Pseudomonas aeruginosa cell-to-cell signaling is required for virulence in a model of acute pulmonary infection

Cell-to-cell signaling controls many virulence genes in Pseudomonas aeruginosa. We tested the virulence of las and rhl quorum-sensing mutants in neonatal mice. A lasI rhlI double mutant was nearly avirulent, and the respective single mutant strains were reduced in virulence compared with the wild-type strain. Quorum sensing plays a role in P. aeruginosa pneumonia in neonatal mice.     

lasI rhlI 基因缺陷对铜绿假单胞菌生物膜形成影响的体外实验研究

 目的 研究群体感应系统在铜绿假单胞菌（PA）生物膜形成中的作用。 方法 采用生理盐水-吸痰管系统进行群体感应系统完整的PAO1 野生型菌株与群体感应系统缺陷（lasI rhlI 基因缺陷）型菌株的体外培养，分别于培养第 3、7 天用扫描电镜观察两种类型 PAO1 的生物膜形成情况。 结果 培养第 3 天 PAO1 野生型菌株可形成较厚、有孔状通道的成熟生物膜结构，而 lasI rhlI 基因缺陷型菌株仅形成明显稀薄的早期生物膜结构；培养第 7 天 PAO1 野生型菌株的生物膜增厚，lasI rhlI 基因缺陷型菌株所形成的生物膜结构呈薄膜状，显著薄于野生型菌株。 结论 lasI rhlI 基因缺陷明显影响 PA 的生物膜形成能力，群体感应系统在 PA 生物膜形成中发挥重要作用。     

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.     

Contribution of quorum-sensing systems to virulence of Pseudomonas aeruginosa in an experimental pyelonephritis model.

BACKGROUND AND PURPOSE: Pseudomonas aeruginosa has been reported to monitor its cell density as well as expression of virulence determinants by quorum-sensing signal mechanisms operative through autoinducers. In the present investigation, we studied the contribution of quorum-sensing signals during the course of P. aeruginosa-induced pyelonephritis in mice. METHODS: The standard parent strain of P. aeruginosa (PAO1), possessing functional las and rhl quorum-sensing systems and its isogenic mutant strains, PAO-JP1 (single mutant), harboring a mutated lasI gene and PAO-JP3 (double mutant), harboring mutated lasI and rhlR genes were employed. One uroisolate of P. aeruginosa belonging to serotype O8 and deficient in production of quorum-sensing signals was also used. RESULTS: The parent strain of P. aeruginosa was significantly more virulent compared to its isogenic mutant strains and quorum-sensing negative clinical strain, as assessed by neutrophil influx, malondialdehyde production, renal bacterial load and pathology induced in experimental animals. CONCLUSIONS: Quorum-sensing systems play an important role in the pathogenicity of P. aeruginosa in pyelonephritis. Both the las and rhl quorum-sensing systems are important for the virulence of P. aeruginosa in the development of pyelonephritis.     

Characterization of N-butanoyl-L-homoserine lactone (C4-HSL) deficient clinical isolates of Pseudomonas aeruginosa

In the opportunistic pathogen Pseudomonas aeruginosa, the production of several virulence factors such as elastase, rhamnolipids and pyocyanin depends on cell-to-cell signaling or quorum sensing (QS) involving N-acylhomoserine lactone (AHL) signal molecules. In vitro studies with laboratory strains and virulence studies in animals with these same strains have demonstrated the contribution of QS to the pathogenesis of P. aeruginosa. However, the importance of P. aeruginosa QS systems in the development of human infections is not clearly known. In order to determine if deficiency within the QS system compromises the ability of P. aeruginosa to cause infections in humans, we collected 50 P. aeruginosa clinical isolates. Phenotypic characterization showed that isolates I-457, I-458, I-459 and I-461 were defective in the production of N-butanoyl-l-homoserine lactone (C4-HSL) signaling molecule and virulence factors elastase, protease, pyocyanin and rhamnolipids. Analysis of the sequences of the lasR, lasI, rhlR and rhlI genes of these four isolates showed that two of the four isolates had mutational defects in both rhlR and rhlI genes while other two isolates were only mutated in the rhlI gene. The combination of rhlR and rhlI mutations or only rhlI mutation probably explains their C4-HSL and virulence factors deficiencies. These observations suggest that QS deficient P. aeruginosa clinical isolates are able to cause infections and that in addition to known virulence factors, factors yet unidentified may contribute to the pathogenesis of P. aeruginosa.     

