The Pseudomonas aeruginosa rhlG gene encodes an NADPH-dependent beta-ketoacyl reductase which is specifically involved in rhamnolipid synthesis

A Pseudomonas aeruginosa gene homologous to the fabG gene, which encodes the NADPH-dependent beta-ketoacyl-acyl carrier protein (ACP) reductase required for fatty acid synthesis, was identified. The insertional mutation of this fabG homolog (herein called rhlG) produced no apparent effect on the growth rate and total lipid content of P. aeruginosa cells, but the production of rhamnolipids was completely abrogated. These results suggest that the synthetic pathway for the fatty acid moiety of rhamnolipids is separate from the general fatty acid synthetic pathway, starting with a specific ketoacyl reduction step catalyzed by the RhlG protein. In addition, the synthesis of poly-beta-hydroxyalkanoate (PHA) is delayed in this mutant, suggesting that RhlG participates in PHA synthesis, although it is not the only reductase involved in this pathway. Traits regulated by the quorum-sensing response, other than rhamnolipid production, including production of proteases, pyocyanine, and the autoinducer butanoyl-homoserine lactone (PAI-2), were not affected by the rhlG mutation. We conclude that the P. aeruginosa rhlG gene encodes an NADPH-dependent beta-ketoacyl reductase absolutely required for the synthesis of the beta-hydroxy acid moiety of rhamnolipids and that it has a minor role in PHA production. Expression of rhlG mRNA under different culture conditions is consistent with this conclusion.     

Elastase and the LasA protease of Pseudomonas aeruginosa are secreted with their propeptides

Pseudomonas aeruginosa elastase and the LasA protease are synthesized as preproenzymes with long amino-terminal propeptides. The elastase propeptide is cleaved autocatalytically in the periplasm to form a transient, inactive elastase-propeptide complex. In contrast, the processing of proLasA does not involve autoproteolysis. In this study, we analyzed short-term P. aeruginosa cultures under conditions that minimize proteolysis and found that an elastase-propeptide complex is secreted, and then the propeptide is degraded extracellularly, apparently by elastase itself. LasA protease, on the other hand, was found to be secreted in its unprocessed 42-kDa proenzyme form. The processing of proLasA occurred extracellularly, and it involved the transient appearance of a 28-kDa intermediate and the respective 14-kDa LasA propeptide fragment. The processing of proLasA in P. aeruginosa strain FRD740, which does not express elastase, also proceeded via the 28-kDa intermediate, but the rate of processing was greatly reduced. This low rate of proLasA processing was further reduced when the activity of a secreted lysine-specific protease was blocked. Purified secreted proteases of P. aeruginosa (i.e. elastase, the lysine-specific protease, and alkaline proteinase) converted proLasA to the active enzyme. Processing by elastase and the lysine-specific enzyme, but not by alkaline proteinase, proceeded via the 28-kDa intermediate, and both were far more effective than alkaline proteinase in converting proLasA to the mature enzyme. We conclude that LasA protease and elastase are secreted with their propeptides, which are then degraded by secreted proteases of P. aeruginosa. In addition to their other functions, the propeptides may play a role in targeting their respective enzymes across the outer membrane.     

Enhanced octadecane dispersion and biodegradation by a Pseudomonas rhamnolipid surfactant (biosurfactant)

A microbial surfactant (biosurfactant) was investigated for its potential to enhance bioavailability and, hence, the biodegradation of octadecane. The rhamnolipid biosurfactant used in this study was extracted from culture supernatants after growth of Pseudomonas aeruginosa ATCC 9027 in phosphate-limited proteose peptone-glucose-ammonium salts medium. Dispersion of octadecane in aqueous solutions was dramatically enhanced by 300 mg of the rhamnolipid biosurfactant per liter, increasing by a factor of more than 4 orders of magnitude, from 0.009 to > 250 mg/liter. The relative enhancement of octadecane dispersion was much greater at low rhamnolipid concentrations than at high concentrations. Rhamnolipid-enhanced octadecane dispersion was found to be dependent on pH and shaking speed. Biodegradation experiments done with an initial octadecane concentration of 1,500 mg/liter showed that 20% of the octadecane was mineralized in 84 h in the presence of 300 mg of rhamnolipid per liter, compared with only 5% octadecane mineralization when no surfactant was present. These results indicate that rhamnolipids may have potential for facilitating the bioremediation of sites contaminated with hydrocarbons having limited water solubility.     

