Pseudomonas aeruginosa-derived rhamnolipids subvert the host innate immune response through manipulation of the human beta-defensin-2 expression

Pseudomonas aeruginosa is a well‐known cause of infections especially in compromised patients. To neutralize this pathogen, the expression of antimicrobial factors in epithelial cells is crucial. In particular the human beta‐defensin hBD‐2 is especially active against P. aeruginosa. In this study, we identified rhamnolipids in P. aeruginosa culture supernatants that are able to prevent the pathogen‐induced hBD‐2 response in keratinocytes. The presence of rhamnolipids within the host cells and inhibition assays suggest that calcium‐regulated pathways and protein kinase C activation are impaired by rhamnolipids. In consequence, the induction of hBD‐2 in keratinocytes by P. aeruginosa‐derived flagellin as well as the host's own hBD‐2 mediator interleukin IL‐1β is inhibited. Strikingly, rhamnolipids did not affect the release of the proinflammatory mediator interleukin IL‐8 by flagellin. Thus, in addition to their function in establishment and persistence of P. aeruginosa infections, rhamnolipids can be engaged by P. aeruginosa for a targeted attenuation of the innate immunity to manage its survival and colonization on compromised epithelia.     

Implication of neutral polysaccharides associated to alginate in inhibition of murine macrophage response to Pseudomonas aeruginosa

There is evidence that exopolysaccharides (EPS) contribute to the persistence of Pseudomonas aeruginosa in cystic fibrosis lung. However, the relationship between the chemical composition of EPS and the modulation of phagocytic cells is poorly understood. In order to evaluate the role of the chemical composition of EPS in macrophage behavior changes, we pretreated macrophages with characterized EPS and assessed P. aeruginosa phagocytosis and reactive oxygen intermediate (ROI) production. The results showed that alginate and neutral polysaccharides are involved in phagocytic impairment of P. aeruginosa. Moreover, alginates were able to prime macrophages for increased P. aeruginosa-induced macrophage oxidative burst as determined by chemiluminescence. In contrast, neutral polysaccharides are responsible for the decrease of ROI by a scavenging effect evaluated by the xanthine–xanthine oxidase system. This study showed that the content of P. aeruginosa EPS in alginate, but also in neutral polysaccharides, influences the behavior of strains towards phagocytosis and macrophage oxidative burst.     

Helical Carbon Nanotubes Enhance the Early Immune Response and Inhibit Macrophage-Mediated Phagocytosis of Pseudomonas aeruginosa

Aerosolized or aspirated manufactured carbon nanotubes have been shown to be cytotoxic, cause pulmonary lesions, and demonstrate immunomodulatory properties. CD-1 mice were used to assess pulmonary toxicity of helical carbon nanotubes (HCNTs) and alterations of the immune response to subsequent infection by Pseudomonas aeruginosa in mice. HCNTs provoked a mild inflammatory response following either a single exposure or 2X/week for three weeks (multiple exposures) but were not significantly toxic. Administering HCNTs 2X/week for three weeks resulted in pulmonary lesions including granulomas and goblet cell hyperplasia. Mice exposed to HCNTs and subsequently infected by P. aeruginosa demonstrated an enhanced inflammatory response to P. aeruginosa and phagocytosis by alveolar macrophages was inhibited. However, clearance of P. aeruginosa was not affected. HCNT exposed mice depleted of neutrophils were more effective in clearing P. aeruginosa compared to neutrophil-depleted control mice, accompanied by an influx of macrophages. Depletion of systemic macrophages resulted in slightly inhibited bacterial clearance by HCNT treated mice. Our data indicate that pulmonary exposure to HCNTs results in lesions similar to those caused by other nanotubes and pre-exposure to HCNTs inhibit alveolar macrophage phagocytosis of P. aeruginosa. However, clearance was not affected as exposure to HCNTs primed the immune system for an enhanced inflammatory response to pulmonary infection consisting of an influx of neutrophils and macrophages.     

<Emphasis Type="Italic">Pseudomonas</Emphasis> siderophores in the sputum of patients with cystic fibrosis

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.     