Virulence of different Pseudomonas species in a burned mouse model: tissue colonization by Pseudomonas cepacia.

The virulence of Pseudomonas aeruginosa and other pseudomonads was examined in a burned mouse model. P. aeruginosa M-2 was highly virulent causing 100% mortality by 38 h with an injection of 10(2) CFU by either a subcutaneous or intraperitoneal route. Subcutaneous injection of 10(2) CFU revealed rapid multiplication of the bacteria at the burn wound with 10(8) CFU/g detectable in the burned skin by 28 h postinjection, 10(5) CFU/g of liver, and 10(3) CFU/ml of blood. Non-P. aeruginosa clinical isolates were markedly less virulent; an injection of greater than or equal to 10(7) CFU caused less than or equal to 60% lethality. P. cepacia SMH colonized the burned skin of thermally injured mice, persisting at levels of 10(7) to 10(8) CFU/g of burned skin after an initial injection of 10(5) CFU. P. cepacia persisted in the burn wound for at least 3 weeks. No organ invasion was detectable throughout this period. Studies with an additional clinical isolate of P. cepacia yielded similar results. An injection of a 10(2) CFU dose revealed that the level of persistence is dose dependent. Results suggest that the tenacious persistence of P. cepacia in the burn wound may provide a model for the study of persistent colonization and infection in a compromised host.     

Gr-1(+)CD11b(+) cells as an accelerator of sepsis stemming from Pseudomonas aeruginosa wound infection in thermally injured mice

Using a mouse model of thermal injury, we studied why antimicrobial peptides are not produced at the burn-site tissues and how this defect contributes to the increased susceptibility to Pseudomonas aeruginosa burn-wound infection. Logarithmic growth of P. aeruginosa was demonstrated locally (at the burn site) and systemically (in circulation) in thermally injured mice exposed to 10(2) CFU/mouse of the pathogen beneath the burn wound. However, neither systemic nor local growth of the pathogen was observed in sham burn mice when they were infected intradermally with 10(6) CFU/mouse P. aeruginosa. Murine beta-defensins (MBDs) were detected in the skin homogenates of sham burn mice. However, the amounts of MBDs were reduced greatly in the same tissue homogenates from thermally injured mice. Gr-1(+)CD11b(+) cells, with an ability to suppress antimicrobial peptide production by skin keratinocytes, were isolated from tissues surrounding the burn areas, and these cells were not obtained from skin tissues of sham burn mice. After intradermal inoculation of Gr-1(+)CD11b(+) cells, which were isolated from burn-site tissues, the production of antimicrobial peptides around the cell-inoculation site of sham burn mice decreased. Also, like thermally injured mice, these mice were shown to be susceptible to P. aeruginosa intradermal infection. These results indicate that sepsis stemming from P. aeruginosa burn-wound infection is accelerated by burn-induced Gr-1(+)CD11b(+) cells with abilities to suppress antimicrobial peptide production by epidermal keratinocytes.     

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.     

Experimental studies of the pathogenesis of Pseudomonas aeruginosa infection: evidence for the in-vivo production of a lethal toxin.

Evidence is presented that a lethal toxin is produced by P. aeruginosa growing in the burned skin of experimental mice. After injection of approximately 100 P. aeruginosa cells into the burned skin there was a rapid proliferation of organisms at the site of inoculation. When the organisms in the burned skin tissue reached a critical concentration, there was generalised toxaemia with subsequent mortality; the process was not reversible at this stage, even by reducing substantially the numbers of infecting organisms. However, when the reduction was accompanied by administration of rabbit serum prepared against filter-sterilised extracts of infected burned tissues, approximately 40% of the animals survived for at least 96 h. The data suggests that the antiserum afforded protection by inactivating a toxin produced by the organisms growing in the infected burned tissues rather than by further reducing the numbers of infecting organisms.     