Biofilms on indwelling urethral catheters produce quorum-sensing signal molecules in situ and in vitro

Acylated homoserine lactones (AHLs) are chemical signals that mediate population density-dependent (quorum-sensing) gene expression in numerous gram-negative bacteria. In this study, gram-negative bacilli isolated from catheters were screened for AHL production by a cross-feeding assay utilizing an AHL-responsive Agrobacterium tumefaciens reporter strain. Positive reactions were obtained from 14 isolates of Pseudomonas aeruginosa; negative or weakly positive reactions were recorded for isolates of five other species. P. aeruginosa biofilms were then produced on catheters in a physical model of the bladder. Sections of colonized all-silicone catheters gave positive reactions for the quorum-sensing signal molecules as did sections that had been cleaned of biofilm and autoclaved. Control sections of unused catheters were negative in the tests. Sections from four of nine catheters that had been freshly removed from patients gave positive reactions for AHLs. Cleaned autoclaved sections of three of these catheters also gave strongly positive reactions for AHLs. These results demonstrate that AHLs are produced by biofilms as they develop on the catheters both in vitro in the model and in vivo in the patient's bladder. They represent the first demonstration of AHL production by biofilms in a clinical setting.     

Influence of growth media on potential virulence factors of Pseudomonas aeruginosa

Potential virulence factors of three Pseudomonas aeruginosa strains after growth in three complex media (CM) and in one mineral medium (MM) were evaluated. Cell surface hydrophobicity demonstrated by adherence of bacteria to xylene as well as enzymatic activity (elastase, protease, lipase) of the strains grown in CM varied with composition of CM and with strain. All strains cultivated in CM showed higher hydrophobicity and higher elastase, protease and lipase (with the exception of one strain) activity in comparison with bacteria incubated in MM. Even no production of elastase was detected in the strains after growth in MM. Motility of bacteria was affected by culture media the least. In vitro composition of growth media influenced some potential virulence factors of P. aeruginosa.     

Production of acyl-homoserine lactone quorum-sensing signals by gram-negative plant-associated bacteria

Many gram-negative bacteria regulate expression of specialized gene sets in response to population density. This regulatory mechanism, called autoinduction or quorum-sensing, is based on the production by the bacteria of a small, diffusible signal molecule called the autoinducer. In the most well-studied systems the autoinducers are N-acylated derivatives of L-homoserine lactone (acyl-HSL). Signal specificity is conferred by the length, and the nature of the substitution at C-3, of the acyl side-chain. We evaluated four acyl-HSL bioreporters, based on tra of Agrobacterium tumefaciens, lux of Vibrio fischeri, las of Pseudomonas aeruginosa, and pigment production by Chromobacterium violaceum, for their ability to detect sets of 3-oxo acyl-HSLs, 3-hydroxy acyl-HSLs, and alkanoyl-HSLs with chain lengths ranging from C4 to C12. The traG::lacZ fusion reporter from the A. tumefaciens Ti plasmid was the single most sensitive and versatile detector of the four. Using this reporter, we screened 106 isolates representing seven genera of bacteria that associate with plants. Most of the Agrobacterium, Rhizobium, and Pantoea isolates and about half of the Erwinia and Pseudomonas isolates gave positive reactions. Only a few isolates of Xanthomonas produced a detectable signal. We characterized the acyl-HSLs produced by a subset of the isolates by thin-layer chromatography. Among the pseudomonads and erwinias, most produced a single dominant activity chromatographing with the properties of N-(3-oxo-hexanoyl)-L-HSL. However, a few of the erwinias, and the P. fluorescens and Ralstonia solanacearum isolates, produced quite different signals, including 3-hydroxy forms, as well as active compounds that chromatographed with properties unlike any of our standards. The few positive xanthomonas, and almost all of the agrobacteria, produced small amounts of a compound with the chromatographic properties of N-(3-oxo-octanoyl)-L-HSL. Members of the genus Rhizobium showed the greatest diversity, with some producing as few as one and others producing as many as seven detectable signals. Several isolates produced extremely nonpolar compounds indicative of very long acyl side-chains. Production of these compounds suggests that quorum-sensing is common as a gene regulatory mechanism among gram-negative plant-associated bacteria.     

