Role of elastase as a virulence factor in experimental Pseudomonas aeruginosa infection in mice.

The role of elastase and alkaline protease in the pathogenesis of fatal infections caused by Pseudomonas aeruginosa was determined in mice treated with calcium chloride. Mortality increased significantly when solutions containing elastase were injected together with non-lethal inocula of strain PA 103, which does not produce proteolytic enzyme. In contrast, solutions containing alkaline protease did not increase mortality. In mice injected intramuscularly with strain PA 103 and calcium chloride, the organisms grew rapidly in the injected muscle but not in the liver. However, when elastase was injected together with strain PA 103 and calcium chloride, viable bacteria were also found in the liver. Moreover, the survival rate of mice challenged with elastase-producing strain 5 and calcium chloride was enhanced, and colonization of the liver prevented, by immunization with elastase toxoid. These results suggest that elastase contributes to the invasiveness of the organism.     

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.     

Calcium and magnesium enhance the production of Pseudomonas aeruginosa protease IV, a corneal virulence factor

The effect of calcium and magnesium on protease IV production during the growth of Pseudomonas aeruginosa was investigated. Strain PA103 was grown to stationary phase in medium containing various concentrations of either calcium or magnesium. Culture supernatants were concentrated, standardized relative to cell density, and the pyoverdine concentrations were measured. Overall extracellular protease activity and specific protease IV (lysine endoproteinase) activity were measured with or without TLCK, a serine protease inhibitor effective against protease IV activity. Protease IV activity was also observed by casein zymography. Calcium and magnesium were quantified in the corneas and aqueous humor of rabbits that were inoculated intrastromally with strain PA103. Pyoverdine production was not significantly different in cultures grown in medium with added calcium or magnesium, but extracellular caseinase activity increased in these cultures. Susceptibility of caseinase activity to TLCK inhibition and a specific assay for protease IV indicated that protease IV activity increased in cultures grown in calcium or magnesium. Casein zymography supported the observation that protease IV activity increased in the cultures with added calcium and magnesium. Addition of calcium or magnesium to the protease IV-specific assay had no effect on the catalytic activity of pure protease IV. Infection of rabbit corneas with PA103 did not change the magnesium concentration in either corneas or aqueous humor, but significantly increased the concentration of calcium in corneas. These results indicate that calcium and magnesium enhance the production of protease IV, but not pyoverdine production. Calcium increases in the cornea following infection with P. aeruginosa could favor production of protease IV.     

Passive protection against Pseudomonas aeruginosa infection in an experimental leukopenic mouse model.

An experimental leukopenic mouse model was used to evaluate the protective capacities of immunoglobulin G (IgG) fractions directed against toxin A (AT-IgG), elastase (AE-IgG), and lipopolysaccharide (ALPS-IgG) against fatal Pseudomonas aeruginosa infection. Statistically significant protection, as measured by long-term survival, was observed only when mice were treated with serotype-specific ALPS-IgG. The mean lethal dose for P. aeruginosa could be increased by as much as 6,600-fold for mice given ALPS-IgG as compared to mice which received only normal rabbit IgG. ALPS-IgG afforded high levels of protection, even when administered up to 6 h postchallenge. Experiments designed to monitor the growth and spread of a locally administered challenge showed that ALPS-IgG prevented bacteremia and organ colonization, which were pronounced in control animals. The effectiveness of combined antibiotic and immune therapy was tested. Gentamicin alone or in combination with AT-IgG or AE-IgG provided no detectable protection. However, its use with ALPS-IgG afforded substantially higher levels of protection than ALPS-IgG alone.     

Production of protease and elastase by Pseudomonas aeruginosa strains isolated from patients.

Using 20 strains of Pseudomonas aeruginosa isolated from patients, production of protease, elastase, and collagenase was determined by shaking culture in either complex or semisynthetic medium. No collagenase was produced by any strain of P. aeruginosa. According to their production of protease and elastase in different media, the P. aeruginosa strains were divided into three groups: the first group can produce elastase in complex medium and both protease and elastase in semisynthetic medium; the second group cannot produce any proteolytic enzymes in complex medium but can produce any proteolytic enzymes in either medium; and the third group cannot produce any proteolytic enzymes in either medium. In spite of the differences in the ability of the strains to produce the enzymes, depending upon the origin of the strains, the protease or elastases produced in different broths were regarded as identical.     

Age-dependent changes in protease and elastase production in cultures of Pseudomonas aeruginosa

The intracellular and extracellular protease and elastase contents of Pseudomonas aeruginosa were studied in relation to the age of the culture. The intracellular protease and elastase content of the organisms decreased as the culture grew older, whereas extracellular protease and elastase greatly increased 24 h after incubation commenced, suggesting that host tissue injury by P. aeruginosa infection may increase in proportion to the duration of tissue colonisation.     

Passive protection by antitoxin in experimental Pseudomonas aeruginosa burn infections.

