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.

Vitamin E supplementation and in vivo immune response in healthy elderly subjects. A randomized controlled trial.

OBJECTIVE: To determine whether long-term supplementation with vitamin E enhances in vivo, clinically relevant measures of cell-mediated immunity in healthy elderly subjects. DESIGN: Randomized, double-blind, placebo-controlled intervention study. SETTING AND PARTICIPANTS: A total of 88 free-living, healthy subjects at least 65 years of age. INTERVENTION: Subjects were randomly assigned to a placebo group or to groups consuming 60, 200, or 800 mg/d of vitamin E for 235 days. MAIN OUTCOME MEASURES: Delayed-type hypersensitivity skin response (DTH); antibody response to hepatitis B, tetanus and diphtheria, and pneumococcal vaccines; and autoantibodies to DNA and thyroglobulin were assessed before and after supplementation. RESULTS: Supplementation with vitamin E for 4 months improved certain clinically relevant indexes of cell-mediated immunity in healthy elderly. Subjects consuming 200 mg/d of vitamin E had a 65% increase in DTH and a 6-fold increase in antibody titer to hepatitis B compared with placebo (17% and 3-fold, respectively), 60-mg/d (41% and 3-fold, respectively), and 800-mg/d (49% and 2.5-fold, respectively) groups. The 200-mg/d group also had a significant increase in antibody titer to tetanus vaccine. Subjects in the upper tertile of serum alpha-tocopherol (vitamin E) concentration (>48.4 micromol/L [2.08 mg/dL]) after supplementation had higher antibody response to hepatitis B and DTH. Vitamin E supplementation had no effect on antibody titer to diphtheria and did not affect immunoglobulin levels or levels of T and B cells. No significant effect of vitamin E supplementation on autoantibody levels was observed. CONCLUSIONS: Our results indicate that a level of vitamin E greater than currently recommended enhances certain clinically relevant in vivo indexes of T-cell-mediated function in healthy elderly persons. No adverse effects were observed with vitamin E supplementation.

A simplified new assay for assessment of fungal cell damage with the tetrazolium dye, (2,3)-bis-(2-methoxy-4-nitro-5-sulphenyl)-(2H)-tetrazolium-5-carboxanil ide (XTT).

Studies of antimycotic host defenses have been limited by the paucity of rapid, reproducible quantitative assays for fungal cell damage. Prior studies defined a colorimetric method that uses MTT, a tetrazolium dye, to quantify polymorphonuclear leukocyte (PMNL)-mediated damage to fungi. These relatively simple, rapid, and reproducible assays require cumbersome extraction of precipitated MTT-formazan and high cell densities to overcome relatively low sensitivity. In experiments that compared assays with MTT and another tetrazolium dye, 2,3-bis-(2-methoxy-4-nitro-5-sulphenyl)-(2H)-tetrazolium-5-+ ++carboxanilide (XTT), estimates of damage to Aspergillus fumigatus hyphae by human PMNL were similar with both dyes. However, XTT reduction was more rapid and sensitive, yielding accurate results with fewer organisms and PMNL. The water-soluble XTT-formazan product also simplified measurements by eliminating the need for solvent-extraction steps that are obligatory in MTT assays. Thus, XTT is advantageous for quantitative assessment of fungal cell damage, although MTT remains useful for assessing fungal cell viability by direct microscopic visualization of precipitated formazan.

Cytoplasts generate oxidants but require added neutrophil granule constituents for fungicidal activity against Candida albicans hyphae.

Killing of Candida albicans hyphae requires oxidant generation by neutrophils (PMNL), but it is uncertain whether hyphal killing is mediated by PMNL oxidants alone or requires contributions by granule constituents. This was assessed using U-cytoplasts (U-CYT), anucleate PMNL fragments depleted of cytoplasmic granules but retaining motility and respiratory burst activity. Granule-depleted U-CYT killed Staphylococcus aureus, but hyphae remained viable despite targeted generation of putatively fungicidal oxidants by avidly adherent U-CYT. Hyphal killing occurred by combining U-CYT with sublethal concentrations of purified PMNL granule extracts approximating those present in equivalent numbers of intact PMNL. Contributions of granule constituents were not entirely attributable to purified granule constituents with known antimicrobial activity (lactoferrin, cathepsin G, myeloperoxidase, and human neutrophil peptide defensins 1-3) individually or combined. Thus, oxidant generation by intact PMNL may be necessary but not always sufficient to mediate hyphal killing without complementary nonoxidative mechanisms.

