lifestyle, where cells share resources and are protected from harmful conditions to the planktonic state. Here, we demonstrate that within minutes of leaving the biofilm, dispersed cells express genes and display phenotypic traits that are distinct from biofilms and planktonic cells. Our findings suggest that dispersed cells quickly adapt to a less structured and protected but more nutrient-rich environment, with this trade-off in environment coinciding with an awakening and a switch in virulence, specifically a switch from directly intoxicating host cells and potential competitors toward more broadly active virulence factors and strategies of evasion. To our knowledge, this is the first report of dispersed cells’ distinct (trade-off) phenotype and their enhanced resilience so soon after egressing from the biofilm.

The global rise of antibiotic resistance calls for new drugs against bacterial pathogens. A common approach is to search for natural compounds deployed by microbes to inhibit competitors. Here, we show that the iron-chelating pyoverdines, siderophores produced by environmental Pseudomonas spp., have strong antibacterial properties by inducing iron starvation and growth arrest in pathogens. A screen of 320 natural Pseudomonas isolates used against 12 human pathogens uncovered several pyoverdines with particularly high antibacterial properties and distinct chemical characteristics. The most potent pyoverdine effectively reduced growth of the pathogens Acinetobacter baumannii, Klebsiella pneumoniae, and Staphylococcus aureus in a concentration- and iron-dependent manner. Pyoverdine increased survival of infected Galleria mellonella host larvae and showed low toxicity for the host, mammalian cell lines, and erythrocytes. Furthermore, experimental evolution of pathogens combined with whole-genome sequencing revealed limited resistance evolution compared to an antibiotic. Thus, pyoverdines from environmental strains have the potential to become a new class of sustainable antibacterials against specific human pathogens.

Iron (Fe) deficiency in crops significantly reduces yield, impacting agricultural productivity worldwide. Synthetic Fe chelates are commonly applied as fertilizers to address this issue, but their synthetic nature and prolonged use poses environmental risks. Thus, inoculation of plant growth-promoting bacteria rises as an alternative to enhance Fe uptake in crops while minimizing reliance on synthetic chelates. This study aimed to examine the influence of Pseudomonas RMC4 inoculation and pyoverdine application on cucumber plants cultivated hydroponically under Fe deficiency conditions. Evaluations included the SPAD index, plant biomass, root morphology, Fe-chelate reductase activity, gene expression, and ionomic analysis. Following Fe deficiency, Pseudomonas RMC4 inoculation improved the SPAD index, increased dry weight, enhanced root development, and facilitated Fe acquisition mechanisms, thus, fostering the endogenous resilience of the plant to the limited Fe availability. This improvement was observed with bacterial inoculation or pyoverdine application alongside an insoluble Fe source (ferrihydrite). Overall, the results suggest the beneficial impact of Pseudomonas RMC4 inoculation in alleviating symptoms of Fe deficiency. Future studies will investigate bacterial application under field conditions to assess its potential in replacing synthetic Fe-chelates fertilizers in crop production in favor of more sustainable agriculture.

ABSTRACT The human pathogen Pseudomonas aeruginosa (Pa) poses a major risk for a range of severe infections, particularly lung infections in patients suffering from cystic fibrosis (CF). As previously reported, the virulent behavior of this pathogen is enhanced by elevated levels of Ca2+ that are commonly present in CF nasal and lung fluids. In addition, a Ca2+-binding EF-hand protein, EfhP (PA4107), was partially characterized and shown to be critical for the Ca2+-regulated virulence in P. aeruginosa. Here, we describe the rapid (10 min, 60 min), and adaptive (12 h) transcriptional responses of PAO1 to elevated Ca2+ detected by genome-wide RNA sequencing and show that efhP deletion significantly hindered both rapid and adaptive Ca2+ regulation. The most differentially regulated genes included multiple Fe sequestering mechanisms, a large number of extracytoplasmic function sigma factors (ECFσ), and several virulence factors, such as the production of pyocins. The Ca2+ regulation of Fe uptake was also observed in CF clinical isolates and appeared to involve the global regulator Fur. In addition, we showed that the efhP transcription is controlled by Ca2+ and Fe, and this regulation required a Ca2+-dependent two-component regulatory system CarSR. Furthermore, the efhP expression is significantly increased in CF clinical isolates and upon pathogen internalization into epithelial cells. Overall, the results established for the first time that Ca2+ controls Fe sequestering mechanisms in P. aeruginosa and that EfhP plays a key role in the regulatory interconnectedness between Ca2+ and Fe signaling pathways, the two distinct and important signaling pathways that guide the pathogen’s adaptation to the host.IMPORTANCEPseudomonas aeruginosa (Pa) poses a major risk for severe infections, particularly in patients suffering from cystic fibrosis (CF). For the first time, kinetic RNA sequencing analysis identified Pa rapid and adaptive transcriptional responses to Ca2+ levels consistent with those present in CF respiratory fluids. The most highly upregulated processes include iron sequestering, iron starvation sigma factors, and self-lysis factors pyocins. An EF-hand Ca2+ sensor, EfhP, is required for at least 1/3 of the Ca2+ response, including the majority of the iron uptake mechanisms and the production of pyocins. Transcription of efhP itself is regulated by Ca2+ and Fe, and increases during interactions with host epithelial cells, suggesting the protein’s important role in Pa infections. The findings establish the regulatory interconnectedness between Ca2+ and iron signaling pathways that shape Pa transcriptional responses. Therefore, understanding Pa’s transcriptional response to Ca2+ and associated regulatory mechanisms will serve in the development of future therapeutics targeting Pa’s dangerous infections.

