Pyoverdin is essential for virulence of Pseudomonas aeruginosa.

The role of pyoverdin, the main siderophore in iron-gathering capacity produced by Pseudomonas aeruginosa, in bacterial growth in vivo is controversial, although iron is important for virulence. To determine the ability of pyoverdin to compete for iron with the human iron-binding protein transferrin, wild-type P. aeruginosa ATCC 15692 (PAO1 strain) and PAO pyoverdin-deficient mutants were grown at 37 degrees C in bicarbonate-containing succinate medium to which apotransferrin had been added. Growth of the pyoverdin-deficient mutants was fully inhibited compared with that of the wild type but was restored when pyoverdin was added to the medium. Moreover, when growth took place at a temperature at which no pyoverdin production occurred (43 degrees C), the wild-type PAO1 strain behaved the same as the pyoverdin-deficient mutants, with growth inhibited by apotransferrin in the presence of bicarbonate and restored by pyoverdin supplementation. Growth inhibition was never observed in bicarbonate-free succinate medium, whatever the strain and the temperature for growth. In vivo, in contrast to results obtained with the wild-type strain, pyoverdin-deficient mutants demonstrated no virulence when injected at 10(2) CFU into burned mice. However, virulence was restored when purified pyoverdin originating from the wild-type strain was supplemented during the infection. These results strongly suggest that pyoverdin competes directly with transferrin for iron and that it is an essential element for in vivo iron gathering and virulence expression in P. aeruginosa. Rapid removal of iron from [59Fe]ferritransferrin by pyoverdin in vitro supports this view.     

Experimental infections with protease-deficient mutants of Pseudomonas aeruginosa in mice.

The virulence of a protease-producing strain of Psuedomonas aeruginosa was compared with that of mutants that had lost the ability to produce proteases and other extracellular enzymes. Lethal infections were produced by inoculating mice intraperitoneally with bacteria in mucin, or by inoculating mice intraperitoneally or intravenously with bacteria 4 days after treatment with cyclophosphamide, 200 mg per kg body weight. No significant difference in virulence between the wild-type parent strain and some of its protease-deficient mutants was found. Histopatholoical examination of different organs in the cyclophosphamide-treated and infected mice showed striking fatty infiltration and focal necrosis of liver, multiple necrotic foci in the spleen and haemorrhagic cystitis with necorsis. The cystitis was produced by cyclophosphamide alone but was aggravated by the infection. In conclusion, no correlation between the production of protease in broth culture and the ability to produce lethal septicaemia in mice was found, and extracellular proteases probably didnot contribute to the virulence of P. aeruginosa. However, the histopathological changes in the liver suggested a role for exotoxin A insystemic infections.     

Evaluation of Pseudomonas aeruginosa exotoxin A and elastase as virulence factors in acute lung infection.

Acute Pseudomonas aeruginosa pneumonia was established in guinea pigs by intratracheal instillation of bacteria. Challenge strains included PAO-1, a strain known to produce exotoxin A, alkaline protease, and elastase, and several PAO-1 mutants deficient in either biologically active exotoxin A or elastase production. Survival, intrapulmonary killing of bacteria, and blood cultures were compared among the groups. Strains of P. aeruginosa deficient in active elastase production appeared to be less virulent than the parent strain and were more easily cleared from the lung. Opposite results were obtained for the exotoxin A-deficient mutants. These data suggest that elastase, but not exotoxin A, was an important virulence factor during acute pneumonia due to P. aeruginosa.     

Contribution of Quorum Sensing to the Virulence of Pseudomonas aeruginosa in Burn Wound Infections