Pseudomonas aeruginosa UG2 rhamnolipid biosurfactants: structural characterization and their use in removing hydrophobic compounds from soil

The structure of two rhamnolipid biosurfactants produced by Pseudomonas aeruginosa UG2 was studied. Analyses by gas chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy showed these two rhamnolipids to be alpha-L-rhamnopyranosyl-beta-hydroxydecanyol-beta-hydroxydecanoate and 2-O-alpha-L-rhamnopyranosyl-alpha-L-rhamnopyranosyl-beta-hydroxydecan oyl-beta- hydroxydecanoate. The ability of UG2 rhamnolipid biosurfactants to enhance removal of naphthalene, anthracene, phenanthrene, fluorene, 2,2',5,5'-tetrachlorobiphenyl, and 3,3',4,4',5,5'-hexachlorobiphenyl into the aqueous phase was affected by soil type, hydrocarbon equilibration time, and biosurfactant adsorption to soil. Partially purified UG2 biosurfactants at a concentration of 5 g/L removed approximately 10% more hydrocarbon from a sandy loam soil than slit loam soil. High levels of UG2 rhamnolipids adsorbed to soil. In 18% (w/v) soil slurries 74, 49, 38, and 20% of 0.5, 1, 2, and 5 g UG2 rhamnolipids/L, respectively, were bound to the soil phase. Sodium dodecyl sulphate recovered lower levels and Witconol SN70 higher levels of phenanthrene and 2,2',5,5'-tetrachlorobiphenyl than UG2 biosurfactants.     

Influence on Pseudomonas aeruginosa elastase, proteinase and alginate by supra-inhibitory and supra-subinhibitory concentrations of quinolones

Suppression of elastase, proteinase and alginate of Pseudomonas aeruginosa after the exposure of a bacterial suspension (30 min) to supra-inhibitory concentrations (2x or 4x MIC) of norfloxacin was found. The norfloxacin at 4x MIC was more effective, and this concentration depressed elastase to 13.0%, proteinase to 14.6% and alginate to 0% of the control values. Enoxacin at 2x MIC did not affect the virulence factors studied, and at a concentration of 4x MIC only elastase was effectively reduced to 61% of the control value. Supra-subinhibitory concentrations of the quinolones tested (2x or 4x MIC + sub-MICs) severely diminished all of the activities tested compared with supra-inhibitory concentrations alone. Combinations of 4x MIC + sub-MICs were more effective in the majority of cases.     

Adherence to and penetration of human intestinal Caco-2 epithelial cell monolayers by Pseudomonas aeruginosa

Clinical isolates of Pseudomonas aeruginosa from blood adhered to and penetrated intestinal Caco-2 cell monolayers to a greater degree than did isolates from sputum, with a concomitant drastic decrease in transepithelial electrical resistance. PAO-PR1, an avirulent exotoxin A mutant of PAO1, did not cause a decrease in the resistance. The Caco-2 monolayer system may be useful for the evaluation of certain P. aeruginosa virulence factor activities.     