The protective effect of intravenously administered rabbit antitoxin serum was studied in lethal Pseudomonas aeruginosa burn infections in mice. Survival after infection with 2 median lethal doses of a toxigenic, low-protease-producing strain (PA103) was enhanced in antitoxin-treated mice, as compared with controls that had received anti-bovine serum albumin serum (P = 0.0004). Survival time was prolonged in other antitoxin-treated mice infected with toxigenic, high-protease-producing strains (PA86 and PA220, P = 0.0003 and P = 0.01, respectively). In contrast, antitoxin had no protective effect in mice challenged with a nontoxigenic strain (WR 5, P = 0.57). There were fewer viable bacteria in blood and liver of antitoxin-treated mice than in those of anti-bovine serum albumin-treated controls after infection with toxigenic organisms, whereas there were no significant differences between the two groups after challenge with the nontoxigenic strain. These data suggest that P. aeruginosa exotoxin A contributes to lethality in this burn infection model, and this effect is diminished by passive immunization with antitoxin.     

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.     

Comparison of the Vibrio cholerae hemagglutinin/protease and the Pseudomonas aeruginosa elastase.

The soluble hemagglutinin/protease (HA/protease) produced by Vibrio cholerae and the elastase of Pseudomonas aeruginosa are both zinc/calcium-dependent proteases. In the present study the two enzymes are compared immunologically and functionally. The N-terminal amino acid sequences of the proteins had 65% identity within the first 20 amino acids. Polyclonal antisera against each purified protein recognized the enzyme of the other species in enzyme-linked immunosorbent assay, checkerboard immunoblot, and Western blot analyses and inhibited the protease activity of both enzymes in milk and elastin agars. Like the HA/protease, the elastase hemagglutinated "responder" but not "nonresponder" chicken erythrocytes, degraded ovomucin, lactoferrin, and fibronectin, and nicked the A subunit of the cholera toxin-related heat-labile enterotoxin from Escherichia coli. Whereas none of the three proteases tested (elastase, HA/protease, or pronase E) had any obvious effect in ileal loop tests in rabbits at doses up to 50 micrograms, all three produced some detectable skin reactions at a dose of 0.1 micrograms and necrosis at a higher dose (i.e., 5 micrograms). We conclude that the V. cholerae HA/protease and the P. aeruginosa elastase are structurally, functionally, and immunologically related.     

Interaction of Pseudomonas aeruginosa alkaline protease and elastase with human polymorphonuclear leukocytes in vitro.

Little is known about the interaction of Pseudomonas aeruginosa extracellular products and human polymorphonuclear leukocytes. The present study was designed to examine the effect of alkaline protease and elastase purified from P. aeruginosa on human neutrophil function. Neutrophil chemotaxis, oxygen consumption, glucose oxidation, superoxide production, and nitro blue tetrazolium reduction were studied. It was found that alkaline protease and elastase at fairly low concentrations (0.05 and 0.0025 micrograms/ml, respectively) inhibited chemotaxis. The inhibitory effect of both enzymes was increased at higher concentrations. The chemotaxis of preincubated and washed cells was also inhibited. Alkaline protease but not elastase inhibited opsonized zymosan-stimulated neutrophil oxygen consumption, whereas neither of the enzymes had any effect on glucose oxidation and nitro blue tetrazolium-reducing activity of stimulated neutrophils. The data on superoxide production ability of the cells indicated that the cells preincubated with enzyme and washed were capable of producing superoxide equal to the amount produced by untreated cells when they were stimulated with phorbol myristate acetate or zymosan. However, when elastase was present in the reaction mixture, the reduction of cytochrome c as a measure of superoxide production was inhibited. Inhibition of neutrophil function, particularly chemotaxis, will have important bearing on the escape of the microorganism from the phagocytic defense system of the host. The role of these products in localized infections and avascular areas such as skin burns, cornea, and, at least initially, in chronic lung colonization in cystic fibrosis patients becomes important.     

Effect of experimental Pseudomonas aeruginosa infection on the activity of mouse phagocytes

It has been stated that experimental infection with 0.01 LD50 Pseudomonas aeruginosa 74 decreases the phagocytic activity of mouse neutrophiles and peritoneal macrophages in relation to latex particles. In the course of infection suppression of phagocytic activity of peritoneal macrophages was delayed for two days as compared with that of neutrophiles. During the dying out of infection phagocytic activity of neutrophils and macrophages returned to the control values. Possible mechanisms of these events were discussed.     

Pseudomonas elastase acts as a virulence factor in burned hosts by Hageman factor-dependent activation of the host kinin cascade.

Purified Pseudomonas elastase injected subcutaneously into the skin of an Evans blue dye-injected (intravenously) guinea pig caused dye leakage similar to that observed when histamine or bradykinin was injected in the same animal. The histamine-induced dye leakage was ablated in antihistamine-treated guinea pigs, but elastase- and bradykinin-induced dye leakages were not. Local injections of specific inhibitors of the host Hageman factor-dependent bradykinin-generating pathway given immediately prior to elastase injection reduced dye leakage in a dose-related manner. Elastase-related dye release was enhanced when angiotension-converting enzyme inhibitor, a substance which prevents host enzymes from breaking down bradykinin, was injected prior to elastase injection. We conclude that Pseudomonas elastase generates bradykinin in the infected host via a Hageman factor-dependent pathway.     