Iron acquisition by Pseudomonas aeruginosa in the lungs of patients with cystic fibrosis

The bacterium Pseudomonas aeruginosa is commonly isolated from the general environment and also infects the lungs of patients with cystic fibrosis (CF). Iron in mammals is not freely available to infecting pathogens although significant amounts of extracellular iron are available in the sputum that occurs in the lungs of CF patients. P. aeruginosa has a large number of systems to acquire this essential nutrient and many of these systems have been characterised in the laboratory. However, which iron acquisition systems are active in CF is not well understood. Here we review recent research that sheds light on how P. aeruginosa obtains iron in the lungs of CF patients.     

Glutathione exhibits antibacterial activity and increases tetracycline efficacy against Pseudomonas aeruginosa

Glutathione (GSH) plays important roles in pulmonary diseases, and inhaled GSH therapy has been used to treat cystic fibrosis (CF) patients in clinical trials. The results in this report revealed that GSH altered the sensitivity of Pseudomonas aeruginosa to different antibiotics through pathways unrelated to the oxidative stress as generally perceived. In addition, GSH and its oxidized form inhibited the growth of P. aeruginosa. Supported by the National Natural Science Foundation of China (Grant Nos. 30870097 and 30611120520)     

Binding of protegrin-1 to Pseudomonas aeruginosa and Burkholderia cepacia

Background  

Pseudomonas aeruginosa and Burkholderia cepacia infections of cystic fibrosis patients' lungs are often resistant to conventional antibiotic therapy. Protegrins are antimicrobial peptides with potent activity against many bacteria, including P. aeruginosa. The present study evaluates the correlation between protegrin-1 (PG-1) sensitivity/resistance and protegrin binding in P. aeruginosa and B. cepacia.     

Monorhamnolipids and 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs) production using Escherichia coli as a heterologous host

Pseudomonas aeruginosa produces the biosurfactants rhamnolipids and 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs). In this study, we report the production of one family of rhamnolipids, specifically the monorhamnolipids, and of HAAs in a recombinant Escherichia coli strain expressing P. aeruginosa rhlAB operon. We found that the availability in E. coli of dTDP-l-rhamnose, a substrate of RhlB, restricts the production of monorhamnolipids in E. coli. We present evidence showing that HAAs and the fatty acid dimer moiety of rhamnolipids are the product of RhlA enzymatic activity. Furthermore, we found that in the recombinant E. coli, these compounds have the same chain length of the fatty acid dimer moiety as those produced by P. aeruginosa. These data suggest that it is RhlAB specificity, and not the hydroxyfatty acid relative abundance in the bacterium, that determines the profile of the fatty acid moiety of rhamnolipids and HAAs. The rhamnolipids level produced in recombinant E. coli expressing rhlAB is lower than the P. aeruginosa level and much higher than those reported by others in E. coli, showing that this metabolic engineering strategy lead to an increased rhamnolipids production in this heterologous host.     

ISPa20 advances the individual evolution of Pseudomonas aeruginosa clone C subclone C13 strains isolated from cystic fibrosis patients by insertional mutagenesis and genomic rearrangements

Pseudomonas aeruginosa clone C strains, which chronically colonize the lungs of cystic fibrosis patients reorganize their genome structure. In this study, a novel member of the IS3 subfamily of IS elements, ISPa20, was detected which was specific for clone C subclone C13 strains. ISPa20, which was present in high copy number, mediated events of genomic reorganization. ISPa20 was inserted into P. aeruginosa backbone genes leading to adaptation to the cystic fibrosis lung habitat and into DNA acquired through horizontal gene transfer. Further on, large chromosomal inversions were mediated by ISPa20. In contrast to strains of other subclonal linages high rates of genomic rearrangements of subclone C13 strains were observed in vitro. The acquisition of mobile elements by P. aeruginosa clone C strains in the lungs of cystic fibrosis patients supports the chronic colonization by insertional mutagenesis and chromosome restructuring leading to microevolution within clone C that reflects macroevolution observed on the species level.     