The Pseudomonas aeruginosa quorum-sensing systems, las and rhl, control the production of numerous virulence factors. In this study, we have used the burned-mouse model to examine the contribution of quorum-sensing systems to the pathogenesis of P. aeruginosa infections in burn wounds. Different quorum-sensing mutants of P. aeruginosa PAO1 that were defective in the lasR, lasI, or rhlI gene or both the lasI and rhlI genes were utilized. The following parameters of the P. aeruginosa infection were examined: (i) lethality to the burned mouse, (ii) dissemination of the P. aeruginosa strain within the body of the infected mouse (by determining the numbers of CFU of P. aeruginosa within the liver and spleen), and (iii) spread of the P. aeruginosa strain within the burned skin (by determining the numbers of CFU of P. aeruginosa at the inoculation site and at a site about 15 mm from the inoculation site [distant site]). In comparison with that of PAO1, the in vivo virulence of lasI, lasR, and rhlI mutants was significantly reduced. However, the most significant reduction in in vivo virulence was seen with the lasI rhlI mutant. The numbers of CFU that were recovered from the livers, spleens, and skin of mice infected with different mutants were significantly lower than those of PAO1. At 8 and 16 h post burn infection, comparable numbers of CFU of PAO1 and lasI and rhlI mutants were obtained from both the inoculation and distant sites of the burned skin of infected mice. In contrast, CFU of the lasR mutant and the lasI rhlI double mutant were recovered only from the inoculation site of infected mice at 8 and 16 h post burn infection. The ability of a plasmid carrying either the lasI or rhlI gene or the lasI and rhlI genes to complement the defect of the lasI rhlI double mutant was also examined. The presence of any of these plasmids within the lasI rhlI double mutant significantly enhanced its in vivo virulence, as well as its ability to spread within the burned skin. These results suggest that the quorum-sensing systems play an important role in the horizontal spread of P. aeruginosa within burned skin and in the dissemination of P. aeruginosa within the bodies of burned-and-infected mice and contributed to the overall virulence of P. aeruginosa in this animal model.     

Construction and characterization of chromosomal insertional mutations of the Pseudomonas aeruginosa exoenzyme S trans-regulatory locus.

Exoenzyme S is an ADP-ribosyltransferase produced by Pseudomonas aeruginosa. Synthesis of exoenzyme S depends on an intact trans-regulatory locus encoding three protein products, ExsC, ExsB, and ExsA. To identify the phenotype of ExsC, -B, and -A mutants in exoenzyme S production, specific insertional mutations with the streptomycin resistance-encoding omega interposon were introduced into cloned DNA and returned to the chromosomes of P. aeruginosa PA103, PAO1, and PAK. Southern blot analysis was used to confirm insertion of omega and resolution of vector sequences. Exoenzyme S expression was measured in parental and mutant derivatives by Western blot (immunoblot) analysis and ADP-ribosyltransferase activity measurement. A complete set of mutations were obtained in strains PAK and PAO1, but in strain PA103, only an insertion in the exsA coding region was identified. Southern blot analysis demonstrated that extensive duplication and rearrangement of the PA103 chromosomal trans-regulatory locus occurred when exsC::omega or exsB::omega recombination events were attempted. Exoenzyme S antigen was not detectable in the supernatant or lysate fractions of mutant strains by Western blot analysis. ADP-ribosyltransferase activity was detected in the lysate but not in the supernatant fractions of mutant derivatives. The general secretion pathway appeared to function normally in mutant strains, as elastase, exotoxin A, and phospholipase C were measured in the supernatants of parental and mutant strains. Several differences were noted when the extracellular protein profiles of parental strains were compared with similar samples from the insertional mutant strains. Some of these differences appeared to be unrelated to exoenzyme S. These data suggest that insertional inactivation of the exoenzyme S trans-regulatory locus may affect a subset of other extracellular proteins.     

Mutations in the cueA gene encoding a copper homeostasis P-type ATPase reduce the pathogenicity of Pseudomonas aeruginosa in mice

A sequencing project identified a putative copper homeostasis gene, cueA, in Pseudomonas aeruginosa strain PAO1. Strains with mutations of the cueA gene, encoding a P-type ATPase linked to copper homeostasis in P. putida, displayed greater sensitivity to copper compared to wild-type bacteria using MIC determinations and in vitro passage in growth media with different concentrations of copper added. An LD50 assay showed a cueA deletion mutant was 50-fold more attenuated than wild-type strain PAO1 bacteria. Complementation of the cueA mutation restored in vitro tolerance to copper and virulence in a systemic model of infection to near wild-type levels. Competition assays between cueA mutants and wild-type P. aeruginosa strains demonstrated 20-fold attenuation by the cueA mutants within spleens of mice. This data suggests the P. aeruginosa CueA protein may be important in maintaining copper homeostasis both in vitro and in vivo.     

