pyochelin and Pseudomonas-Infections

During chronic cystic fibrosis (CF) infections, evolved Pseudomonas aeruginosa antibiotic resistance is linked to increased pulmonary exacerbations, decreased lung function, and hospitalizations. However, the virulence mechanisms underlying worse outcomes caused by antibiotic resistant infections are poorly understood. Here, we investigated evolved aztreonam resistant P. aeruginosa virulence mechanisms. Using a macrophage infection model combined with genomic and transcriptomic analyses, we show that a compensatory mutation in the rne gene, encoding RNase E, increased pyoverdine and pyochelin siderophore gene expression, causing macrophage ferroptosis and lysis. We show that iron-bound pyochelin was sufficient to cause macrophage ferroptosis and lysis, however, apo-pyochelin, iron-bound pyoverdine, or apo-pyoverdine were insufficient to kill macrophages. Macrophage killing could be eliminated by treatment with the iron mimetic gallium. RNase E variants were abundant in clinical isolates, and CF sputum gene expression data show that clinical isolates phenocopied RNase E variant functions during macrophage infection. Together these data show how P. aeruginosa RNase E variants can cause host damage via increased siderophore production and host cell ferroptosis but may also be targets for gallium precision therapy.

Topics: Humans; Iron; Pseudomonas aeruginosa; Pseudomonas Infections; Siderophores; Virulence

Topics: Animals; Mice; Peritonitis; Phenols; Pseudomonas aeruginosa; Pseudomonas Infections

Siderophores are iron chelators produced by bacteria to access iron, an essential nutrient. The pathogen Pseudomonas aeruginosa produces two siderophores, pyoverdine and pyochelin, the former with a high affinity for iron and the latter with a lower affinity. Furthermore, the production of both siderophores involves a positive auto-regulatory loop: the presence of the ferri-siderophore complex is essential for their large production. Since pyochelin has a lower affinity for iron it was hard to consider the role of pyochelin in drastic competitive environments where the host or the environmental microbiota produce strong iron chelators and may inhibit iron chelation by pyochelin. We showed here that the pyochelin pathway overcomes this difficulty through a more complex regulating mechanism for pyochelin production than previously described. Indeed, in the absence of pyoverdine, and thus higher difficulty to access iron, the bacteria are able to produce pyochelin independently of the presence of ferri-pyochelin. The regulation of the pyochelin pathway appeared to be more complex than expected with a more intricate tuning between repression and activation. Consequently, when the bacteria cannot produce pyoverdine they are able to produce pyochelin even in the presence of strong iron chelators. Such results support a more complex and varied role for this siderophore than previously described, and complexify the battle for iron during P. aeruginosa infection.

Topics: Humans; Iron; Oligopeptides; Phenols; Pseudomonas aeruginosa; Pseudomonas Infections; Siderophores; Thiazoles

Pseudomonas aeruginosa is a problematic human pathogen resistant to almost all available antibiotics. The important prerequisite for these drugs to target this bacterium is an efficient delivery system. Siderophore-mediated drug delivery system is a promising approach to carry out antibiotics to the cells. Pyochelin, a siderophore of P. aeruginosa, was successfully synthesized in a five-step procedure. PEGylated liposomal pyochelin-antibiotic (L-Pch-Ab) carrier was fabricated by thin-film hydration method. L-Pch-Ab had an average size of 90.31 ± 0.11 nm holding a negative zeta potential at -54.12 ± 0.03 mV (PDI <2). The MIC determined by broth dilution method against three clinical strains isolated from burn wounds showed that L-Pch-Ab significantly reduced (≤16 µg/ml) the MIC values than those of free antibiotics. In the time kill assay, L-Pch-Ab was bactericidal against all strains at most time intervals at 2 × and 4 × MIC up to 24 h. TEM observations revealed that L-Pch-Ab was actively taken up by P. aeruginosa and exhibited membrane deformation within 2 h. Developed L-Pch-Ab fused intimately with the outer membrane of MDRPa and exhibited effective antibacterial activity than free Ab. Furthermore, L-Pch-Ab kills MDRPa within infected HaCaT keratinocytes without any cytotoxic effects at 4× MIC concentrations after 72 h. Thus, the specific targeting of L-Pch-Ab with its higher efficacy to deliver drug by limiting the toxicity will be a novel approach to fight infections caused by P. aeruginosa.