Pseudomonas toxin pyocyanin triggers autophagy: Implications for pathoadaptive mutations

Pseudomonas aeruginosa can establish life-long chronic infection in patients with cystic fibrosis by generating genetic loss-of-function mutations, which enhance fitness of the bacterium in the airways. However, the precise role of the pathoadaptive mutations in persistence in chronic airways infection remains largely unknown. Here we demonstrate that pyocyanin, a well-described P. aeruginosa virulence factor that plays an important role in the initial infection, promotes autophagy in bronchial epithelial cells. Disruption of phzM, which is required for pyocyanin biosynthesis, leads to a significant reduction in autophagy in Beas-2B cells and lung tissues. Pyocyanin-induced autophagy is mediated by the EIF2AK4/GCN2-EIF2S1/eIF2α-ATF4 pathway. Interestingly, rats infected with the phzMΔ mutant strain have high mortality rate and numbers of colony-forming units, compared to those infected with wild-type (WT) P. aeruginosa PA14 strain, during chronic P. aeruginosa infection. In addition, the phzMΔ mutant strain induces more extensive alveolar wall thickening than the WT strain in the pulmonary airways of rats. As autophagy plays an essential role in suppressing bacterial burden, our findings provide a detailed understanding of why reduction of pyocyanin production in P. aeruginosa in chronic airways infections has been associated with better host adaptation and worse outcomes in cystic fibrosis.     

Mechanistic understanding of Phenyllactic acid mediated inhibition of quorum sensing and biofilm development in Pseudomonas aeruginosa

Pseudomonas aeruginosa depends on its quorum sensing (QS) system for its virulence factors’ production and biofilm formation. Biofilms of P. aeruginosa on the surface of indwelling catheters are often resistant to antibiotic therapy. Alternative approaches that employ QS inhibitors alone or in combination with antibiotics are being developed to tackle P. aeruginosa infections. Here, we have studied the mechanism of action of 3-Phenyllactic acid (PLA), a QS inhibitory compound produced by Lactobacillus species, against P. aeruginosa PAO1. Our study revealed that PLA inhibited the expression of virulence factors such as pyocyanin, protease, and rhamnolipids that are involved in the biofilm formation of P. aeruginosa PAO1. Swarming motility, another important criterion for biofilm formation of P. aeruginosa PAO1, was also inhibited by PLA. Gene expression, mass spectrometric, functional complementation assays, and in silico data indicated that the quorum quenching and biofilm inhibitory activities of PLA are attributed to its ability to interact with P. aeruginosa QS receptors. PLA antagonistically binds to QS receptors RhlR and PqsR with a higher affinity than its cognate ligands N-butyryl-l-homoserine lactone (C₄–HSL) and 2-heptyl-3,4-dihydroxyquinoline (PQS; Pseudomonas quinolone signal). Using an in vivo intraperitoneal catheter-associated medaka fish infection model, we proved that PLA inhibited the initial attachment of P. aeruginosa PAO1 on implanted catheter tubes. Our in vitro and in vivo results revealed the potential of PLA as anti-biofilm compound against P. aeruginosa.

     

Mycoflora in cystic fibrosis: Some ecologic aspects of pseudomonas aeruginosa and Candida albicans

The mycotic and bacterial flora of 65 patients with cystic fibrosis was studied.C. albicans andP. aeruginosa were present in 33% and 43% of sputa samples, respectively; only 6.5% harbored both organisms. The mycotic flora of the nasopharynx, rectum and skin of the cystic fibrosis patients was similar to that of children with other chronic lung diseases and to that of normal children.In vitro studies clearly revealed inhibition ofC. albicans byP. aeruginosa. It is suggested thatP. aeruginosa, so prevalent with cystic fibrosis, has an inhibitory effect onC. albicans and that this interaction is effective to some extent in preventing candidal infection.Supported in part by Cancer Center Training Grant CA-08480 and Clinical Training Grant CA-08151 from the National Cancer Institute, and by ALSAC.     

Production of rhamnolipids by Pseudomonas aeruginosa

Pseudomonas aeruginosa produces glycolipidic surface-active molecules (rhamnolipids) which have potential biotechnological applications. Rhamnolipids are produced by P. aeruginosa in a concerted manner with different virulence-associated traits. Here, we review the rhamnolipids biosynthetic pathway, showing that it has metabolic links with numerous bacterial products such as alginate, lipopolysaccharide, polyhydroxyalkanoates, and 4-hydroxy-2-alkylquinolines (HAQs). We also discuss the factors controlling the production of rhamnolipids and the proposed roles this biosurfactant plays in P. aeruginosa lifestyle.     