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. [ABSTRACT FROM AUTHOR]

Microbial secondary metabolites are a rich source for pharmaceutical discoveries and play crucial ecological functions. While tools exist to identify secondary metabolite clusters in genomes, precise sequence-to-function mapping remains challenging because neither function nor substrate specificity of biosynthesis enzymes can accurately be predicted. Here, we developed a knowledge-guided bioinformatic pipeline to solve these issues. We analyzed 1928 genomes of Pseudomonas bacteria and focused on iron-scavenging pyoverdines as model metabolites. Our pipeline predicted 188 chemically different pyoverdines with nearly 100% structural accuracy and the presence of 94 distinct receptor groups required for the uptake of iron-loaded pyoverdines. Our pipeline unveils an enormous yet overlooked diversity of siderophores (151 new structures) and receptors (91 new groups). Our approach, combining feature sequence with phylogenetic approaches, is extendable to other metabolites and microbial genera, and thus emerges as powerful tool to reconstruct bacterial secondary metabolism pathways based on sequence data.

Pyoverdines are high affinity siderophores produced by most Pseudomonas with a wide role in microbial interspecies interactions. They are primarily composed of a conserved chromophore moiety, an acyl side chain and a peptide backbone which may be highly variable among strains. Upon ferric iron sequestration, pyoverdines are internalized through specialized receptors. The peptide precursor of pyoverdine, termed ferribactin, is synthesized by a set of non-ribosomal peptide synthetase (NRPS) enzymes and further modified by tailoring enzymes. While PvdL, the NRPS responsible for the synthesis of the peptide moiety that derives into the chromophore is conserved, the NRPSs for the peptide backbone are different across fluorescent Pseudomonas. Although the variation of pyoverdine is a widely recognized characteristic within the genus, the evolutionary events associated with the diversity and distribution of this trait remain mostly unknown. This study analyzed the NRPSs clusters for the biosynthesis of the peptide backbone of ferribactin in the genomes of a representative subset of strains of the Pseudomonas fluorescens complex. Bioinformatic analysis of the specificity of adenylation domains of the NRPSs allowed the prediction of 30 different pyoverdine variants. Phylogenetic reconstruction and mapping of the NRPS clusters pinpointed two different general levels of modifications. In the first level, a complete replacement of the set of NRPRs by horizontal transfer occurs. In the second level, the original set of NRPSs is modified through different mechanisms, including partial substitution of the NRPS genes by horizontal transfer, adenylation domain specificity change or NRPS accessory domain gain/loss.

Pseudomonas aeruginosa is a common pathogen of acute and chronic infections that can increase risk to burned patients and those with chronic wounds, especially when forming biofilms. These complex ecosystems are formed by several bacteria that communicate, cooperate, and compete with each other. To enhance its survival capacity in such a competitive environment, P. aeruginosa produces secondary metabolites with a range of biological activities, among other factors. Here, we evaluated how such bioactive molecules mediate intraspecies interactions to modulate P. aeruginosa biofilm formation. An overall decrease in biofilm formation on the surface of stainless steel coupons was observed for both dual-strain and single-strain biofilms formed in the presence of secondary metabolites extracted from other P. aeruginosa strains. Modulation of biofilm formation by these metabolites during P. aeruginosa intraspecies interactions suggests a role for these molecules in strain competition. The most likely mechanism by which it occurs might be modulation of iron uptake. Among the genes analyzed, those related to import (tonB1) and biosynthesis (pvdS) of the siderophore pyoverdine were the most downregulated during exposure to the metabolite extracts. Exploring P. aeruginosa intraspecies interactions during biofilm formation might be an interesting approach to understand its lifestyle better and, thus, the mechanisms involved in the species’ biofilm formation.

The emergence of multidrug-resistant (MDR) microorganisms combined with the ever-draining antibiotic pipeline poses a disturbing and immensely growing public health challenge that requires a multidisciplinary approach and the application of novel therapies aimed at unconventional targets and/or applying innovative drug formulations. Hence, bacterial iron acquisition systems and bacterial Fe2+/3+-containing enzymes have been identified as a plausible target of great potential. The intriguing "Trojan horse" approach deprives microorganisms from the essential iron. Recently, gallium's potential in medicine as an iron mimicry species has attracted vast attention. Different Ga3+ formulations exhibit diverse effects upon entering the cell and thus supposedly have multiple targets. The aim of the current study is to specifically distinguish characteristics of great significance in regard to the initial gallium-based complex, allowing the alien cation to effectively compete with the native ferric ion for binding the siderophores pyochelin and pyoverdine secreted by the bacterium P. aeruginosa. Therefore, three gallium-based formulations were taken into consideration: the first-generation gallium nitrate, Ga(NO3)3, metabolized to Ga3+-hydrated forms, the second-generation gallium maltolate (tris(3-hydroxy-2-methyl-4-pyronato)gallium), and the experimentally proven Ga carrier in the bloodstream—the protein transferrin. We employed a reliable in silico approach based on DFT computations in order to understand the underlying biochemical processes that govern the Ga3+/Fe3+ rivalry for binding the two bacterial siderophores. [ABSTRACT FROM AUTHOR]

The increasing incidence of antimicrobial resistance exhibited by biofilm-forming microbial pathogens has been recognized as one of the major issues in the healthcare sector. In the present study, nanomaterial-based controlling the biofilm and virulence properties has been considered an alternative approach. Pyoverdine (PVD) isolated from the Pseudomonas aeruginosa was utilized as a biological corona to synthesize silver nanoparticles (AgNPs), which will be helpful in a targeted action to microbial pathogens due to the recognition of the corona of the nanoparticles by the pathogenic membrane. Synthesized PVD-AgNPs were spherical to irregular, with an average size value of 251.87 ± 21.8 nm and zeta potential with a value of −36.51 ± 0.69 mV. The MIC value of PVD-AgNPs towards P. aeruginosa, Listeria monocytogenes, Staphylococcus aureus, Streptococcus mutans, Escherichia coli, and Candida albicans in the standard and host-mimicking media were observed in decreasing order in a multi-fold, such as standard growth media > sputum > synthetic human urine > saliva. Both the initial stage and the well-established biofilms of these microbial pathogens have been effectively inhibited and eradicated by PVD-AgNPs. PVD-AgNPs increase the susceptibility of tetracycline, PVD, and amphotericin B towards established mature mono- and mixed-species biofilms of S. aureus and C. albicans. Additionally, PVD-AgNPs attenuate several virulence properties, such as inhibition of protease activity, motility, and PVD and pyocyanin production in P. aeruginosa. The inhibition of gene expression of biofilm and virulence-associated genes in P. aeruginosa validates its phenotypic effects.