Identification of an additional member of the secretin superfamily of proteins in Pseudomonas aeruginosa that is able to function in type II protein secretion

Pseudomonas aeruginosa is a prolific exporter of virulence factors and contains three of the four protein secretion systems that have been described in gram-negative bacteria. The P. aeruginosa type II general secretory pathway (GSP) is used to export the largest number of proteins from this organism, including lipase, phospholipase C, alkaline phosphatase, exotoxin A, elastase and LasA. Although these exoproteins contain no sequence similarity, they are specifically and efficiently transported by the secretion apparatus. Bacterial homologues of XcpQ (GspD), the only outer membrane component of this system, have been proposed to play the role of gatekeeper, by presumably interacting and recognizing the exported substrates to allow their passage through the outer membrane. While determining the phenotype of nonpolar deletions in each of the xcp genes, we have shown that a deletion of the P. aeruginosa strain K xcpQ does not completely abolish protein secretion. As the proposed function of XcpQ should be requisite for secretion, we searched for additional factors that could carry out this role. A cosmid DNA library from a PAK strain deleted for xcpP-Z was tested for its ability to increase protein secretion by screening for enhanced growth on lipid agar, a medium that selects for the secretion of lipase. In this manner, we have identified an XcpQ homologue, XqhA, that is solely responsible for the residual export observed in a deltaxcpQ strain, although it is not required for efficient secretion in wild-type P. aeruginosa. We have also demonstrated that this protein is capable of recognizing all of the exoproteins of P. aeruginosa, arguing against the proposed role of members of the secretin family as determinants of specificity.     

Pseudomonas keratitis. The role of an uncharacterized exoprotein, protease IV, in corneal virulence

PURPOSE: The role of exoproteins in the pathogenesis of Pseudomonas aeruginosa keratitis was investigated in three animal models by assessing the relationship between corneal virulence and the activities of exotoxin A, elastase, alkaline protease, and an uncharacterized protease, protease IV. METHODS: The four Pseudomonal strains tested included a prototype strain (ATCC 27853) producing exotoxin A, elastase, and alkaline protease; a parent strain (PA103) producing only exotoxin A and protease IV; a mutant (PA103-29) producing only protease IV; and a mutant (PA103-AP1) producing exotoxin A and having only approximately 5% of the protease IV activity of its parent. Corneal virulence was evaluated in the mouse scratch, rabbit scratch, and rabbit intrastromal models in terms of clinical signs (slit lamp examination, slit lamp examination), and viable bacteria. RESULTS: Protease IV, the only protease produced by PA103 and PA103-29, was found to produce a unique band on zymograms (120 kDa) and to react distinctively with a synthetic substrate. Evidence for the role of protease IV in corneal virulence included two findings: PA103-29,which produced protease IV but not the other exoproteins, caused infections that were as severe as those caused by the prototype strain (ATCC 27853) in all three models (P>0.24); and PA103-AP1, the strain deficient in 95% of the parent protease IV activity, mediated infections characterized by slit lamp examination scores significantly lower than those of infections caused by the parent (PA103) or the prototype strain (ATCC 27853) in the rabbit and mouse scratch models (P<0.02). CONCLUSIONS: Protease IV was found to be a novel Pseudomonas protease contributing to corneal virulence in rabbits and mice when infections were initiated at the corneal surface. Furthermore, production of protease IV in low quantities was sufficient for virulence when the topical stages of keratitis were bypassed by an intrastromal injection of Pseudomonas.     

Use of model plant hosts to identify Pseudomonas aeruginosa virulence factors

We used plants as an in vivo pathogenesis model for the identification of virulence factors of the human opportunistic pathogen Pseudomonas aeruginosa. Nine of nine TnphoA mutant derivatives of P. aeruginosa strain UCBPP-PA14 that were identified in a plant leaf assay for less pathogenic mutants also exhibited significantly reduced pathogenicity in a burned mouse pathogenicity model, suggesting that P. aeruginosa utilizes common strategies to infect both hosts. Seven of these nine mutants contain TnphoA insertions in previously unknown genes. These results demonstrate that an alternative nonvertebrate host of a human bacterial pathogen can be used in an in vivo high throughput screen to identify novel bacterial virulence factors involved in mammalian pathogenesis.     