Surface expression of ferripyochelin-binding protein is required for virulence of Pseudomonas aeruginosa.

Pseudomonas aeruginosa mutants which do not express ferripyochelin-binding protein (FBP) on the cell surface have previously been isolated. These mutants were used to assess the role of FBP in virulence in an acute and a systemic animal infection model. In a mouse corneal infection model, the pathology of eyes infected with the mutant strains was significantly less than that of eyes infected with the parent strain. The mutants were also cleared more rapidly from the eye. In a burn infection model, the mortality rate in mice infected with mutant FBP-28 was much less than that of mice infected with the parent strain at an inoculum of 10(2) CFU. At higher inocula (10(4) CFU), the mortality rate was not significantly different but the survival time was dramatically longer with the mutant strain. Quantitative bacteriology of blood and tissue homogenates revealed that P. aeruginosa PAO could multiply in the skin and could also be cultured from the blood, livers, and spleens of infected mice. FBP-28 could only be cultured from the skin. Therefore, this mutant could colonize the skin but could not disseminate. These data indicate that functional, exposed FBP is required for virulence of PAO.     

Effect of pyochelin on the virulence of Pseudomonas aeruginosa.

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.     

Pseudomonas aeruginosa virulence factors delay airway epithelial wound repair by altering the actin cytoskeleton and inducing overactivation of epithelial matrix metalloproteinase-2

To investigate the role of P. aeruginosa virulence factors in the repair of human airway epithelial cells (HAEC) in culture, we evaluated the effect of stationary-phase supernatants from the wild-type strain PAO1 on cell migration, actin cytoskeleton distribution, epithelial integrity during and after repair of induced wounds, and the balance between matrix metalloproteinases (MMP) and their tissue inhibitors (TIMP). PAO1 supernatant altered wound repair by slowing the migration velocity in association with altered actin cytoskeleton polymerization in the lamellipodia of migrating airway epithelial cells and delaying or inhibiting the restoration of epithelial integrity after wound closure. PAO1 virulence factors overactivated two of the gelatinolytic enzymes, MMP-2 and MMP-9, produced by HAEC during repair. During HAEC repair in the presence of PAO1 virulence factors, enhanced MMP-2 activation was associated with decreased rates of its specific inhibitor TIMP-2, whereas enhanced MMP-9 activation was independent of changes of its specific inhibitor TIMP-1. These inhibitory effects were specific to P. aeruginosa elastase-producing strains (PAO1 and lipopolysaccharide-deficient AK43 strain); supernatants from P. aeruginosa strain elastase-deficient PDO240 and Escherichia coli strain DH5alpha had no inhibitory effect. To mimic the effects of P. aeruginosa, we further analyzed HAEC wound closure in the presence of increasing concentrations of activated MMP-9 or MMP-2. Whereas increasing concentrations of active MMP-9 accelerated repair, excess activated MMP-2 generated a lower migration velocity. All these data demonstrate that P. aeruginosa virulence factors, especially elastase, may impede airway epithelial wound closure by altering cell motility and causing an imbalance between pro- and activated forms of MMP-2.     

Phenotypic comparison of Pseudomonas aeruginosa strains isolated from a variety of clinical sites.

Pseudomonas aeruginosa elaborates a number of extracellular products which have been shown to play a role in the pathogenesis of disease caused by this organism. In this study, we showed that the host environment markedly affects the levels of exoproducts produced. We compared the phenotypes of a number of P. aeruginosa strains obtained from a variety of clinical sources, including burn wounds, skin wounds, urine, cystic fibrosis sputum, acute pneumonia sputum, and blood. The clinical isolates were examined quantitatively for levels of total protease, elastase, phospholipase C, exotoxin A, and exoenzyme S produced in vitro under defined conditions. The exoproduct levels varied significantly, depending on the site of isolation. Elevated levels of elastase were demonstrated in strains isolated from acute lung infections, phospholipase C levels were elevated in urinary tract and blood isolates, exotoxin A levels were elevated in blood isolates, and exoenzyme S levels were increased in acute pneumonia isolates. Isolates from cystic fibrosis sputum produced low amounts of virtually all of the tested exoproducts, particularly as compared with sputum isolates from acute P. aeruginosa lung infections.     