Relationship of phospholipase C- and phospholipase D-mediated phospholipid remodeling pathways to respiratory burst activation in human neutrophils stimulated by Candida albicans hyphae.

Neutrophil (PMN) oxidant release, a key component of defenses against disseminated candidiasis, was preceded by oxidant generation after stimulation by Candida albicans hyphae. Opsonized or unopsonized hyphae triggered phospholipase D (PLD) activation within 5 or 30 s, respectively, forming 1-O-alkyl-phosphatidic acid (alkyl-PA) or 1-O-alkyl-phosphatidyl-ethanol in the presence of ethanol. Ethanol, which competitively lowers phosphatidic acid (PA) production, caused dose-dependent inhibition of superoxide (O2-) generation after hyphal stimulation but altered neither baseline-unstimulated O2- production nor responses to phorbol myristate acetate. PA rises evoked by unopsonized hyphae began 2 min before significant O2- release, also preceding both phospholipase C activation and cytosolic Ca2+ rises. Diacylglycerol (DAG) rose in two distinct phases after stimulation by opsonized or unopsonized hyphae, peaking briefly after 60 or 120 s, respectively, followed by prolonged secondary rises. Initial DAG rises preceded inositol triphosphate elevations evoked by unopsonized hyphae. Though PA rose before DAG, no dephosphorylation of PA to form 1-O-alkyl-DAG was noted. Propranalol, which increases PA accumulation by inhibiting PA phosphohydrolase, lowered PMN O2- responses to hyphae. Early DAG rises temporally overlapped respiratory burst initiation but PMN responses to hyphae were unchanged by a DAG kinase inhibitor, R59022, which blocks phosphorylation of DAG to PA and enhances DAG accumulation. Thus, neither PA nor DAG accumulation individually accounted for triggering PMN O2- responses to hyphae. PLD activation and PA production may facilitate PMN fungicidal responses to hyphae but play an indirect role in initiating the respiratory burst.

Interactions of phagocytic cells with Candida and other opportunistic fungi.

Phagocytic host defense mechanisms play a pivotal role in the prevention and containment of invasive, disseminated candidiasis, aspergillosis, and other opportunistic mycoses. The specific processes involved depend upon both the species, growth phase, and surface properties of the fungal target and the type and differentiation of the effector phagocyte. The nature and outcome of phagocyte-fungal interactions are also influenced by definable surface features of the organisms, binding of opsonic serum components, triggering of particular phagocyte plasma membrane receptors, and modulating effects of both fungal products and host cytokines. Broadening insights into specific interactive cellular mechanisms promise to provide new strategies for identifying high risk patients and preventing or successfully treating potentially fatal, progressive fungal infections.

Flow cytometric kinetic measurements of neutrophil phospholipase A activation.

The interrelationships between activation of phospholipases and neutrophil stimulus-induced Ca2+ responses remain unclear. We report here that immune complexes activate a phosphatidylcholine-specific phospholipase A in a neutrophil only after the cytoplasmic Ca2+ transient has been initiated in the same cell, while chemotactic peptide activation does not proceed via such a phospholipase A-mediated mechanism. Measurements of [Ca2+] changes and of phosphatidylcholine-specific phospholipase A activity were made by flow cytometry, using Indo-1 for Ca2+ indication, and a new fluorescent probe, bis-BODIPY-phosphatidylcholine, localized in the inner leaflet of the plasma membrane, to measure phospholipase A activation. Both 100 nM formyl-methionyl-leucyl-phenylalanine (with or without cytochalasin B) and 60 micrograms/ml insoluble immune complexes elicited cytoplasmic Ca2+ transients, but only insoluble immune complexes stimulated phospholipase A activation in a subpopulation of cells exhibiting an elevation of [Ca2+]in. Phospholipase A activation followed the Ca2+ transient, starting, in each cell, after [Ca2+]in had begun to decrease as Ca2+ redistributed in the activated cell. The products of this phospholipase activation were confirmed by thin layer chromatography. We conclude that neutrophils respond to immune complexes with an elevated cytoplasmic Ca(2+)-requiring phosphatidylcholine-specific phospholipase A activation and to chemotactic peptides by a different mechanism.

In vitro, Candida albicans releases the immune modulator adenosine and a second, high-molecular weight agent that blocks neutrophil killing.

