Comparisons of virulence between a Pseudomonas parent strain and an isogenic mutant devoid of protease IV have demonstrated a significant role for this enzyme during infection. We have characterized purified Pseudomonas aeruginosa protease IV in terms of its biochemical and enzymatic properties, and found it to be a unique extracellular protease. The N-terminal decapeptide sequence of protease IV is not homologous with any published protein sequence. Protease IV has a molecular mass of 26 kDa, an isoelectric point of 8.70, and optimum enzymatic activity at pH 10.0 and 45°C. Purified protease IV demonstrates activity for the carboxyl side of lysine-containing peptides and can digest a number of biologically important proteins, including immunoglobulin, complement components, fibrinogen, and plasminogen. Protease IV is not inhibited by thiol-, carboxyl-, or metalloproteinase inhibitors. The total loss of enzyme activity in the presence of N-p-tosyl-L-chloromethyl ketone and the partial inhibition of enzyme activity by diisopropyl fluorophosphate or phenylmethylsulfonyl fluoride imply that protease IV is a serine protease. Inhibition by dithiothreitol and ␤-mercaptoethanol suggests that intramolecular disulfide bonds are essential for enzyme activity. The characteristics of this enzyme suggest that inhibitors of serine proteases could be developed into a medication designed to arrest tissue damage during Pseudomonas infection.

No abstract

Pulmonary surfactant has two distinct functions within the lung: reduction of surface tension at the air-liquid interface and participation in innate host defense. Both functions are dependent on surfactant-associated proteins. Pseudomonas aeruginosa is primarily responsible for respiratory dysfunction and death in cystic fibrosis patients and is also a leading pathogen in nosocomial pneumonia. P. aeruginosa secretes a number of proteases that contribute to its virulence. We hypothesized that P. aeruginosa protease IV degrades surfactant proteins and results in a reduction in pulmonary surfactant host defense and biophysical functions. Protease IV was isolated from cultured supernatant of P. aeruginosa by gel chromatography. Incubation of cell-free bronchoalveolar lavage fluid with protease IV resulted in degradation of surfactant proteins (SP)-A, -D, and -B. SPs were degraded in a time- and dose-dependent fashion by protease IV, and degradation was inhibited by the trypsin-like serine protease inhibitor Nalpha-p-tosyl-L-lysine-chloromethyl ketone (TLCK). Degradation by protease IV inhibited SP-A- and SP-D-mediated bacterial aggregation and uptake by macrophages. Surfactant treated with protease IV was unable to reduce surface tension as effectively as untreated surfactant, and this effect was inhibited by TLCK. We speculate that protease IV may be an important contributing factor to the development and propagation of acute lung injury associated with P. aeruginosa via loss of surfactant function within the lung.

Staphylococcus aureus corneal infection results in extensive inflammation and tissue damage. Our previous studies of bacterial mutants have demonstrated a role for alpha-toxin in corneal virulence. This study analyzes, by genetic rescue experiments, the virulence of mutants affecting alpha-toxin and beta-toxin activity and demonstrates the ocular toxicity of these purified staphylococcal proteins. Three types of isogenic mutants were analyzed: (i) mutants specifically deficient in alpha-toxin (Hla) or beta-toxin (Hlb), (ii) a mutant deficient in both Hla and Hlb, and (iii) a regulatory mutant, deficient in the accessory gene regulator (agr), that produces reduced quantities of multiple exoproteins, including alpha-and beta-toxins. Plasmids coding for Hla and Hlb (pDU1212 and pCU1hlb, respectively) were used to restore toxin activity to mutants specifically deficient in each of these toxins. Either corneas were injected intrastromally with logarithmic-phase S. aureus or purified alpha-or beta-toxins were administered to normal eyes. Ocular pathology was evaluated by slit lamp examination and myeloperoxidase activity of infiltrating polymorphonuclear leukocytes. Corneal homogenates were cultured to determine the CFU per cornea. Eyes infected with the wild-type strain developed significantly greater corneal damage than eyes infected with Agr ؊ , Hlb ؊ , or Hla ؊ strains. Epithelial erosions produced by parent strains were not produced by Agr ؊ or Hla ؊ strains. Hlb ؉ strains, unlike Hlb ؊ strains, caused scleral edema. Plasmid pDU1212 restored corneal virulence to strain DU1090 (Hla ؊), and plasmid pCU1hlb restored corneal virulence to strain DU5719 (Hlb ؊). Application of purified alpha-toxin produced corneal epithelial erosions and iritis, while application of beta-toxin caused scleral inflammation. These studies confirm the role of alpha-toxin as a major virulence factor during S. aureus keratitis and implicate beta-toxin, a mediator of edema, as a lesser contributor to ocular damage.

Staphylococcus aureus is a major pathogen of the eye able to infect the tear duct, eyelid, conjunctiva, cornea, anterior and posterior chambers, and the vitreous chamber. Of these infections, those involving the cornea (keratitis) or the inner chambers of the eye (endophthalmitis) are the most threatening because of their potential to cause a loss in visual acuity or even blindness. Each of these ocular sites is protected by the constitutive expression of a variety of antimicrobial factors and these defenses are augmented by a protective host response to the organism. Such infections often involve a predisposing factor that weakens the defenses, such as the use of contact lenses prior to the development of bacterial keratitis or, for endophthalmitis, the trauma caused by cataract surgery or intravitreal injection. The structural carbohydrates of the bacterial surface induce an inflammatory response able to reduce the bacterial load, but contribute to the tissue damage. A variety of bacterial secreted proteins including alpha-toxin, beta-toxin, gamma-toxin, Panton-Valentine leukocidin and other two-component leukocidins mediate tissue damage and contribute to the induction of the inflammatory response. Quantitative animal models of keratitis and endophthalmitis have provided insights into the S. aureus virulence and host factors active in limiting such infections.

PASP is a protease that has not been previously identified. It causes corneal epithelial erosions, indicating its likely activity as a virulence-promoting factor in Pseudomonas keratitis.

Lysostaphin is effective in treating experimental endophthalmitis mediated by MRSA.

The present study demonstrates for the first time that protease IV is synthesized as a large precursor that is processed intracellularly through an intermediate form and secreted into the extracellular milieu as a mature protease. The results also confirm a significant correlation between production of protease IV and corneal virulence.