Accumulation of ceramide in the trachea and intestine of cystic fibrosis mice causes inflammation and cell death

Cystic fibrosis is a hereditary metabolic disorder caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene and characterized by severe intestinal and pulmonary symptoms, in particular intestinal obstruction, pancreatic insufficiency, chronic pulmonary inflammation, and microbial lung infections. Recent studies have demonstrated an accumulation of ceramide in the lungs of cystic fibrosis patients and in several mouse models. These findings showed that pulmonary ceramide concentrations play an important role in pulmonary inflammation and infection. In this study we investigated whether ceramide concentrations are also altered in the trachea and the intestine of cystic fibrosis mice and whether an accumulation of ceramide in these organs has functional consequences that are typical of cystic fibrosis. Our findings demonstrate a marked accumulation of ceramide in tracheal and intestinal epithelial cells of cystic fibrosis mice. When acid sphingomyelinase activity is inhibited by treating cystic fibrosis mice with amitriptyline or by genetic heterozygosity of acid sphingomyelinase in cystic fibrosis mice, ceramide concentrations in the trachea and the intestine are normalized. Moreover, increased rates of cell death and increased cytokine concentrations in the trachea, the intestine, or both were normalized by the inhibition of acid sphingomyelinase activity and the concomitant normalization of ceramide concentrations. These findings suggest that ceramide plays a crucial role in inflammation and increased rates of cell death in several organs of cystic fibrosis mice.     

Biofilm inhibitory effect of alginate lyases on mucoid P. aeruginosa from a cystic fibrosis patient

Chronic mucoid Pseudomonas aeruginosa infections are a major scourge in cystic fibrosis patients. Mucoid P. aeruginosa displays structured alginate-rich biofilms that are resistant to antibiotics. Here, we have assessed the efficacy of a panel of alginate lyases in combating mucoid P. aeruginosa biofilms in cystic fibrosis. Albeit we could not demonstrate alginate degradation by alginate lyases in sputum, we demonstrate that the endotypic alginate lyases, CaAly (from Cellulophaga algicola) and VspAlyVI (from Vibrio sp. QY101) and the exotypic alginate lyases, FspAlyFRB (from Falsirhodobacterium sp. alg1), and SA1-IV (from Sphingomonas sp. A1), indeed inhibit biofilm formation by a mucoid P. aeruginosa strain isolated from the sputum of a cystic fibrosis patient with comparative effect to that of the glycoside hydrolase PslG, a promising candidate for biofilm treatment. We believe that these enzymes should be explored for in vivo efficacy in future studies.     

Pseudomonas aeruginosa elastase provides an escape from phagocytosis by degrading the pulmonary surfactant protein-A

Pseudomonas aeruginosa is an opportunistic pathogen that causes both acute pneumonitis in immunocompromised patients and chronic lung infections in individuals with cystic fibrosis and other bronchiectasis. Over 75% of clinical isolates of P. aeruginosa secrete elastase B (LasB), an elastolytic metalloproteinase that is encoded by the lasB gene. Previously, in vitro studies have demonstrated that LasB degrades a number of components in both the innate and adaptive immune systems. These include surfactant proteins, antibacterial peptides, cytokines, chemokines and immunoglobulins. However, the contribution of LasB to lung infection by P. aeruginosa and to inactivation of pulmonary innate immunity in vivo needs more clarification. In this study, we examined the mechanisms underlying enhanced clearance of the ΔlasB mutant in mouse lungs. The ΔlasB mutant was attenuated in virulence when compared to the wild-type strain PAO1 during lung infection in SP-A^+/+ mice. However, the ΔlasB mutant was as virulent as PAO1 in the lungs of SP-A^-/- mice. Detailed analysis showed that the ΔlasB mutant was more susceptible to SP-A-mediated opsonization but not membrane permeabilization. In vitro and in vivo phagocytosis experiments revealed that SP-A augmented the phagocytosis of ΔlasB mutant bacteria more efficiently than the isogenic wild-type PAO1. The ΔlasB mutant was found to have a severely reduced ability to degrade SP-A, consequently making it unable to evade opsonization by the collectin during phagocytosis. These results suggest that P. aeruginosa LasB protects against SP-A-mediated opsonization by degrading the collectin.     