Topics: Anti-Bacterial Agents; Cell Line; Drug Delivery Systems; Drug Resistance, Bacterial; Humans; Liposomes; Phenols; Polyethylene Glycols; Pseudomonas aeruginosa; Pseudomonas Infections; Thiazoles

Wound infections are traditionally thought to occur when microbial burden exceeds the innate clearance capacity of host immune system. Here, we introduce the idea that the wound environment itself plays a significant contributory role to wound infection.. We developed a clinically relevant murine model of soft tissue infection to explore the role of activation of microbial virulence in response to tissue factors as a mechanism by which pathogenic bacteria cause wound infections. Mice underwent abdominal skin incision and light muscle injury with a crushing forceps versus skin incision alone followed by topical inoculation of Pseudomonas aeruginosa. Mice were sacrificed on postoperative Day 6, and abdominal tissues were analyzed for clinical signs of wound infection. To determine if specific wound tissue components induce bacterial virulence, P. aeruginosa was exposed to the skin, fascia, and muscle.. Gross wound infection caused by P. aeruginosa was observed to be significantly increased in injured tissues versus noninjured (80% vs.10%) tissues (n = 20 per group, p < 0.0001). Exposure of P. aeruginosa to individual tissue components demonstrated that fascia significantly induced bacterial virulence as judged by the production of pyocyanin, a redox-active phenazine compound known to kill immune cells. Whole-genome transcriptional profiling of P. aeruginosa exposed to the fascia demonstrated activation of multiple genes responsible for the synthesis of the iron scavenging molecule pyochelin.. We conclude that wound elements, in particular fascia, may play a significant role in enhancing the virulence of P. aeruginosa and may contribute to the pathogenesis of clinical wound infection.

Topics: Abdominal Muscles; Animals; Disease Models, Animal; Fascia; Gene Expression Profiling; Mice; Mice, Inbred C57BL; Microarray Analysis; Phenols; Pseudomonas aeruginosa; Pseudomonas Infections; Surgical Wound Infection; Thiazoles; Virulence

Pseudomonas aeruginosa establishes airway infections in Cystic Fibrosis patients. Here, we investigate the molecular interactions between P. aeruginosa and airway mucus secretions (AMS) derived from the primary cultures of normal human tracheal epithelial (NHTE) cells. PAO1, a prototype strain of P. aeruginosa, was capable of proliferating during incubation with AMS, while all other tested bacterial species perished. A PAO1 mutant lacking PA4834 gene became susceptible to AMS treatment. The ΔPA4834 mutant was grown in AMS supplemented with 100 μM ferric iron, suggesting that the PA4834 gene product is involved in iron metabolism. Consistently, intracellular iron content was decreased in the mutant, but not in PAO1 after the AMS treatment. Importantly, a PAO1 mutant unable to produce both pyoverdine and pyochelin remained viable, suggesting that these two major siderophore molecules are dispensable for maintaining viability during incubation with AMS. The ΔPA4834 mutant was regrown in AMS amended with 100 μM nicotianamine, a phytosiderophore whose production is predicted to be mediated by the PA4836 gene. Infectivity of the ΔPA4834 mutant was also significantly compromised in vivo. Together, our results identify a genetic element encoding a novel iron acquisition system that plays a previously undiscovered role in P. aeruginosa airway infection.