Role of exotoxin A and elastase in the pathogenicity of Pseudomonas aeruginosa strain PAO experimental mouse burn infection

We examined the virulence of Pseudomonas aeruginosa strain PAO and xcp (extracellular proteins deficient) and xch (extracellular proteins hyperproducing) mutants derived from strain PAO in an experimental mouse burn infection model. The results showed that xcp mutants, which produced little or no extracellular elastase and exotoxin A, were as virulent as their corresponding xcp+ strains. The xch mutants produced more elastase and exotoxin A than the wild type strain, however, they had significantly lower virulence, probably due to reduced ability of these strains to take up iron. Treatment of mice with ferric ammonium citrate had no effect on the wild type strain but enhanced mortality in mice challenged with xch mutants. Neither elastase nor exotoxin A seem to play any role in burn infections with P. aeruginosa strain PAO. However, ability for iron uptake is an important virulence factor.     

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.     

Contribution of specific Pseudomonas aeruginosa virulence factors to pathogenesis of pneumonia in a neonatal mouse model of infection.

We sought to identify which Pseudomonas aeruginosa products are involved initiating respiratory tract infection. Defined mutants derived from strain PAO i.e., PAOR1 (lasR),PAO-pmm (algC) (an LPS mutant), and AK1152 (which is Fla- and lacks functional pili), were significantly less virulent than PAO1 in a BALBc/ByJ neonatal mouse model of infection as measured by their abilities to cause acute pneumonia, bacteremia, and death. All three mutants were also less adherent to epithelial cells in an in vitro binding assay. PAOR1 and AK1152 were less able to elicit epithelial production of interleukin-8 than PAO1. LasR was found to be required for the optimal expression of neuraminidase under conditions of increased osmolarity, as might be present in certain pathological conditions. PAO-exsA::omega,, which lacks exoenzyme S expression, was fully virulent, causing at least as much pathology as PAO1. The expression of several P. aeruginosa virulence factors appears to be required to establish pulmonary infection in the neonatal mouse.     

Experimental candidiasis after thermal injury.

The ability of Candida albicans to infect thermally injured mice was studied. Female mice were either left unburned or given a 20% total body surface area 2-s or 7-s scald burn. The wound or skin surface was then inoculated with a human burn wound isolate of C. albicans. At 4 h postburn, approximately 10(2) to 10(3) CFU/g of tissue could be recovered from the skin of burned and unburned animals. Unburned mice cleared the organisms from the skin by 72 h, whereas in 7-s-burned animals, the candida increased in numbers to approximately 10(7) CFU/g of tissue. The ability of the organisms to invade systemically after wound surface inoculation was examined in mice given either a 2-s or a 7-s scald burn. Each injury was histologically confirmed as a full-thickness (third degree) burn, with slightly deeper tissue damage observed with the 7-s burn. At each time period examined (1, 4, 7, and 10 days), there were significantly fewer organisms in the wounds of mice given the 2-s injury than in wounds of mice burned for 7 s (P less than 0.05). In 3 of 33 mice given a 7-s injury, organisms were recovered from the kidneys at the time of sacrifice, whereas no evidence of invasion into the kidneys was noted in mice given a 2-s thermal injury. This study demonstrated that thermal injury enhances the ability of C. albicans to infect mice and that the depth of burn appears to be an important factor in determining whether the organisms can invade the burn wound to cause systemic infection. This animal model should be valuable in elucidating the virulence factors of C. albicans that play a role in the pathogenesis of candidiasis after thermal injury.     

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.     

Mutations in the hemolytic-phospholipase C operon result in decreased virulence of Pseudomonas aeruginosa PAO1 grown under phosphate-limiting conditions.

The phospholipase C (PLC) operon of Pseudomonas aeruginosa consists of plcS, which encodes a heat-labile secreted hemolysin, and two in-phase, overlapping genes, plcR1 and plcR2, which may encode Pi-regulatory genes. A 2.8-kilobase-pair deletion mutation in this operon was constructed, and a tetracycline resistance (Tcr) cartridge replaced the deleted sequences. A deletion mutant of strain PAO1 was obtained through recombination between the flanking regions of the mutated cloned PLC operon and the homologous chromosomal regions. The deletion of the chromosomal PLC operon and its replacement by the Tcr cartridge was confirmed by Southern hybridization. The deletion strain, PLC SR, is nonhemolytic. However, it retains PLC activity when measured on a synthetic substrate. A second mutant strain, PLC R, contains a deletion in the plcR genes. This mutant is more hemolytic and produces more enzymatic activity than PAO1. The virulence of both of these mutants was compared with that of PAO1 in the mouse burn model of infection. When mice were infected with cultures grown in a high-Pi medium, there was a 10-fold increase in the 50% lethal dose of the mutants compared with PAO1. In contrast, when the inoculum originated from low-Pi cultures, there was a 200- to 10,000-fold increase in the 50% lethal dose of the mutants over PAO1.     