Serum antibody response to Pseudomonas aeruginosa antigens during corneal infection.

Previous studies in our laboratory have indicated that naturally resistant, inbred DBA/2J mice mount a greater serum antibody response to Pseudomonas aeruginosa 19660 than susceptible C57BL/6J mice. However, the specificity of the antibody produced was not known. The present study examines the specificity and kinetics of the humoral response of these mouse strains to potential virulence factors produced by the organism during both a primary and a secondary corneal infection administered 4 weeks after the primary infection. Serum antibody levels specific for lipopolysaccharide (LPS), exotoxin A, phospholipase C (PLC), alkaline protease, elastase, and flagella were measured by enzyme-linked immunosorbent assay. Little or no antibody to either alkaline protease or elastase was detected during either primary or secondary infection. Immunoglobulin G (IgG) antibodies specific to exotoxin A, PLC, and flagella were detected 2 weeks after primary infection, and a rapid response to these antigens was measured 1 week after secondary infection. During primary infection, detectable LPS-specific antibody was only IgM, while IgG appeared only after secondary infection. The kinetics of the humoral response in susceptible C57BL/6J mice were similar to those in resistant DBA/2J mice, although the magnitude of the response varied according to the antigen tested. These results indicate that LPS, exotoxin A, PLC, and flagella are present or produced in amounts that are immunogenic during corneal infection by P. aeruginosa 19660 in the mouse strains tested.     

Effect of passive immunotherapy on murine gut-derived sepsis caused by Pseudomonas aeruginosa.

The effect of passive immunotherapy with antisera against heat-killed Pseudomonas aeruginosa and three of its exo-enzymes (elastase, alkaline protease and exotoxin A) in gut-derived P. aeruginosa sepsis was evaluated. Mice were given a suspension of P. aeruginosa strain D4 in their drinking water, together with ampicillin (200 mg/kg) to disrupt the normal bacterial flora. Cyclophosphamide was then administered to induce translocation of P. aeruginosa that had colonised the gastrointestinal tract so that gut-derived septicaemia was produced. In this model, intraperitoneal administration of antiserum against heat-killed bacteria, 100 microl/mouse, twice a day for 3 consecutive days significantly increased the survival rate over that of mice treated with normal rabbit serum. Antiserum against elastase, alkaline protease, or a combination of these two antisera, failed to provide significant protection. In contrast, antiserum against exotoxin A significantly increased the survival rate over that of mice treated with normal rabbit serum. These results indicate that passive immunisation with antiserum against heat-killed bacteria and exotoxin A, but not with antiserum against either elastase or alkaline protease, protects mice against gut-derived sepsis caused by P. aeruginosa.     

Effects of FP2 and a mercury resistance plasmid from Pseudomonas aeruginosa PA103 on exoenzyme production.

Plasmids encoding mercury resistance carried by Pseudomonas aeruginosa PAO1161 and PA103 were found to be involved in regulating the secretion of protease, phospholipase C, and alkaline phosphatase. Previously, mutations in Pseudomonas strains that caused pleiotropic effects on the production of extracellular enzymes were mapped to the bacterial chromosome. We show that pleiotropic changes in extracellular enzyme production can also be regulated by plasmids. In this study, the effects on secretion of exoenzymes by two mercury resistance plasmids, FP2 from PAO1161 and pRLW103 from PA103, were assayed in P. aeruginosa PAO1 and PAO18. The introduction of either plasmid into PAO1 resulted in a significant decrease in exoprotease production. Additionally, pRLW103 significantly increased the production of alkaline phosphatase by both strains. Phospholipase C was produced only in strain PAO18 containing the pRLW103 plasmid. FP2 had no effect on alkaline phosphatase or phospholipase C production in either strain and was found to decrease exoprotease secretion only in strain PAO1. The results indicate the P. aeruginosa mercury resistance plasmids vary in their ability to modify exoenzyme expression, and this ability is influenced by the host strain.     