Cationic Peptides Facilitate Iron-induced Mutagenesis in Bacteria

Pseudomonas aeruginosa is the causative agent of chronic respiratory infections and is an important pathogen of cystic fibrosis patients. Adaptive mutations play an essential role for antimicrobial resistance and persistence. The factors that contribute to bacterial mutagenesis in this environment are not clear. Recently it has been proposed that cationic antimicrobial peptides such as LL-37 could act as mutagens in P. aeruginosa. Here we provide experimental evidence that mutagenesis is the product of a joint action of LL-37 and free iron. By estimating mutation rate, mutant frequencies and assessing mutational spectra in P. aeruginosa treated either with LL-37, iron or a combination of both we demonstrate that mutation rate and mutant frequency were increased only when free iron and LL-37 were present simultaneously. Colistin had the same effect. The addition of an iron chelator completely abolished this mutagenic effect, suggesting that LL-37 enables iron to enter the cells resulting in DNA damage by Fenton reactions. This was also supported by the observation that the mutational spectrum of the bacteria under LL-37-iron regime showed one of the characteristic Fenton reaction fingerprints: C to T transitions. Free iron concentration in nature and within hosts is kept at a very low level, but the situation in infected lungs of cystic fibrosis patients is different. Intermittent bleeding and damage to the epithelial cells in lungs may contribute to the release of free iron that in turn leads to generation of reactive oxygen species and deterioration of the respiratory tract, making it more susceptible to the infection.     

Relationship between cystic fibrosis respiratory tract bacterial communities and age,genotype, antibiotics and Pseudomonas aeruginosa

Polymicrobial bronchopulmonary infections in cystic fibrosis (CF) cause progressive lung damage and death. Although the arrival of Pseudomonas aeruginosa often heralds a more rapid rate of pulmonary decline, there is significant inter‐individual variation in the rate of decline, the causes of which remain poorly understood. By coupling culture‐independent methods with ecological analyses, we discovered correlations between bacterial community profiles and clinical disease markers in respiratory tracts of 45 children with CF. Bacterial community complexity was inversely correlated with patient age, presence of P. aeruginosa and antibiotic exposure, and was related to CF genotype. Strikingly, bacterial communities lacking P. aeruginosa were much more similar to each other than were those containing P. aeruginosa, regardless of antibiotic exposure. This suggests that community composition might be a better predictor of disease progression than the presence of P. aeruginosa alone and deserves further study.     

Reactive-Oxygen-Species-Mediated P. aeruginosa Killing Is Functional in Human Cystic Fibrosis Macrophages

Pseudomonas aeruginosa is the most common pathogen for chronic lung infection in cystic fibrosis (CF) patients. About 80% of adult CF patients have chronic P. aeruginosa infection, which accounts for much of the morbidity and most of the mortality. Both bacterial genetic adaptations and defective innate immune responses contribute to the bacteria persistence. It is well accepted that CF transmembrane conductance regulator (CFTR) dysfunction impairs the airways-epithelium-mediated lung defence; however, other innate immune cells also appear to be affected, such as neutrophils and macrophages, which thus contribute to this infectious pathology in the CF lung. In macrophages, the absence of CFTR has been linked to defective P. aeruginosa killing, increased pro-inflammatory cytokine secretion, and reduced reactive oxygen species (ROS) production. To learn more about macrophage dysfunction in CF patients, we investigated the generation of the oxidative burst and its impact on bacterial killing in CF macrophages isolated from peripheral blood or lung parenchyma of CF patients, after P. aeruginosa infection. Our data demonstrate that CF macrophages show an oxidative response of similar intensity to that of non-CF macrophages. Intracellular ROS are recognized as one of the earliest microbicidal mechanisms against engulfed pathogens that are activated by macrophages. Accordingly, NADPH inhibition resulted in a significant increase in the intracellular bacteria survival in CF and non-CF macrophages, both as monocyte-derived macrophages and as lung macrophages. These data strongly suggest that the contribution of ROS to P. aeruginosa killing is not affected by CFTR mutations.