Aspergillus fumigatus (Af) and Pseudomonas aeruginosa (Pa) are pathogens inhabiting the lungs of persons with cystic fibrosis (CF), or immune-compromised patients, causing or aggravating disease. We previously investigated their microbial interaction as well as susceptibility to anti-fungal drugs using RPMI medium (contains undetectable iron concentrations), as is standard for susceptibility testing. Here we investigated microbial interaction in synthetic sputum medium (SSPM), a complex mixture designed to mimic the milieu in CF lungs. SSPM contains Fe2+. Pa laboratory strain PA14 or PA14 siderophore mutant planktonic culture filtrate, prepared in RPMI or SSPM, were compared for inhibition of Af biofilm formation. SSPM enhanced bacterial and fungal growth and the production of the Pa molecules pyoverdine, phenazines, and rhamnolipids. Af was more susceptible to these molecules in SSPM (with the exception of pyoverdine). SSPM interfered with fungal susceptibility to pyoverdine. Studies with the mutant helped to reveal that the reduced anti-fungal activity of pyoverdine in SSPM appears to be compensated by higher production of other anti-fungal molecules, e.g., rhamnolipids, phenazines, and PQS, and higher Af sensitivity to these molecules. In summary, SSPM better defines Pa–Af intermicrobial competition in the milieu of CF lungs.

Abstract The majority of the root microbiota formation derives from soil-dwelling microorganisms. The limited extent of thorough investigation leads to a dearth of knowledge concerning the intricate mechanisms of microbe-microbe interaction implicated in the establishment of root microbiota. Therefore, the taxonomic signatures in bacterial inhibition profiles were determined by in vitro testing of 39,204 binary interbacterial interactions. However, findings from genetic and metabolomic studies elucidated that co-functioning of the antimicrobial 2,4-d iacetylphloroglucinol (DAPG) and the iron chelator pyoverdine as exometabolites has significantly contributed to the potent inhibitory activities of the highly antagonistic Pseudomonas brassicacearum R401. Microbiota restoration with a core of Arabidopsis thaliana root commensals showed that these exometabolites possess a root niche-specific function in establishing root competence and inducing anticipated changes in root surroundings. Both biosynthetic operons are abundant in roots in natural habitats, indicating that these exometabolites co-functioning is an adaptive feature that helps Pseudomonad dominate the root microbiota.

ABSTRACTThe multidrug-resistant pathogen Pseudomonas aeruginosa is a common nosocomial respiratory pathogen that continues to threaten the lives of patients with mechanical ventilation in intensive care units and those with underlying comorbidities such as cystic fibrosis or chronic obstructive pulmonary disease. For over 20 years, studies have repeatedly demonstrated that the major siderophore pyoverdine is an important virulence factor for P. aeruginosa in invertebrate and mammalian hosts in vivo. Despite its physiological significance, an in vitro, mammalian cell culture model that can be used to characterize the impact and molecular mechanisms of pyoverdine-mediated virulence has only been developed very recently. In this study, we adapt a previously-established, murine macrophage-based model to use human bronchial epithelial (16HBE) cells. We demonstrate that conditioned medium from P. aeruginosa induced rapid 16HBE cell death through the pyoverdine-dependent secretion of cytotoxic rhamnolipids. Genetic or chemical disruption of pyoverdine biosynthesis decreased rhamnolipid production and mitigated cell death. Consistent with these observations, chemical depletion of lipids or genetic disruption of rhamnolipid biosynthesis abrogated the toxicity of the conditioned medium. Furthermore, we also examine the effects of exposure to purified pyoverdine on 16HBE cells. While pyoverdine accumulated within cells, it was largely sequestered within early endosomes, resulting in minimal cytotoxicity. More membrane-permeable iron chelators, such as the siderophore pyochelin, decreased epithelial cell viability and upregulated several pro-inflammatory genes. However, pyoverdine potentiated these iron chelators in activating pro-inflammatory pathways. Altogether, these findings suggest that the siderophores pyoverdine and pyochelin play distinct roles in virulence during acute P. aeruginosa lung infection.IMPORTANCEMultidrug-resistant Pseudomonas aeruginosa is a versatile bacterium that frequently causes lung infections. This pathogen is life-threatening to mechanically-ventilated patients in intensive care units and is a debilitating burden for individuals with cystic fibrosis. However, the role of P. aeruginosa virulence factors and their regulation during infection are not fully understood. Previous murine lung infection studies have demonstrated that the production of siderophores (e.g., pyoverdine and pyochelin) is necessary for full P. aeruginosa virulence. In this report, we provide further mechanistic insight into this phenomenon. We characterize distinct and novel ways these siderophores contribute to virulence using an in vitro human lung epithelial cell culture model.

Antibiotic resistance is a growing concern for global health, demanding innovative and effective strategies to combat pathogenic bacteria. Pyoverdines, iron-chelating siderophores produced by environmental Pseudomonas spp. present a novel class of promising compounds to induce growth arrest in pathogens through iron starvation. While we previously demonstrated the efficacy of pyoverdines as antibacterials, our understanding of how these molecules interact with antibiotics and impact resistance evolution remains unknown. Here, we investigated the propensity of three Escherichia coli strains to evolve resistance against pyoverdine, the cephalosporin antibiotic ceftazidime, and their combination. We used a naive E. coli wildtype strain and two isogenic variants carrying the bla TEM-1 β-lactamase gene on either the chromosome or a costly multicopy plasmid to explore the influence of genetic background on selection for resistance. We found that strong resistance against ceftazidime and weak resistance against pyoverdine evolved in all E. coli variants under single treatment. Ceftazidime resistance was linked to mutations in outer membrane porin genes (envZ and ompF), whereas pyoverdine resistance was associated with mutations in the oligopeptide permease (opp) operon. In contrast, ceftazidime resistance phenotypes were attenuated under combination treatment, especially for the E. coli variant carrying bla TEM-1 on the multicopy plasmid. Altogether, our results show that ceftazidime and pyoverdine interact neutrally and that pyoverdine as an antibacterial is particularly potent against plasmid-carrying E. coli strains, presumably because iron starvation compromises both cellular metabolism and plasmid replication. [ABSTRACT FROM AUTHOR]