A His19 to Ala mutant of melanoma growth-stimulating activity is a partial antagonist of the CXCR2 receptor

Melanoma growth stimulating activity (MGSA) and IL-8 are related chemokines that are potent chemoattractants and activators of neutrophils both in vitro and in vivo. Increasing evidence suggests that these molecules play an important role in inflammation; thus, antagonists of their action could be useful therapeutically as antiinflammatory agents. We have generated an MGSA mutant, H19A, that shows a dissociation between receptor binding and biologic activity. The biologic activity of the H19A mutant is between 133-fold and 282-fold less potent than that of wild-type MGSA measured by three independent assays of neutrophil function, i.e., elastase release chemotaxis and the up-regulation of CD18. In addition, pretreatment of cells with the H19A mutant inhibited the ability of MGSA to induce elastase release and chemotaxis and to increase intracellular calcium. However, competition binding studies in cells transfected with the CXCR2 receptor and in neutrophils demonstrate that the receptor affinity of the H19A mutant is only 13-fold less than that of wild-type MGSA. These studies suggest that the mutant MGSA is defective in activating signaling through the receptor and indicate that binding to the receptor is not sufficient to activate a biologic response. The dissociation between receptor binding and activation for this mutant suggests that it should be possible to design antagonists of MGSA that may be of clinical utility.     

Evaluation of multiple Pseudomonas aeruginosa strains with different characteristics in clinical specimens; combined results from serotyping, drug susceptibility and enzyme production

Multiple Pseudomonas aeruginosa strains with different characteristics are occasionally isolated from a clinical specimen. Therefore, more than five isolated colonies of P. aeruginosa obtained at random from each clinical specimen (47 sputa, 18 urine, 10 pus and 8 others). These were investigated for serotype, drug susceptibility to eight antimicrobial agents and productivity of enzymes, such as protease and elastase. The specimens with multiple serotype colonies were shown in 17% of the sputa, 11% of the urine and 10% of the pus. 45.7% of the specimens with single serotype colonies exhibited more than two different patterns of enzyme productivity and so did 47.1% different patterns of drug susceptibility. Single serotype strains of P. aeruginosa with different characteristics of these tests were demonstrated in 81.3% of the urine, 73.6% of the sputum, 50.0% of the pus and 66.7% of others. We conclude that it is important to recognize the possible existence of multiple P. aeruginosa strains with different patterns of the enzyme productivity and drug susceptibility, regardless of single serotype, in clinical specimens.     

Extended durability of external metal mirror reflector of side-firing Nd:YAG laser fiber used for prostatic ablation

The effect on Pseudomonas aeruginosa virulence factors (elastase and proteinase) after short-time treatment (30 min) with suprainhibitory (2x or 4x MIC) as well as with supra-subinhibitory concentrations (2x MIC + sub-MICs or 4x MIC + sub-MICs) of pefloxacin was studied. An effective decrease of elastase activity (to 42.6% of the control value) was observed only after higher suprainhibitory antibiotic concentration. More significant depression of the activities tested was found after the postantibiotic effect of subinhibitory concentrations. Elastase as well as proteinase activity were suppressed by supra-subinhibitory concentration of 4x MIC + 0.1x MIC to 13.1% or 37.9%, as compared to controls.     

Immunologic aspects of lung diseases and cystic fibrosis

Immunologic lung disorders are accompanied by an array of laboratory abnormalities, some of which contribute to disease pathogenesis. Allergic bronchopulmonary aspergillosis, which complicates asthma and cystic fibrosis, causes mucous plugging of airways, eosinophilic pneumonia, and bronchiolitis obliterans. Aspergillus fumigatus, growing saprophytically in bronchial mucus, is responsible for most cases, and prednisone, not antifungal agents, is the drug of choice because it controls the immunologic responses of the lung. In cystic fibrosis, epithelial surface fluid from the lung does not kill Pseudomonas aeruginosa, in part because antibodies to P aeruginosa are plentiful but ineffective in opsonizing bacteria. Neutrophil-derived elastase cleaves immunoglobulins and digests the C3b receptor on neutrophils, which limits phagocytosis of pathogens. In helminth infections and infestations, pulmonary and peripheral blood eosinophilia can be accompanied by increases in total and antiparasite IgE concentrations and generate T(H)2 CD4+ T-lymphocyte responses. Understanding the immunologic abnormalities of lung disorders may lead to more effective therapies.