Elastase, a potential virulence factor from the opportunistic pathogen Aspergillus fumigatus, was purified 220-fold from culture broth by fast-performance liquid chromatography employing anion exchange (Q Sepharose fast flow), cation exchange (S Sepharose fast flow), and gel filtration (Superose 12). Purified to near homogeneity, the elastase had an apparent molecular mass of 32 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (silver stain) but a mass of about 19.1 kDa as determined by gel filtration on Superdex 75. The elastase is not glycosylated and is positively charged at neutral pH, having a pI of 8.75. Inhibition by 0.2 mM phenylmethylsulfonyl fluoride (100%) and 0.21 mM leupeptin (60%) implies that the elastase is a serine protease. However, the enzyme is also inhibited by 5 mM EDTA (100%) and 10 mM 1,10-orthophenanthroline (30%), suggesting a requirement for divalent cations. The enzyme acts optimally at pH 7.4 and 45°C in 50 mM sodium borate buffer, but in Tris HCI, the pH optimum shifts to 8.8. While strains of Aspergillus species can cause allergic reactions upon inhalation of high spore concentrations, proliferation of the fungus is generally not a problem in individuals with normal immune status. Invasive aspergillosis occurs in individuals predisposed to infection through diseases such as acute leukemia and diabetes mellitus and in patients undergoing cancer chemotherapy or immunosuppressive therapy following organ transplants. Several Aspergillus species, including Aspergillus flavus, A. niger, A. glaucus, andA. terreus, can cause invasive aspergillosis, but A. fumigatus is the predominant causative organism (2). Elastases have been implicated in the pathogenesis of bacteria, fungi, and schistosomes (9, 13, 14, 19). In 1984, we showed that environmental strains of A. fumigatus were capable of producing an extracellular elastase (8). Production of this enzyme was correlated with the ability to cause invasive aspergillosis in immunocompromised mice. In 1988, Rhodes et al. (21) examined 38 clinical specimens and reported that all isolates of Aspergillus causing invasive aspergillosis produced elastase. The elastase from an isolate of A. flavus was purified and characterized (20). However, there is no direct evidence in these reports that the fungal elastase is a virulence factor actually required for invasion of Aspergillus spp. into lung tissue. By determining whether murine antibodies to this enzyme can ameliorate invasive aspergillosis in immunocompromised mice, our research aims to determine if the elastase plays a direct role in pathogenesis. The present report deals with the purification and characterization of the A. fiumigatus elastase used in preparation of monoclonal and polyclonal antibodies (6) for this study. MATERIALS AND METHODS Screening method for elastase production. The strains ofA. fiumigatus described in 1984 (8), which were isolated from air samples collected near a sewage sludge-composting facility * Corresponding author. t New Jersey Agricultural Experiment St...

The postantibiotic subminimum inhibitory concentration effect (PA SME) may simulate in vivo drug exposure more accurately than the postantibiotic effect (PAE) since subinhibitory concentrations of drug persist between antibiotic dosings. In this study, we compared the PAEs and PA SMEs of levofloxacin and ciprofloxacin for clinical isolates of fluoroquinolone-susceptible Staphylococcus aureus and Streptococcus pneumoniae. At two times the MIC, PAEs of levofloxacin were an average of 0.6 h longer than the PAEs obtained for ciprofloxacin for methicillin-susceptible and methicillin-resistant S. aureus strains. The PAEs of levofloxacin and ciprofloxacin ranged from 1.8 to 3.1 and 1.1 to 2.4 h, respectively. Continued exposure of the methicillin-resistant strain to 1/16, 1/8, and 1/4 the MIC resulted in PA SMEs of 6.5, 15.3, and >22.3 h, respectively, for levofloxacin and 3.8, 8.0, and 12.3 h, respectively, for ciprofloxacin. For isolates of S. pneumoniae, at two times the MIC of both fluoroquinolones, the average PAEs of levofloxacin and ciprofloxacin were equivalent: 1.3 h for the penicillin-susceptible isolate and 0.6 h for the penicillin-resistant isolate. Continued exposure of the penicillin-susceptible S. pneumoniae strain to 1/16, 1/8, and 1/4 the MIC resulted in average PA SMEs of 1.0, 1.4, and 2.8 h, respectively, for levofloxacin and 1.8, 2.0, and 2.5 h, respectively, for ciprofloxacin. Continued exposure of penicillin-resistant S. pneumoniae to 1/16, 1/8, and 1/4 the MIC of the same fluoroquinolones resulted in average PA SMEs of 0.6, 1.1, and 2.9 h, respectively, for levofloxacin and 0.6, 1.1, and 1.5 h, respectively, for ciprofloxacin. The PA SMEs observed demonstrate the superior activity of levofloxacin against methicillin-susceptible or methicillin-resistant S. aureus. Although PAEs were similar for the penicillin-susceptible and penicillin-resistant S. pneumoniae strains, the PA SME of levofloxacin at one-fourth the MIC was longer for penicillin-resistant S. pneumoniae.

The discovery of novel classes of antifungal drugs depends to a certain extent on the identification of new, unexplored targets that are essential for growth of fungal pathogens. Likewise, the broad-spectrum capacity of future antifungals requires the target gene(s) to be conserved among key fungal pathogens. Using a genome comparison (or concordance) tool, we identified 240 conserved genes as candidates for potential antifungal targets in 10 fungal genomes. To facilitate the identification of essential genes in Candida albicans, we developed a repressible C. albicans MET3 (CaMET3) promoter system capable of evaluating gene essentiality on a genome-wide scale. The CaMET3 promoter was found to be highly amenable to controlled gene expression, a prerequisite for use in target-based whole-cell screening. When the expression of the known antifungal target C. albicans ERG1 was reduced via down-regulation of the CaMET3 promoter, the CaERG1 conditional mutant strain became hypersensitive, specifically to its inhibitor, terbinafine. Furthermore, parallel screening against a small compound library using the CaERG1 conditional mutant under normal and repressed conditions uncovered several hypersensitive compound hits. This work therefore demonstrates a streamlined process for proceeding from selection and validation of candidate antifungal targets to screening for specific inhibitors.

Elastase has been implicated as a potential virulence factor involved in the invasion process of the opportunistic pathogen, Aspergillus fumigatus. Monoclonal and polyclonal antibodies, known to inhibit elastase in vitro, were employed in an immunocompromised mouse model of invasive aspergillosis to determine if the antibodies could protect mice from fatal infection. Individual monoclonal antibodies, known to inhibit elastase partially (13 to 23%), or combinations of monoclonal antibodies, known to inhibit elastase 70 to 100%, were tested in the mouse model. No individual nor combination of monoclonal antibodies protected immunosuppressed, infected mice in the doses tested. Similarly, elastase-specific polyclonal antibodies, raised in mice or rabbits, did not exhibit a protective effect, nor did immunization of mice with elastase prior to immunosuppression and infection. Histological examination of the lungs of immunosuppressed, infected mice showed no amelioration of fungal invasiveness by treatment with elastase-specific monoclonal or polyclonal antibodies. However, immunocompetent mice, instilled with a spore inoculum much higher than used in the preceding studies and treated with antibodies, survived, while control mice not treated with antibodies were overwhelmed by the massive spore dose and died. Nevertheless, overall evidence suggests that elastase may not be the primary virulence factor involved in invasive pulmonary aspergillosis.

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