Contribution of toxin A and elastase to virulence of Pseudomonas aeruginosa in chronic lung infections of rats

A chronic pulmonary infection model in rats was employed to assess the role of individual Pseudomonas aeruginosa exoproducts in disease due to this organism. Groups of rats were inoculated transtracheally with agar beads in which were embedded approximately 10(4) colony-forming units of P. aerugijnosa PAO and the PAO derivatives PR1, T1, E64, and a mixture of T1 and E64 in equal numbers (10(4)). Eight animals from each group were sacrificed at 3, 9, and 30 days after challenge, and their lungs were examined for histopathological changes, bacterial numbers, and the presence of P. aeruginosa exoproducts. The Tox- mutant T1 and the PR1 mutant, which produces enzymatically inactive toxin A, were both found to be less virulent in the rat lung model than was the toxigenic parental strain PAO. Pathological changes seen in animals infected with these mutants were restricted to intra- and peribronchial inflammation, whereas the toxigenic parental strain caused parenchymal changes, including a dense mononuclear-cell infiltration in the alveolar spaces in addition to intra- and peribronchial inflammation. Additionally, mutant E64, which produces a temperature-sensitive elastase, was also found to be less virulent in the rat lung model than was the parental strain. These data demonstrate that both active toxin A and elastase are required for maximum virulence of P. aeruginosa in this model.     

Requirement of the Pseudomonas aeruginosa tonB gene for high-affinity iron acquisition and infection

To investigate the contribution of the TonB protein to high-affinity iron acquisition in Pseudomonas aeruginosa, we constructed tonB-inactivated mutants from strain PAO1 and its derivative deficient in producing the siderophores pyoverdin and pyochelin. The tonB mutants could not grow in a free-iron-restricted medium prepared by apotransferrin addition, even though the medium was supplemented with each purified siderophore or with a heme source (hemoglobin or hemin). The tonB inactivation was shown to make P. aeruginosa unable to acquire iron from the transferrin with either siderophore. Introduction of a plasmid carrying the intact tonB gene restored growth of the tonB mutant of PAO1 in the free-iron-restricted medium without any supplements and restored growth of the tonB mutant of the siderophore-deficient derivative in the medium supplemented with pyoverdin, pyochelin, hemoglobin, or hemin. In addition, animal experiments showed that, in contrast to PAO1, the tonB mutant of PAO1 could not grow in vivo, such as in the muscles and lungs of immunosuppressed mice, and could not kill any of the animals. The in vivo growth ability and lethal virulence were also restored by introduction of the tonB-carrying plasmid in the tonB mutant. These results indicate clearly that the intact tonB gene-and, therefore, the TonB protein encoded by it-is essential for iron acquisition mediated by pyoverdin and pyochelin and via heme uptake in P. aeruginosa and suggest that the TonB-dependent iron acquisition may be essential for P. aeruginosa to infect the animal host.     

Role of lipopolysaccharide in virulence of Pseudomonas aeruginosa

The role of lipopolysaccharide (LPS) in the virulence of Pseudomonas aeruginosa was studied. The virulence of several P. aeruginosa strains for burned mice was found to be directly related to the dispersion of LPS into either the phenol or the water phase after extraction. Virulence decreased as the proportion of LPS recovered from the phenol phase increased. No similar correlation was observed when several other strain characteristics were investigated. This phenomenon was studied in greater detail by using the "smooth"-specific phage E79 to select mutants altered in LPS structure. One such mutant, PA220-R2, was extensively characterized. LPS isolated from PA220-R2 was found to be completely deficient in high-molecular-weight polysaccharide material. This alteration rendered the strain serum sensitive and dramatically changed the reaction with O-specific typing sera and sensitivity to typing phages. However, motility, toxin A and elastase production, and 22 metabolic functions remained unchanged. PA220-R2 was found to be comparatively nonvirulent, with a 50% lethal dose more than 1,000-fold higher than that of its parent for burned mice. This was due to the inability of PA220-R2 to establish an infection in burned skin.