Cloning and sequencing of a Candida albicans catalase gene and effects of disruption of this gene.

Catalase plays a key role as an antioxidant, protecting aerobic organisms from the toxic effects of hydrogen peroxide, and in some cases has been postulated to be a virulence factor. To help elucidate the function of catalase in Candida albicans, a single C. albicans-derived catalase gene, designated CAT1, was isolated and cloned. Degenerate PCR primers based on highly conserved areas of other fungal catalase genes were used to amplify a 411-bp product from genomic DNA of C. albicans ATCC 10261. By using this product as a probe, catalase clones were isolated from genomic libraries of C. albicans. Nucleotide sequence analysis revealed an open reading frame encoding a protein of 487 amino acid residues. Construction of a CAT1-deficient mutant was achieved by using the Ura-blaster technique for sequential disruption of multiple alleles by integrative transformation using URA3 as a selectable marker. Resulting mutants exhibited normal morphology and comparable growth rates of both yeast and mycelial forms. Enzymatic analysis revealed an abundance of catalase in the wild-type strain but decreasing catalase activity in heterozygous mutants and no detectable catalase in a homozygous null mutant. In vitro assays showed the mutant strains to be more sensitive to damage by both neutrophils and concentrations of exogenous peroxide that were sublethal for the parental strain. Compared to the parental strain, the homozygous null mutant strain was far less virulent for mice in an intravenous infection model of disseminated candidiasis. Definitive linkage of CAT1 with virulence would require restoration of activity by reintroduction of the gene into mutants. However, initial results in mice, taken together with the enhanced susceptibility of catalase-deficient hyphae to damage by human neutrophils, suggest that catalase may enhance the pathogenicity of C. albicans.

Human platelets damage Aspergillus fumigatus hyphae and may supplement killing by neutrophils.

Neutropenia is considered a significant risk factor for invasive aspergillosis but is almost always associated with concurrent thrombocytopenia. Studies determined that platelets, like neutrophils, attached to cell walls of the invasive hyphal form of Aspergillus fumigatus. Organisms were damaged as shown by loss of cell wall integrity in scanning laser confocal microscopy and release of defined hyphal surface glycoproteins. Rapid expression appearance of surface antigen CD63 and release of markers of platelet degranulation confirmed activation during attachment to hyphae. Optimal platelet activation required opsonization of hyphae with fresh or heat-inactivated whole plasma. These effects of opsonization with whole plasma could not be duplicated by pooled human serum, immunoglobulin G, or fibrinogen, whether used separately or combined. Thus, platelets in the presence of whole plasma have the potential to play an important role in normal host defenses against invasive aspergillosis.

Mechanisms and target sites of damage in killing of Candida albicans hyphae by human polymorphonuclear neutrophils.

Target sites of fungal cell damage were studied to define mechanisms of neutrophil-mediated killing of Candida albicans hyphae. Neutrophils induced hyphal cell wall damage, as evidenced by release of cell wall glycoproteins and confocal microscopic changes. Damage occurred in the presence of neutrophil granule extracts and did not require oxidants. However, oxidation of hyphal surface glycoproteins correlated strongly with parallel increments in fungicidal activity, suggesting that oxidants did contribute to maximal cell wall damage. Neutrophil oxidants also induced hyphal DNA fragmentation, primarily single-strand breakage, as shown by increased electrophoretic migration after nuclease-S1 DNA digestion at single-strand break sites. The onset of damage to hyphal cell walls and DNA preceded detectable neutrophil-mediated fungicidal effects. Likewise, hyphal amino acid and nucleotide turnover as well as ATP initially rose, then declined as lethal effects became detectable. Thus, preceding detectable fungal cell death, neutrophil oxidative and oxygen-independent mechanisms damaged defined targets.

Disparate effects of interferon-gamma and tumor necrosis factor-alpha on early neutrophil respiratory burst and fungicidal responses to Candida albicans hyphae in vitro.