Inhibition of human natural killer cell activity by Pseudomonas aeruginosa alkaline protease and elastase.

The present study was designed to examine the effect of Pseudomonas aeruginosa alkaline protease (AP) and elastase (Ela) on human natural killer (NK) cell activity in vitro. AP and Ela were found to inhibit NK cell function. Addition of alpha interferon and interleukin-2 did not abolish this inhibition of NK cell activity. Adhesion of effector to target cells was studied in a single-cell agarose assay of monocyte-depleted NK-cell-enriched cell populations. AP and Ela were shown to inhibit effector/target cell conjugate formation. Furthermore, AP and Ela inhibited the binding of the monoclonal antibody Leu-11, which reacts with the Fc receptor of NK cells. The inhibition of NK cell binding to the target cell by P. aeruginosa proteases is most likely due to proteolytic cleavage of the surface receptors involved in the binding of the effector cell to the target cell.     

Evaluation of Pseudomonas aeruginosa toxin A in experimental rat burn wound sepsis.

The search for methods to achieve control of Pseudomonas aeruginosa infection continues with the introduction of aluminum-absorbed toxoid developed from P. aeruginosa exotoxin. This toxoid induces significant titers of neutralizing and precipitating antibodies for toxin A when given with appropriate adjuvants. These experiments show that immunization with aluminum phosphate-absorbed toxoid failed to protect burned rats infected with P. aeruginosa. These and previous experiments show that active immunization with live P. aeruginosa provides good strain-specific protection in the same model. No cross-protection was demonstrated between strains of P. aeruginosa in these experiments.     

CLearance of Pseudomonas aeruginosa from the lungs in experimental burn trauma

Experiments carried out in rats demonstrated that a burn of degree III of 20% of the body surface disturbs considerably the process of lung clearance from bacterial dissemination (5 million bodies of Pseudomonas pyocyanea). Not only decreased but even negative clearance may be observed indicating multiplication of the bacteria in pulmonary tissues for the first 3 days after burn, and intratracheal infection of the animals. Reduced general antibacterial resistance as a result of burn facilitates rapid penetration of P. pyocyanea into the lymph nodes, blood, kidneys, liver, spleen where it is found 6 hours after infection.     

Urease as a virulence factor in experimental cryptococcosis

Urease catalyzes the hydrolysis of urea to ammonia and carbamate and has been found to be an important pathogenic factor for certain bacteria. Cryptococcus neoformans is a significant human pathogenic fungus that produces large amounts of urease; thus we wanted to investigate the importance of urease in the pathogenesis of cryptococcosis. We cloned and sequenced the genomic locus containing the single-copy C. neoformans urease gene (URE1) and used this to disrupt the native URE1 in the serotype A strain H99. The ure1 mutant strains were found to have in vitro growth characteristics, phenoloxidase activity, and capsule size similar to those of the wild type. Comparison of a ure1 mutant with H99 after intracisternal inoculation into corticosteroid-treated rabbits revealed no significant differences in colony counts recovered from the cerebrospinal fluid. However, when these two strains were compared in both the murine intravenous and inhalational infection models, there were significant differences in survival. Mice infected with a ure1 strain lived longer than mice infected with H99 in both models. The ure1 strain was restored to urease positivity by complementation with URE1, and two resulting transformants were significantly more pathogenic than the ure1 strain. Our results suggest that urease activity is involved in the pathogenesis of cryptococcosis but that the importance may be species and/or infection site specific.     

Pseudomonas aeruginosa invades corneal epithelial cells during experimental infection.

Pseudomonas aeruginosa is considered an extracellular pathogen. Using assays to determine intracellular survival in the presence of gentamicin, we have demonstrated that some strains of P. aeruginosa are able to invade corneal cells during experimental bacterial keratitis in mice. Although intracellular bacteria were detectable 15 min after inoculation, the number of intracellular bacteria increased in a time-dependent manner over a 24-h period. Levels of invasion were similar when bacteria were grown as a biofilm on solid medium and when they were grown in suspension. Intracellular bacteria survived in vitro for at least 24 h, although only minimal bacterial multiplication within cells was observed. P. aeruginosa PAK and Escherichia coli HB101 did not cause disease in this model and were not isolated from corneas after 24 h even when an inoculum of 10(8) CFU was applied. Transmission electron microscopy of corneal epithelium from eyes infected for 8 h revealed that intracellular bacteria were present within membrane-bound vacuoles, which suggests that bacterial entry was an endocytic process. At 24 h, the observation of many bacteria free in the cytoplasm indicated that P. aeruginosa was able to escape the endocytic vacuole. The ability of some P. aeruginosa strains to invade corneal epithelial cells may contribute to the pathogenesis or to the progression of disease, since intracellular bacteria can evade host immune effectors and antibiotics commonly used to treat infection.