Topics: Animals; Azetidinecarboxylic Acid; Chlorides; Coculture Techniques; Epithelial Cells; Ferric Compounds; Gene Expression Regulation, Bacterial; Genes, Bacterial; Host-Pathogen Interactions; Humans; Iron; Male; Mice; Mice, Inbred C57BL; Microarray Analysis; Microbial Viability; Mucus; Mutation; Oligopeptides; Phenols; Primary Cell Culture; Pseudomonas aeruginosa; Pseudomonas Infections; Siderophores; Thiazoles; Trachea

Genomic islands are foreign DNA blocks inserted in so-called regions of genomic plasticity (RGP). Depending on their gene content, they are classified as pathogenicity, symbiosis, metabolic, fitness or resistance islands, although a detailed functional analysis is often lacking. Here we focused on a 34-kb pathogenicity island of Pseudomonas aeruginosa PA14 (PA14GI-6), which is inserted at RGP5 and carries genes related to those for pyochelin/enantiopyochelin biosynthesis. These enantiomeric siderophores of P. aeruginosa and certain strains of Pseudomonas protegens are assembled by a thiotemplate mechanism from salicylate and two molecules of cysteine. The biochemical function of several proteins encoded by PA14GI-6 was investigated by a series of complementation analyses using mutants affected in potential homologs. We found that PA14_54940 codes for a bifunctional salicylate synthase/salicyl-AMP ligase (for generation and activation of salicylate), that PA14_54930 specifies a dihydroaeruginoic acid (Dha) synthetase (for coupling salicylate with a cysteine-derived thiazoline ring), that PA14_54910 produces a type II thioesterase (for quality control), and that PA14_54880 encodes a serine O-acetyltransferase (for increased cysteine availability). The structure of the PA14GI-6-specified metabolite was determined by mass spectrometry, thin-layer chromatography, and HPLC as (R)-Dha, an iron chelator with antibacterial, antifungal and antitumor activity. The conservation of this genomic island in many clinical and environmental P. aeruginosa isolates of different geographical origin suggests that the ability for Dha production may confer a selective advantage to its host.

Topics: Bacterial Proteins; Biosynthetic Pathways; Conserved Sequence; Environmental Microbiology; Genes, Bacterial; Genetic Complementation Test; Genomic Islands; Humans; Lyases; Phenols; Pseudomonas aeruginosa; Pseudomonas Infections; Serine O-Acetyltransferase; Thiazoles; Thiolester Hydrolases

Emergence of antimicrobial resistance in Pseudomonas aeruginosa has led to the search for alternative agents for infections control. Natural products have been a good alternative to present antibiotics. The present study was undertaken to evaluate the effectiveness of cranberry in attenuation of virulence of P. aeruginosa in experimental urinary tract infection (UTI) in mouse model. Efforts were also directed to explore the action of cranberry towards virulence of P. aeruginosa through quorum sensing (QS) inhibition.. Efficacy of cranberry was evaluated in an experimental UTI mouse model and on production of QS signals, alginate, pyochelin, haemolysin, phospholipase-C, cell-surface hydrophobicity, uroepithelial cell-adhesion assay and biofilm formation by already standardized methods.. Presence of cranberry showed significant decline in the production of QS signals, biofilm formation and virulence factors of P. aeruginosa in vitro (P<0.001). Further, cranberry was found to be useful in prevention of experimental UTI in mouse model as indicated by reduced renal bacterial colonization and kidney tissues destruction.. The findings of the present study indicated that cranberry inhibited QS and hence elaboration of virulence factors of P. aeruginosa. It also affected the adherence ability of this pathogen. This approach can lead to the discovery of new category of safe anti-bacterial drugs from dietary sources such as cranberry with reduced toxicity without the risk of antibiotic resistance.