We examined effects of priming with recombinant human interferon-gamma (IFN) or tumor necrosis factor-alpha (TNF) on neutrophil responses to Candida albicans hyphae. Both cytokines increased early superoxide generation after hyphal stimulation. The more pronounced effects of TNF were accompanied by an augmented surface membrane depolarization rate and were insensitive to both pertussis toxin and calcium ion chelation, but were negated by concomitant incubation with puromycin or cycloheximide during priming. IFN augmented hyphal killing despite its only minor enhancement of early respiratory burst responses, but TNF reduced neutrophil fungicidal activity to nearly 40% below those by unprimed control cells even though it enhanced early superoxide responses more dramatically. Though TNF-primed neutrophils killed hyphae at normal initial rates, IFN-primed or even unprimed cells manifested more fungicidal sustained activity. These disparate consequences of cytokine priming on hyphal destruction were paralleled by differences in late generation of potentially candidacidal oxidants, hydrogen peroxide, and hypochlorous acid. IFN added during priming failed to correct TNF-associated functional defects in neutrophil anti-Candida responses. Thus, augmentation of early respiratory burst responses to oxidant-sensitive organisms need not necessarily reflect concomitant salutary effects on microbicidal activity.

Independence of neutrophil respiratory burst oxidant generation from the early cytosolic calcium response after stimulation with unopsonized Candida albicans hyphae.

We previously noted differences between neutrophil responses to unopsonized Candida albicans hyphae and responses to other particulate stimuli such as opsonized hyphae or zymosan; these differences include delayed rises in cytosolic calcium [( Ca2+]i), 1,4,5-inositol trisphosphate, and superoxide release and the total absence of early membrane depolarization. Respiratory burst stimulation is required for killing of C. albicans hyphae. Since an early rise in [Ca2+]i may act as a second messenger for burst activation by most agonists, we chelated (Ca2+)i and extracellular Ca2+ [( Ca2+)e] to compare requirements for superoxide responses to hyphae and other stimuli. Intracellular chelation, which ablated early [Ca2+]i rises, eliminated the fMet-Leu-Phe-induced respiratory burst and profoundly reduced that response to opsonized zymosan (by 96.7%), but chelation of both (Ca2+)i and (Ca2+)e only partially inhibited responses to opsonized and unopsonized hyphae (60.5 and 23.3%, respectively; the latter exceeded absolute responses evoked by opsonized zymosan, a 12-fold-more-potent stimulus for unchelated cells). Simultaneous (Ca2+)i and (Ca2+)e chelation further decreased superoxide responses to opsonized zymosan and hyphae (99.4 and 90.4%, respectively) but not to unopsonized hyphae (26.7% inhibition). Though both ingestible (zymosan) and uningestible (hyphae) opsonized particulate stimuli elicited reduced but significant respiratory bursts without early [Ca2+]i rises, the greater superoxide responses and sensitivity to chelation with opsonized zymosan suggest important differences in initiation and/or regulation of responses to these particulate stimuli. In contrast, the respiratory burst elicited by unopsonized hyphae appeared largely Ca2+ independent. If different events mediate neutrophil activation by opsonized and unopsonized hyphae, candidacidal activity in vivo may vary under divergent conditions with specific localized sites of infection.

Temporal association of calcium mobilization, inositol trisphosphate generation, and superoxide anion release by human neutrophils activated by serum opsonized and nonopsonized particulate stimuli.

We have investigated the involvement of phospholipase C mediated polyphosphoinositide turnover in activation of polymorphonuclear leukocytes by particulate stimuli. Results showed that stimulation of leukocytes with serum opsonized zymosan (ingestible particle), serum opsonized Candida albicans hyphae (noningestible particle), or nonopsonized hyphae was followed by a transient rise in cellular inositol phosphates as has been described for neutrophil activation via the formyl peptide receptor. The kinetics of inositol trisphosphate generation paralleled both the time course of changes in cytosolic calcium concentration and the onset of superoxide anion generation, suggesting that these may be related events.

Electrophoretic and immunoblot analyses of Rhizopus arrhizus antigens.

Four antigen preparations from Rhizopus arrhizus were made and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and column chromatography. Electrophoretic analyses of these antigens indicated that there are 18 to 28 component bands with a molecular mass range of approximately 10,500 to 83,000 daltons. Seven of these bands appear to be components common to three antigen preparations. Several of the bands identified by SDS-PAGE were composed of glycoproteins or carbohydrates as determined by their affinity for concanavalin A. Western blots, using sera from five patients with mucormycosis, consistently identified five different determinants in the R. arrhizus antigens separated by SDS-PAGE. This suggests that several of the Rhizopus antigens are present during mucormycosis. Four of the antigenic determinants recognized by patient sera reacted with the concanavalin A-peroxidase stain, indicating that they are composed of glycoproteins or carbohydrate. Enzyme-linked immunosorbent assays of sera from five patients with mucormycosis and with rabbit antisera resulted in antibody titers ranging from 1:64 to 1:32,000 for the R. arrhizus antigens.