The airways of cystic fibrosis (CF) patients are colonized by many pathogens and the most common is Pseudomonas aeruginosa, an environmental pathogen that is able to infect immunocompromised patients thanks to its ability to develop resistance to conventional antibiotics. Over 12% of all patients colonized by P. aeruginosa harbour multi-drug resistant species. During airway infection in CF, P. aeruginosa adopts various mechanisms to survive in a hostile ecological niche characterized by low oxygen concentration, nutrient limitation and high osmotic pressure. To this end, P. aeruginosa uses a variety of virulence factors including pigment production, biofilm formation, motility and the secretion of toxins and proteases. This study represents the first report that systematically analyzes the differences in virulence features, in normoxia and anoxia, of clinical P. aeruginosa isolated from CF patients, characterized by multi- or pan-drug antibiotic resistance compared to antibiotic sensitive strains. The virulence features, such as biofilm formation, protease secretion and motility, are highly diversified in anaerobiosis, which reflects the condition of chronic CF infection. These findings may contribute to the understanding of the real-world lifestyle of pathogens isolated during disease progression in each particular patient and to assist in the design of therapeutic protocols for personalized medicine. [ABSTRACT FROM AUTHOR]

The application of synthetic iron chelates to overcome iron deficiency in crops is leading to a high impact on the environment, making it necessary to find more friendly fertilizers. A promising alternative is the application of biodegradable iron chelates, such as those based on siderophores. In the present work, seven bacterial strains of the genus Pseudomonas were selected for their ability to secrete pyoverdine, a siderophore with a high affinity for iron, which could be used as a biofertilizer. The concentration of siderophores secreted by each bacterium expressed as desferrioxamine B equivalents, and the pyoverdine concentration was determined. Their potential as Fe biofertilizers was determined based on their capacity to complex Fe, determining the maximum iron complexation capacity at alkaline pH and selecting the RMC4 strain. The biostimulant capacity of the RMC4 strain was evaluated through the secretion of organic acids such as the hormone Indol-3-acetic acid or glutamic acid, among others, in a kinetic assay. Finally, the genome of RMC4 was determined, and the strain was identified as Pseudomonas monsensis. The annotated genome was screened for genes and gene clusters implicated in biofertilization and plant growth promotion. Besides iron mobilization, genes related to phosphorus solubilization, production of phytohormones and biological control, among others, were observed, indicating the suitability of RMC4 as an inoculant. In conclusion, RMC4 and its siderophores are promising sources for Fe biofertilization in agriculture. [ABSTRACT FROM AUTHOR]

ABSTRACT Tripartite efflux systems transport antimicrobial agents, toxic metabolites, and siderophores from Gram-negative cells into the environment. For example, the main siderophore pyoverdine of the soil bacterium Pseudomonas putida KT2440 is secreted into the environment via the PvdRT-OpmQ and MdtABC-OpmB systems. Here, we looked for efflux systems that might be involved in the secretion of pyoverdine in addition to the latter two systems. Screening of different efflux systems revealed that parX (encoding the periplasmic adapter protein of the ParXY system) is of particular importance for bacterial growth under iron limitation. Further analysis showed that the deletion of parX impairs the production and secretion of pyoverdine, causing the observed growth effect. The effects were dependent on the presence of other tripartite efflux systems and the conditions of iron limitation. The results suggest that ParXY not only plays a role in antibiotic resistance, as shown previously, but also influences the secretion of siderophores in a network of overlapping activities of different tripartite efflux systems. IMPORTANCE Gram-negative bacteria from the Pseudomonas group are survivors in various environmental niches. For example, the bacteria secrete siderophores to capture ferric ions under deficiency conditions. Tripartite efflux systems are involved in the secretion of siderophores, which are also important for antibiotic resistance. For one of these efflux systems, the resistance-nodulation-cell division transporter ParXY from the model organism Pseudomonas putida KT2440, we show that it influences the secretion of the siderophore pyoverdine in addition to its already known involvement in antibiotic resistance. Phenotypically, its role in pyoverdine secretion is only apparent when other pyoverdine secretion systems are inactive. The results confirm that the different tripartite efflux systems have overlapping substrate specificities and can at least partially functionally substitute for each other, especially in important physiological activities such as supplying the cell with iron ions. This fact must be taken into account when developing specific inhibitors for tripartite efflux systems.

The majority of the root microbiota formation derives from soil-dwelling microorganisms. The limited extent of thorough investigation leads to a dearth of knowledge concerning the intricate mechanisms of microbe-microbe interaction implicated in the establishment of root microbiota. Therefore, the taxonomic signatures in bacterial inhibition profiles were determined by in vitro testing of 39,204 binary interbacterial interactions. However, findings from genetic and metabolomic studies elucidated that co-functioning of the antimicrobial 2,4-d iacetylphloroglucinol (DAPG) and the iron chelator pyoverdine as exometabolites has significantly contributed to the potent inhibitory activities of the highly antagonistic Pseudomonas brassicacearum R401. Microbiota restoration with a core of Arabidopsis thaliana root commensals showed that these exometabolites possess a root niche-specific function in establishing root competence and inducing anticipated changes in root surroundings. Both biosynthetic operons are abundant in roots in natural habitats, indicating that these exometabolites co-functioning is an adaptive feature that helps Pseudomonad dominate the root microbiota. [ABSTRACT FROM AUTHOR]

Abstract 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 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.

Abstract 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.