Topics: Animals; Biofilms; Fruit; Hemolysin Proteins; Mice; Microscopy, Electron, Scanning; Phenols; Plant Extracts; Pseudomonas aeruginosa; Pseudomonas Infections; Quorum Sensing; Thiazoles; Urinary Tract Infections; Vaccinium macrocarpon; Virulence; Virulence Factors

The lungs of patients with cystic fibrosis become chronically infected with the bacterium Pseudomonas aeruginosa, which heralds progressive lung damage and a decline in health. Iron is a crucial micronutrient for bacteria and its acquisition is a key factor in infection. P. aeruginosa can acquire this element by secreting pyoverdine and pyochelin, iron-chelating compounds (siderophores) that scavenge iron and deliver it to the bacteria. Siderophore-mediated iron uptake is generally considered a key factor in the ability of P. aeruginosa to cause infection. We have investigated the amounts of pyoverdine in 148 sputum samples from 36 cystic fibrosis patients (30 infected with P. aeruginosa and 6 as negative controls). Pyoverdine was present in 93 samples in concentrations between 0.30 and 51 μM (median 4.6 μM) and there was a strong association between the amount of pyoverdine and the number of P. aeruginosa present. However, pyoverdine was not present, or below the limits of detection (~0.3 μM), in 21 sputum samples that contained P. aeruginosa. Pyochelin was also absent, or below the limits of detection (~1 μM), in samples from P. aeruginosa-infected patients with little or no detectable pyoverdine. Our data show that pyoverdine is an important iron-scavenging molecule for P. aeruginosa in many cystic fibrosis patients, but other P. aeruginosa iron-uptake systems must be active in some patients to satisfy the bacterial need for iron.

Topics: Adult; Cystic Fibrosis; Female; Humans; Iron; Male; Oligopeptides; Phenols; Pseudomonas aeruginosa; Pseudomonas Infections; Siderophores; Sputum; Thiazoles; Young Adult

Nonmucoid Pseudomonas aeruginosa responds to iron deprivation by synthesizing the siderophores pyochelin and pyoverdine. When grown in iron-deficient medium, six mucoid P. aeruginosa strains isolated from cystic fibrosis patients synthesized copious amounts of the exopolysaccharide alginate. A procedure that eliminated the interference of alginate was developed so that siderophores could be extracted from the growth medium. All six isolates were then noted to produce both pyoverdine and pyochelin. This report thus confirms that mucoid P. aeruginosa, like its nonmucoid counterparts, elicits the siderophores commonly cited as those of the microbe.

Topics: Cystic Fibrosis; Iron Chelating Agents; Oligopeptides; Phenols; Pigments, Biological; Pseudomonas aeruginosa; Pseudomonas Infections; Siderophores; Thiazoles

Tissue injury has been linked to neutrophil associated hydroxyl radical (.OH) generation, a process that requires an exogenous transition metal catalyst such as iron. In vivo most iron is bound in a noncatalytic form. To obtain iron required for growth, many bacteria secrete iron chelators (siderophores). Since Pseudomonas aeruginosa infections are associated with considerable tissue destruction, we examined whether iron bound to the Pseudomonas siderophores pyochelin (PCH) and pyoverdin (PVD) could act as .OH catalysts. Purified PCH and PVD were iron loaded (Fe-PCH, Fe-PVD) and added to a hypoxanthine/xanthine oxidase superoxide- (.O2-) and hydrogen peroxide (H2O2)-generating system. Evidence for .OH generation was then sought using two different spin-trapping agents (5.5 dimethyl-pyrroline-1-oxide or N-t-butyl-alpha-phenylnitrone), as well as the deoxyribose oxidation assay. Regardless of methodology, .OH generation was detected in the presence of Fe-PCH but not Fe-PVD. Inhibition of the process by catalase and/or SOD suggested .OH formation with Fe-PCH occurred via the Haber-Weiss reaction. Similar results were obtained when stimulated neutrophils were used as the source of .O2- and H2O2. Addition of Fe-PCH but not Fe-PVD to stimulated neutrophils yielded .OH as detected by the above assay systems. Since PCH and PVD bind ferric (Fe3+) but not ferrous (Fe2+) iron, .OH catalysis with Fe-PCH would likely involve .O2(-)-mediated reduction of Fe3+ to Fe2+ with subsequent release of "free" Fe2+. This was confirmed by measuring formation of the Fe2(+)-ferrozine complex after exposure of Fe-PCH, but not Fe-PVD, to enzymatically generated .O2-. These data show that Fe-PCH, but not Fe-PVD, is capable of catalyzing generation of .OH. Such a process could represent as yet another mechanism of tissue injury at sites of infection with P. aeruginosa.