The role of extracellular products of Pseudomonas aeruginosa in the pathogenesis of infection; an experimental study employing intraperitoneal diffusion chambers.

Exotoxin A is currently thought to be the principal lethal factor in experimental Pseudomonas aeruginosa infection. This belief is founded on the demonstrable toxicity of purified preparations, and on detection of the toxin in the tissues of burned animals infected with Ps. aeruginosa. In the present study, strains of Ps. aeruginosa differing in their ability to produce exotoxin A and other virulence factors in vitro were enclosed within vinyl diffusion chambers and implanted i.p. into mice. Strains which produced much exotoxin A in vitro were not significantly more virulent when enclosed in chambers than strains which produced little exotoxin. In all cases, diffusion of exotoxin A produced within the chamber was impeded by dense adherence of the omentum. It is concluded that, although absorption of exotoxin A may be an important factor in causing death after infected burns, it is not necessarily equally significant in other types of infection.     

Impact of siderophore production on Pseudomonas aeruginosa infections in immunosuppressed mice

Pseudomonas aeruginosa produces siderophores, pyoverdin and pyochelin, for high-affinity iron uptake. To investigate their contribution to P. aeruginosa infections, we constructed allelic exchange mutants from strain PAO1 which were deficient in producing one or both of the siderophores. When inoculated into the calf muscles of immunosuppressed mice, pyochelin-deficient and pyoverdin-deficient mutants grew and killed the animals as efficiently as PAO1. In contrast, the pyochelin- and pyoverdin-deficient (double) mutant did not show lethal virulence, although it did infect the muscles. On the other hand, when inoculated intranasally, all mutants grew in the lungs and killed immunosuppressed mice. Compared with PAO1, however, the pyoverdin-deficient mutant and the double mutant grew poorly in the lungs, and the latter was significantly attenuated for virulence. Irrespective of the inoculation route, the pyoverdin-deficient and doubly deficient mutants detected in the blood were significantly less numerous than PAO1. Additionally, in vitro examination demonstrated that the growth of the double mutant was extremely reduced under a free-iron-restricted condition with apotransferrin but that the growth reduction was completely canceled by supplementation with hemoglobin as a heme source. These results suggest that both pyoverdin and pyochelin are required for efficient bacterial growth and full expression of virulence in P. aeruginosa infection, although pyoverdin may be comparatively more important for bacterial growth and dissemination. However, the siderophores were not always required for infection. It is possible that non-siderophore-mediated iron acquisition, such as via heme uptake, might also play an important role in P. aeruginosa infections.     

Evidence for different pyoverdine-mediated iron uptake systems among Pseudomonas aeruginosa strains.

Fourteen strains of Pseudomonas aeruginosa (P. aeruginosa ATCC 15692, P. aeruginosa ATCC 27853, and 12 clinical isolates) were checked for the production of pyoverdine and for pyoverdine-mediated iron uptake. Under iron restriction, two isolates produced undetectable amounts of pyoverdine, but all the other strains produced a compound with physicochemical properties identical or close to those of the pyoverdine of P. aeruginosa ATCC 15692 (strain PAO1). The pyoverdines were purified and tested for their growth-promoting activity and for their ability to facilitate 59Fe uptake in homologous experiments involving each pyoverdine and its producing strain, as well as in heterologous systems involving all the other strains. The results of both types of experiments suggested the existence of three specificity groups. This was confirmed by analysis of the amino acid composition of the pyoverdines, which differed for each group. A partially purified polyclonal antiserum raised against a major 80-kilodalton (kDa) iron-regulated outer membrane protein (IROMP) of P. aeruginosa PAO1 recognized the 80-kDa IROMPs from P. aeruginosa PAO1 and the clinical isolates belonging to the same group, whereas the IROMPs from the strains belonging to the two other groups were not detected. A second antiserum raised against the P. aeruginosa ATCC 27853 80-kDa IROMP gave similar results by reacting specifically with the 80-kDa IROMP from the strains belonging to this group. Thus, together with the already known pyoverdine from P. aeruginosa PAO1, two new types of pyoverdines produced by strains belonging to this species were characterized.     