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. [ABSTRACT FROM AUTHOR]

The aim of this study was to examine the impact of twenty honey samples, harvested in Mt. Olympus (Greece), on the virulence factors implicated in P. aeruginosa pathogenesis. Six key virulence factors (protease and elastase activity, pyocyanin and pyoverdine concentration, biofilm formation, and swimming motility) were selected in order to assess the effect of the tested honeys compared with Manuka honey. All tested honeys demonstrated a significant inhibition of protease and elastase activity compared with the control. Six and thirteen honeys exerted superior protease (no inhibition zone) and elastase (values lower than 55%) activity, respectively, compared with Manuka honey. Seventeen tested honeys exhibited reduced pyoverdine production compared with the control; all tested honeys, except for one, showed an inhibitory effect on pyocyanin production compared with the control. Regarding swimming motility, nine tested honeys demonstrated significantly higher inhibition compared with Manuka honey. Honey concentrations (6% v/v and 8% v/v) had the most profound impact, as they reduced biofilm formation to less than 20% compared with the control. Overall, our data demonstrate a significant inhibition of the virulence factors in the tested Mt. Olympus honeys, highlighting the strong antimicrobial activity against P. aeruginosa, an antibiotic-resistant pathogen of growing concern, which is implicated in severe nosocomial infections globally. [ABSTRACT FROM AUTHOR]

Tripartite efflux systems of the ABC‐type family transport a variety of substrates and contribute to the antimicrobial resistance of Gram‐negative bacteria. PvdRT‐OpmQ, a member of this family, is thought to be involved in the secretion of the newly synthesized and recycled siderophore pyoverdine in Pseudomonas species. Here, we purified and characterized the inner membrane component PvdT and the periplasmic adapter protein PvdR of the plant growth‐promoting soil bacterium Pseudomonas putida KT2440. We show that PvdT possesses an ATPase activity that is stimulated by the addition of PvdR. In addition, we provide the first biochemical evidence for direct interactions between pyoverdine and PvdRT. [ABSTRACT FROM AUTHOR]

Over the last years, the interkingdom microbial interactions concerning bacteria and fungi cohabiting and/or responsible for human pathologies have been investigated. In this context, the Gram-negative bacterium Pseudomonas aeruginosa and fungal species belonging to the Scedosporium/Lomentospora genera are widespread, multidrug-resistant, emergent, opportunistic pathogens that are usually co-isolated in patients with cystic fibrosis. The available literature reports that P. aeruginosa can inhibit the in vitro growth of Scedosporium/Lomentospora species; however, the complex mechanisms behind this phenomenon are mostly unknown. In the present work, we have explored the inhibitory effect of bioactive molecules secreted by P. aeruginosa (3 mucoid and 3 non-mucoid strains) on S. apiospermum (n = 6 strains), S. minutisporum (n = 3), S. aurantiacum (n = 6) and L. prolificans (n = 6) under cultivation in a cystic fibrosis mimic environment. It is relevant to highlight that all bacterial and fungal strains used in the present study were recovered from cystic fibrosis patients. The growth of Scedosporium/Lomentospora species was negatively affected by the direct interaction with either mucoid or non-mucoid strains of P. aeruginosa. Moreover, the fungal growth was inhibited by the conditioned supernatants obtained from bacteria-fungi co-cultivations and by the conditioned supernatants from the bacterial pure cultures. The interaction with fungal cells induced the production of pyoverdine and pyochelin, 2 well-known siderophores, in 4/6 clinical strains of P. aeruginosa. The inhibitory effects of these four bacterial strains and their secreted molecules on fungal cells were partially reduced with the addition of 5-flucytosine, a classical repressor of pyoverdine and pyochelin production. In sum, our results demonstrated that distinct clinical strains of P. aeruginosa can behave differently towards Scedosporium/Lomentospora species, even when isolated from the same cystic fibrosis patient. Additionally, the production of siderophores by P. aeruginosa was induced when co-cultivated with Scedosporium/Lomentospora species, indicating competition for iron and deprivation of this essential nutrient, leading to fungal growth inhibition. [ABSTRACT FROM AUTHOR]

Computational modeling and simulation of biological systems have become valuable tools for understanding and predicting cellular performance and phenotype generation. This work aimed to construct, model, and dynamically simulate the virulence factor pyoverdine (PVD) biosynthesis in Pseudomonas aeruginosa through a systemic approach, considering that the metabolic pathway of PVD synthesis is regulated by the quorum-sensing (QS) phenomenon. The methodology comprised three main stages: (i) Construction, modeling, and validation of the QS gene regulatory network that controls PVD synthesis in P. aeruginosa strain PAO1; (ii) construction, curating, and modeling of the metabolic network of P. aeruginosa using the flux balance analysis (FBA) approach; (iii) integration and modeling of these two networks into an integrative model using the dynamic flux balance analysis (DFBA) approximation, followed, finally, by an in vitro validation of the integrated model for PVD synthesis in P. aeruginosa as a function of QS signaling. The QS gene network, constructed using the standard System Biology Markup Language, comprised 114 chemical species and 103 reactions and was modeled as a deterministic system following the kinetic based on mass action law. This model showed that the higher the bacterial growth, the higher the extracellular concentration of QS signal molecules, thus emulating the natural behavior of P. aeruginosa PAO1. The P. aeruginosa metabolic network model was constructed based on the iMO1056 model, the P. aeruginosa PAO1 strain genomic annotation, and the metabolic pathway of PVD synthesis. The metabolic network model included the PVD synthesis, transport, exchange reactions, and the QS signal molecules. This metabolic network model was curated and then modeled under the FBA approximation, using biomass maximization as the objective function (optimization problem, a term borrowed from the engineering field). Next, chemical reactions shared by both network models were chosen to combine them into an integrative model. To this end, the fluxes of these reactions, obtained from the QS network model, were fixed in the metabolic network model as constraints of the optimization problem using the DFBA approximation. Finally, simulations of the integrative model (CCBM1146, comprising 1123 reactions and 880 metabolites) were run using the DFBA approximation to get (i) the flux profile for each reaction, (ii) the bacterial growth profile, (iii) the biomass profile, and (iv) the concentration profiles of metabolites of interest such as glucose, PVD, and QS signal molecules. The CCBM1146 model showed that the QS phenomenon directly influences the P. aeruginosa metabolism to PVD biosynthesis as a function of the change in QS signal intensity. The CCBM1146 model made it possible to characterize and explain the complex and emergent behavior generated by the interactions between the two networks, which would have been impossible to do by studying each system's individual components or scales separately. This work is the first in silico report of an integrative model comprising the QS gene regulatory network and the metabolic network of P. aeruginosa. [ABSTRACT FROM AUTHOR]