Topics: Electron Spin Resonance Spectroscopy; Humans; Hydroxides; Hydroxyl Radical; Iron; Iron Chelating Agents; Neutrophils; Phenols; Pseudomonas Infections; Thiazoles

Exogenously supplied pyochelin influenced the virulence of Pseudomonas cepacia pyochelin-negative strains in a chronic pulmonary infection model in rats. Groups of rats were inoculated transtracheally with agar beads containing P. cepacia or P. aeruginosa strains, saturated with either pyochelin or PBS. Supplementation of the inocula with pyochelin had no effect on the number of bacteria recovered from the lungs. The availability of pyochelin, however, increased the degree of pathology observed in lungs infected with pyochelin-negative strains of P. cepacia. The area of pathological involvement in the lung was about 2-fold larger, when pyochelin was present. Inclusion of pyochelin in the inoculum had no effect on the degree of pathology observed in lungs infected with a pyochelin-positive P. aeruginosa strain. Pyochelin was shown to stimulate in vitro growth of P. cepacia, but it had no effect on production of lipase or protease, factors which may be involved in P. cepacia virulence. These studies support our hypothesis that pyochelin may be important for dissemination in P. cepacia infections.

Topics: Animals; Lung Diseases; Phenols; Pseudomonas; Pseudomonas Infections; Rats; Thiazoles

Forty-three Pseudomonas cepacia isolates were screened for the production of pyochelin. Twenty-one (49%) produced pyochelin, and 22 (51%) were pyochelin negative. Of the 21 strains producing pyochelin, 18 were from patients with severe infections, 11 of which resulted in death. Of the 22 strains which did not produce pyochelin, 13 were from patients with mild or moderate infections. Pyochelin production by P. cepacia isolates infecting cystic fibrosis patients correlates with morbidity and mortality in these patients. Pyochelin was shown to stimulate the in vitro growth of P. cepacia in the presence of transferrin. P. cepacia isolates were able to accumulate 59Fe-pyochelin regardless of whether they produced this siderophore.

Topics: Cystic Fibrosis; Humans; Iron Chelating Agents; Phenols; Pseudomonas; Pseudomonas Infections; Siderophores; Thiazoles; Transferrin

Topics: Biological Transport; Blood Bactericidal Activity; Humans; Iron; Iron Chelating Agents; Oligopeptides; Phenols; Pigments, Biological; Pseudomonas aeruginosa; Pseudomonas Infections; Pyocyanine; Siderophores; Thiazoles; Transferrin

A virulent isolate of Pseudomonas aeruginosa PAO1, which had been obtained from eight sequential intraperitoneal infections in mice compromised with iron and methotrexate, expressed greater lethality than the avirulent parent strain when both strains were injected into mice treated with iron. The present study demonstrates that pyochelin, a siderophore produced by P. aeruginosa, also increases the lethality of the virulent bacteria but not of the avirulent bacteria. Analysis of the growth and clearance of both virulent and avirulent strains in mice revealed that pyochelin increased the growth and lethality of virulent bacteria but only increased the survival of the avirulent bacteria. A streptomycin-dependent mutant of strain PAO1 (strd1) was used to demonstrate that pyochelin did not affect the clearance activity of mice. This strongly suggests that the effects of pyochelin in stimulating the persistence of avirulent bacteria and in increasing the lethality of virulent bacteria are due solely to the promotion of bacterial growth. Since the virulent bacteria were equivalent to the avirulent bacteria in utilizing pyochelin during in vitro growth in the presence of transferrin, it appears that the stimulation of growth by pyochelin allows the expression of additional virulence properties by the virulent bacteria.

Topics: Animals; Iron Chelating Agents; Lethal Dose 50; Liver; Mice; Peritoneal Cavity; Phenols; Pseudomonas aeruginosa; Pseudomonas Infections; Sepsis; Spleen; Thiazoles; Transferrin