Multiple virulence factors regulated by quorum sensing may help in establishment and colonisation of urinary tract by Pseudomonas aeruginosa during experimental urinary tract infection

Purpose: Damage caused by an organism during infection is attributed to production of virulence factors. Different virulence factors produced by the organism contribute to its pathogenicity, individually. During infectious conditions, role of virulence factors produced by the pathogen is different, depending upon the site of involvement. Pseudomonas aeruginosa is an opportunistic nosocomial pathogen known to cause infections of the respiratory tract, burn wound, urinary tract and eye. Importance of virulence factors produced by P. Aeruginosa during infections such as keratitis, burn wound and respiratory tract is known. The present study was designed to understand the importance of different virulence factors of P. aeruginosa in urinary tract infection in vivo. Materials and methods: An ascending urinary tract infection model was established in mice using standard parent strain PAO1 and its isogenic mutant, JP2. Mice were sacrificed at different time intervals and renal tissue homogenates were used for estimation of renal bacterial load and virulence factors. Results: Both parent and mutant strains were able to reach the renal tissue. PAO₁ PAO1was isolated from renal tissue till day 5 post-infection. However, the mutant strain was unable to colonise the renal tissue. Failure of mutant strain to colonise was attributed to its inability to produce protease, elastase and rhamnolipid. Conclusion: This study suggests that protease, elastase and rhamnolipid contribute to pathogenesis and survival of P. aeruginosa during urinary tract infection.     

Isolation and characterization of transposon-induced mutants of Pseudomonas aeruginosa deficient in production of exoenzyme S.

Exoenzyme S is an extracellular product of Pseudomonas aeruginosa. This enzyme catalyzes the transfer of ADP-ribose from NAD to a number of as yet unidentified eucaryotic proteins, but it is distinct from toxin A. To evaluate the role of exoenzyme S in the pathogenicity of P. aeruginosa, we isolated transposon-induced mutants of strain 388, a clinical isolate that produces exoenzyme S but no toxin A. The transposon Tn1 was introduced by using a temperature-sensitive derivative of plasmid RP1. A Tn1-induced mutant was found which had no detectable exoenzyme S activity or antigen in culture supernatants or in cell lysates. Except for its lack of exoenzyme S and resistance to carbenicillin, this mutant was indistinguishable from the parent strain. When tested in an experimental mouse burn infection model, this Tn1-induced mutant was reduced in virulence by at least 2,000-fold, suggesting a role for exoenzyme S in the virulence of this strain.     

Pseudomonas aeruginosa degrades pulmonary surfactant and increases conversion in vitro

Although it is known that surfactant lipids and proteins are altered in patients with Pseudomonas aeruginosa infections, the mechanisms and implications of these alterations are not clear. In this study, the effects of P. aeruginosa on the surfactant large aggregate fraction were examined using an in vitro surface area cycling model. Large aggregates were isolated from porcine bronchoalveolar lavage fluid and incubated with supernatants from P. aeruginosa cultures (PAO1, parent strain; PAO1-A1, lasA-negative mutant; PAO1-B1, elastase-negative mutant) or purified elastase. Amounts of surfactant protein (SP)-A and SP-B, phospholipid content, and large aggregate conversion were assessed. In addition, lipid degradation was assessed by incubating a mixture of radiolabeled phospholipids with P. aeruginosa supernatants. The results demonstrated that SP-A was degraded by PAO1 and PAO1-A1 supernatants, and by purified elastase. SP-B was degraded by PAO1 and PAO1-B1 supernatants, but not by elastase. P. aeruginosa supernatants degraded phospholipids, a process inhibited by ZnCl(2). P. aeruginosa supernatants and elastase increased conversion. The data suggest that protein degradation facilitates increased conversion, and that phospholipid degradation and conversion enhance degradation of surfactant proteins. In conclusion, P. aeruginosa secretes multiple virulence factors that cooperate to result in degradation of surfactant components and alteration of large aggregate conversion.