Siderocalin/Lipocalin 2/Neutrophil Gelatinase Associated Lipocalin/24p3 is an innate immune system protein with bacteriostatic activity, acting by tightly binding and sequestering diverse catecholate and mixed-type ferric siderophores from enteric bacteria and mycobacteria. Bacterial virulence achieved through siderophore modifications, or utilization of alternate siderophores, can be explained by evasion of Siderocalin binding. Siderocalin has also been implicated in a wide variety of disease processes, though often in seemingly contradictory ways, and has been proposed to bind to a broader array of ligands beyond siderophores. Using structural, directed mutational, and binding studies, we have sought to rigorously test, and fully elucidate, the Siderocalin recognition mechanism. Several proposed ligands fail to meet rigorous binding criteria, including the bacterial siderophore pyochelin, the iron-chelating catecholamine hormone norepinephrine, and the bacterial second messenger cyclic diguanylate monophosphate. While possessing a remarkably rigid structure, in principle simplifying analyses of ligand recognition, understanding Scn recognition is complicated by the observed conformational and stoichiometric plasticity, and instability, of its bona fide siderophore ligands. Since the role of Siderocalin at the early host/pathogen interface is to compete for bacterial ferric siderophores, we also analyzed how bacterial siderophore binding proteins and enzymes alternately recognize siderophores that efficiently bind to, or evade, Siderocalin sequestration - including determining the crystal structure of Bacillus cereus YfiY bound to schizokinen. These studies combine to refine the potential physiological functions of Siderocalin by defining its multiplexed recognition mechanism.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The application of synthetic iron chelates to overcome iron deficiency in crops is leading to a high impact on the environment, making it necessary to find more friendly fertilizers. A promising alternative is the application of biodegradable iron chelates, such as those based on siderophores. In the present work, seven bacterial strains of the genus Pseudomonas were selected for their ability to secrete pyoverdine, a siderophore with a high affinity for iron, which could be used as a biofertilizer. The concentration of siderophores secreted by each bacterium expressed as desferrioxamine B equivalents, and the pyoverdine concentration was determined. Their potential as Fe biofertilizers was determined based on their capacity to complex Fe, determining the maximum iron complexation capacity at alkaline pH and selecting the RMC4 strain. The biostimulant capacity of the RMC4 strain was evaluated through the secretion of organic acids such as the hormone Indol-3-acetic acid or glutamic acid, among others, in a kinetic assay. Finally, the genome of RMC4 was determined, and the strain was identified as Pseudomonas monsensis. The annotated genome was screened for genes and gene clusters implicated in biofertilization and plant growth promotion. Besides iron mobilization, genes related to phosphorus solubilization, production of phytohormones and biological control, among others, were observed, indicating the suitability of RMC4 as an inoculant. In conclusion, RMC4 and its siderophores are promising sources for Fe biofertilization in agriculture.

The airways of cystic fibrosis (CF) patients are colonized by many pathogens and the most common is Pseudomonas aeruginosa, an environmental pathogen that is able to infect immunocompromised patients thanks to its ability to develop resistance to conventional antibiotics. Over 12% of all patients colonized by P. aeruginosa harbour multi-drug resistant species. During airway infection in CF, P. aeruginosa adopts various mechanisms to survive in a hostile ecological niche characterized by low oxygen concentration, nutrient limitation and high osmotic pressure. To this end, P. aeruginosa uses a variety of virulence factors including pigment production, biofilm formation, motility and the secretion of toxins and proteases. This study represents the first report that systematically analyzes the differences in virulence features, in normoxia and anoxia, of clinical P. aeruginosa isolated from CF patients, characterized by multi- or pan-drug antibiotic resistance compared to antibiotic sensitive strains. The virulence features, such as biofilm formation, protease secretion and motility, are highly diversified in anaerobiosis, which reflects the condition of chronic CF infection. These findings may contribute to the understanding of the real-world lifestyle of pathogens isolated during disease progression in each particular patient and to assist in the design of therapeutic protocols for personalized medicine.

Regional sensitivity analysis is a part of global sensitivity analysis methods. In this study, a multivariate sensitivity analysis was conducted on a qualitative-phenomenological model. Study results allowed us to pinpoint the most influential parameters. Moreover, important measures or ranking gave more insight into factors ordering according to their relative influence and permitted them to follow parameter sensitivities over time. Lastly, a confirmatory procedure using extensive parameter randomisation that covered a broad range of the parameters space allowed more insight into and certainty of each input parameters’ relative importance.

ABSTRACT Iron is essential for many biological functions in bacteria, but its poor solubility is a limiting factor for growth. Bacteria produce siderophores, soluble natural products that bind iron with high affinity, to overcome this challenge. Siderophore-iron complexes return to the cell through specific outer membrane transporters. The opportunistic pathogen Pseudomonas aeruginosa makes multiple transporters that recognize its own siderophores, pyoverdine and pyochelin, and xenosiderophores produced by other bacteria or fungi, which gives it a competitive advantage. Some antibiotics exploit these transporters to bypass the membrane to reach their intracellular targets—including the thiopeptide antibiotic, thiostrepton (TS), which uses the pyoverdine transporters FpvA and FpvB to cross the outer membrane. Here, we assessed TS susceptibility in the presence of various siderophores and discovered that ferrichrome and ferrioxamine B antagonized TS uptake via FpvB. Unexpectedly, we found that FpvB transports ferrichrome and ferrioxamine B with higher affinity than pyoverdine. Site-directed mutagenesis of FpvB coupled with competitive growth inhibition and affinity label quenching studies suggested that the siderophores and antibiotic share a binding site in an aromatic pocket formed by the plug and barrel domains but have differences in their binding mechanism and molecular determinants for uptake. This work describes an alternative uptake pathway for ferrichrome and ferrioxamine B in P. aeruginosa and emphasizes the promiscuity of siderophore transporters, with implications for Gram-negative antibiotic development via the Trojan horse approach. IMPORTANCE Gram-negative bacteria express a variety of outer membrane transporters to import critical nutrients such as iron. Due to its insolubility, iron is taken up while bound to small-molecule chelators called siderophores. Pseudomonas aeruginosa takes up its own siderophores pyoverdine and pyochelin but can also steal siderophores produced by other bacteria and fungi, giving it a competitive advantage in iron-limited environments. Here, we used whole-cell reporter assays to show that FpvB, originally identified as a secondary transporter for pyoverdine, transports the chemically distinct fungal siderophore ferrichrome and the bacterial siderophore ferrioxamine B with high affinity. FpvB is also used by thiopeptide antibiotic thiostrepton for uptake. We predicted that all of these ligands bind to a common hydrophobic pocket in FpvB and used site-directed mutagenesis coupled with phenotypic assays to identify residues required for uptake. These analyses showed that siderophore and antibiotic uptake could be uncoupled. Our data show that FpvB is a promiscuous transporter of multiple chemically distinct ligands and fills in missing details of ferrichrome transport by P. aeruginosa. A clearer picture of the spectrum of outer membrane transporter substrate specificity is useful for the design of novel siderophore-antibiotic conjugates that can exploit nutrient uptake pathways to kill challenging Gram-negative pathogens.

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. [ABSTRACT FROM AUTHOR]

Simple Summary: The human pathogen Pseudomonas aeruginosa (P. aeruginosa) causes several infections, both acute and chronic, mainly in hosts with compromised immunity and in patients suffering from cystic fibrosis. The pathogenesis of this bacterium is caused by several factors. Metallophores, which are molecules for sequestering metal ions, are among these factors. P. aeruginosa secretes three types of metallophores, two for iron chelation (siderophores) named pyoverdine and pyochelin and the third one (pseudopaline) which can chelate zinc, nickel and cobalt. This review sums up in one paper all the important characteristics of these three metallophores, including their biosynthesis process, secretion, uptake after metal chelating and genetic regulation. P. aeruginosa is a common Gram-negative bacterium found in nature that causes severe infections in humans. As a result of its natural resistance to antibiotics and the ability of biofilm formation, the infection with this pathogen can be therapeutic challenging. During infection, P. aeruginosa produces secondary metabolites such as metallophores that play an important role in their virulence. Metallophores are metal ions chelating molecules secreted by bacteria, thus allowing them to survive in the host under metal scarce conditions. Pyoverdine, pyochelin and pseudopaline are the three metallophores secreted by P. aeruginosa. Pyoverdines are the primary siderophores that acquire iron from the surrounding medium. These molecules scavenge and transport iron to the bacterium intracellular compartment. Pyochelin is another siderophore produced by this bacterium, but in lower quantities and its affinity for iron is less than that of pyoverdine. The third metallophore, pseudopaline, is an opine narrow spectrum ion chelator that enables P. aeruginosa to uptake zinc in particular but can transport nickel and cobalt as well. This review describes all the aspects related to these three metallophore, including their main features, biosynthesis process, secretion and uptake when loaded by metals, in addition to the genetic regulation responsible for their synthesis and secretion. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa is an opportunistic pathogen that causes several serious health problems and numerous forms of virulence. During the treatment of P. aeruginosa infections, the development of multidrug-resistant isolates creates significant clinical problems. Using antivirulence compounds to disrupt pathogenicity rather than killing the bacterium may be an interesting strategy to overcome this problem, because less harsh conditions will exist for the development of resistance. To reduce pathogenicity and biofilm formation, newly synthesized analogs of imidazolyl (8n) and previously synthesized analogs (8a–8m) with a similar backbone [the 5-(imidazolyl-methyl) thiazolidinediones] were tested against pyoverdine and pyocyanin production, protease activity, and biofilm formation. Compared to the positive control group, the best compounds reduced the production of pyoverdine (8n) by 89.57% and pyocyanin (8i) by 22.68%, and protease activity (8n) by 2.80% for PAO1 strain, at a concentration of 10 μM. Moreover, the biofilm formation assay showed a reduction of 87.94% (8i) for PAO1, as well as 30.53% (8d) and 44.65% (8m) for 1074 and 1707 strains, respectively. The compounds used in this study did not show any toxicity in the human dermal fibroblasts and 4T1 cells (viability higher than 90%). The in silico study of these compounds revealed that their antivirulence activity could be due to their interaction with the PqsR, PqsE, and LasR receptors. [ABSTRACT FROM AUTHOR]

Phaseolotoxin is a major virulence factor of the bean pathogen bacterium P. savastanoi pv. phaseolicola. This toxin plays a key role in the development of the halo blight disease in bean plants. So far, the signal transduction pathways involved in the synthesis of phaseolotoxin have not been elucidated. The influence of regulation mechanisms related to the oxidative stress response, in particular the OxyR protein, it has been suggested to be involved in this process.. In this study we evaluated the role of OxyR in P. savastanoi pv. phaseolicola, mainly compared to the synthesis of phaseolotoxin and the virulence of this phytopathogen. Generation of the oxyR-mutant, pathogenicity and virulence tests, and analyses of gene expression by RT-PCR assays were performed. The results showed that OxyR exerts an effect on the synthesis of phaseolotoxin and positively influences the expression of the Pht and Pbo cluster genes. Likewise, OxyR influences the production of pyoverdine by the control of the expression of the genes encoding the PvdS sigma factor, involved in the synthesis of this pigment. This study is the first report on members of the OxyR regulon of P. savastanoi pv. phaseolicola NPS3121. [ABSTRACT FROM AUTHOR]

The aim of this study was to examine the impact of twenty honey samples, harvested in Mt. Olympus (Greece), on the virulence factors implicated in P. aeruginosa pathogenesis. Six key virulence factors (protease and elastase activity, pyocyanin and pyoverdine concentration, biofilm formation, and swimming motility) were selected in order to assess the effect of the tested honeys compared with Manuka honey. All tested honeys demonstrated a significant inhibition of protease and elastase activity compared with the control. Six and thirteen honeys exerted superior protease (no inhibition zone) and elastase (values lower than 55%) activity, respectively, compared with Manuka honey. Seventeen tested honeys exhibited reduced pyoverdine production compared with the control; all tested honeys, except for one, showed an inhibitory effect on pyocyanin production compared with the control. Regarding swimming motility, nine tested honeys demonstrated significantly higher inhibition compared with Manuka honey. Honey concentrations (6% v/v and 8% v/v) had the most profound impact, as they reduced biofilm formation to less than 20% compared with the control. Overall, our data demonstrate a significant inhibition of the virulence factors in the tested Mt. Olympus honeys, highlighting the strong antimicrobial activity against P. aeruginosa, an antibiotic-resistant pathogen of growing concern, which is implicated in severe nosocomial infections globally.

Pseudomonas aeruginosa is an opportunistic pathogen that produces two types of siderophores, pyoverdine and pyochelin, that play pivotal roles in iron scavenging from the environment and host cells. P. aeruginosa siderophores can serve as virulence factors and perform various functions. Several bacterial and fungal species are likely to interact with P. aeruginosa due to its ubiquity in soil and water as well as its potential to cause infections in plants, animals, and humans. Siderophores produced by P. aeruginosa play critical roles in iron scavenging for prokaryotic species (bacteria) and eukaryotic hosts (fungi, animals, insects, invertebrates, and plants) as well. This review provides a comprehensive discussion of the role of P. aeruginosa siderophores in interaction with prokaryotes and eukaryotes as well as their underlying mechanisms of action. The evolutionary relationship between P. aeruginosa siderophore recognition receptors, such as FpvA, FpvB, and FptA, and those of other bacterial species has also been investigated. [ABSTRACT FROM AUTHOR]

Computational modeling and simulation of biological systems have become valuable tools for understanding and predicting cellular performance and phenotype generation. This work aimed to construct, model, and dynamically simulate the virulence factor pyoverdine (PVD) biosynthesis in Pseudomonas aeruginosa through a systemic approach, considering that the metabolic pathway of PVD synthesis is regulated by the quorum-sensing (QS) phenomenon. The methodology comprised three main stages: (i) Construction, modeling, and validation of the QS gene regulatory network that controls PVD synthesis in P. aeruginosa strain PAO1; (ii) construction, curating, and modeling of the metabolic network of P. aeruginosa using the flux balance analysis (FBA) approach; (iii) integration and modeling of these two networks into an integrative model using the dynamic flux balance analysis (DFBA) approximation, followed, finally, by an in vitro validation of the integrated model for PVD synthesis in P. aeruginosa as a function of QS signaling. The QS gene network, constructed using the standard System Biology Markup Language, comprised 114 chemical species and 103 reactions and was modeled as a deterministic system following the kinetic based on mass action law. This model showed that the higher the bacterial growth, the higher the extracellular concentration of QS signal molecules, thus emulating the natural behavior of P. aeruginosa PAO1. The P. aeruginosa metabolic network model was constructed based on the iMO1056 model, the P. aeruginosa PAO1 strain genomic annotation, and the metabolic pathway of PVD synthesis. The metabolic network model included the PVD synthesis, transport, exchange reactions, and the QS signal molecules. This metabolic network model was curated and then modeled under the FBA approximation, using biomass maximization as the objective function (optimization problem, a term borrowed from the engineering field). Next, chemical reactions shared by both network models were chosen to combine them into an integrative model. To this end, the fluxes of these reactions, obtained from the QS network model, were fixed in the metabolic network model as constraints of the optimization problem using the DFBA approximation. Finally, simulations of the integrative model (CCBM1146, comprising 1123 reactions and 880 metabolites) were run using the DFBA approximation to get (i) the flux profile for each reaction, (ii) the bacterial growth profile, (iii) the biomass profile, and (iv) the concentration profiles of metabolites of interest such as glucose, PVD, and QS signal molecules. The CCBM1146 model showed that the QS phenomenon directly influences the P. aeruginosa metabolism to PVD biosynthesis as a function of the change in QS signal intensity. The CCBM1146 model made it possible to characterize and explain the complex and emergent behavior generated by the interactions between the two networks, which would have been impossible to do by studying each system’s individual components or scales separately. This work is the first in silico report of an integrative model comprising the QS gene regulatory network and the metabolic network of P. aeruginosa.

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. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa is an opportunistic pathogen that can cause acute and severe infections. Increasing resistance to antibiotics has given rise to the urgent need for an alternative antimicrobial agent. A promising strategy is the inhibition of iron sequestration in the bacteria. The current work aimed to screen for inhibitors of pyoverdine-mediated iron sequestration in P. aeruginosa. As a drug target, we choose l-ornithine-N5-monooxygenase (PvdA), an enzyme involved in the biosynthesis of pyoverdine that catalyzes the FAD-dependent hydroxylation of the side chain amine of ornithine. As drug repurposing is a fast and cost-efficient way of discovering new applications for known drugs, the approach may help to solve emerging clinical problems. In this study, we use data about molecules from drug banks for screening. A total of 15 drugs that are similar in structure to l-ornithine, the substrate of PvdA, and 30 drugs that are sub-structures of l-ornithine were virtually docked against PvdA. N-2-succinyl ornithine and cilazapril were found to be the top binders with a binding energy of −12.8 and −9.1 kcal mol−1, respectively. As the drug-likeness and ADME properties of the drugs were also found to be promising, molecular dynamics studies were performed to further confirm the stability of the complexes. The results of this in silico study indicate that N-2-succinyl ornithine could potentially be explored as a drug for the treatment of P. aeruginosa infections. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]