We previously described a lyophilized supernatant from germinated Candida albicans that blocks human neutrophil (PMN) O2- production and degranulation stimulated by several PMN agonists but does not block stimulation by PMA. In studies to further characterize this Candida hyphal inhibitory product (CHIP), we noted several physicochemical parallels with the purine nucleoside adenosine (Ado). A Sephadex G-10 semipurified fraction of CHIP had an absorption peak near 260 nm, an apparent m.w. of less than 400, and was resistant to boiling and proteases. Maximally effective doses of CHIP (100 micrograms/ml) and Ado (100 microM) blocked 0.1 microM FMLP-stimulated O2- production by 76.8 +/- 4.1 and 81.7 +/- 4.8%, respectively. Ado deaminase, known to inactivate Ado, reversed inhibition by both Ado and CHIP. Results were comparable for the effect of CHIP and Ado on FMLP-stimulated beta-glucuronidase and lactoferrin release. Activation of the respiratory burst by opsonized C. albicans yeast was also inhibited by CHIP and Ado, but the extent of inhibition was less than for FMLP. At yeast:PMN ratios of 4:1, 10:1, and 40:1, CHIP inhibited O2- by -3.8%, 14.3%, and 12.8%, respectively; Ado blocked production by 32.9%, 24.2%, and 11.5%, respectively. The effect of CHIP and Ado on Candida killing by PMN was compared using two viability assays in each of four experiments. Ado (100 microM) had no effect on killing, although CHIP (100 micrograms/ml) inhibited killing in the MTT assay at 15 and 45 min by 81.6 +/- 6.3 and 24.7 +/- 6.2%, respectively; as assayed by CFU, CHIP inhibited killing by 34.1 +/- 6.2 and 10.3 +/- 2.5%, respectively. The ability of CHIP to inhibit killing was not affected by adding Ado deaminase, providing additional evidence that an Ado-like effect by CHIP is not essential for killing inhibition. Killing of opsonized Streptococcus pneumoniae was also inhibited in a concentration-dependent manner. Reverse-phase HPLC of the semipurified fraction revealed a peak, eluting identically to authentic Ado, which was eliminated by adding Ado deaminase. Ado content of the G-10 fraction was sufficient to account fully for the FMLP-inhibitory activity. The antikilling activity was resistant to boiling and proteases but was eliminated by mild periodation. Fractions eluting from a Sephadex CL6B column between 0.8 and 2.0 x 10(6) m.w. had increased sp. act. for killing inhibition. Sp. act. increased as carbohydrate content increased, but killing inhibition by various Candida cell wall constituents was absent to modest compared to inhibition induced by CHIP.(ABSTRACT TRUNCATED AT 400 WORDS)

Anticandidal activity of major human salivary histatins.

We have previously shown that histatins 1, 3, and 5 are homologous, histidine-rich proteins present in human parotid and submandibular secretions which contain 38, 32, and 24 amino acids, respectively. Interest in these proteins stems from the fact that histatins exhibit candidacidal and candidastatic activities. The goal of the present investigation was a detailed functional characterization of these anticandidal activities of histatins at the levels of killing of blastoconidia, killing of germinated cells, and inhibition of germination by using three bioassays. Candidacidal activities were evaluated at several ionic strengths, in the presence of different mono- and divalent ions, and at multiple pH values. In addition, the susceptibility of Candida albicans in different growth phases to histatins was investigated. While all three major human histatins demonstrated candidacidal activities, they differed in their abilities to kill blastoconidia and germinated cells, with histatin 5 being the most active, histatin 3 showing moderate activity, and histatin 1 exhibiting the lowest level of activity. For the inhibition of germination, however, histatin 3 exhibited more activity than either histatin 1 or histatin 5. The candidacidal activity of histatins was inversely proportional to both the ionic strength and the divalent cation concentration in the medium. Stepwise reduction of the pH of the assay medium enhanced the candidacidal activities of histatins 1 and 3, while the activity of histatin 5 was pH independent over the range of pHs 4 to 8. C. albicans in log-phase growth was more susceptible to histatins 1 and 3 than cells in stationary phase. Cells in either growth phase were still more vulnerable to histatin 5 than to histatins 1 and 3. The results obtained establish the functional relationship of the major histatins with respect to both their fungicidal and fungistatic activities and provide insights into their activities under ionic and pH conditions likely to be encountered in vivo in the oral cavity. Moreover, the data point towards possible mechanisms responsible for the anticandidal activities of histatins.

The growing problem of mycoses in patients infected with the human immunodeficiency virus.

With the rapid increase in cases of AIDS over the past 10 years, certain mycoses have dramatically risen in frequency, particularly those contained by T cell-mediated mechanisms of host defense. In this clinical setting mucocutaneous candidiasis as well as systemic cryptococcosis, histoplasmosis, and coccidioidomycosis pose special diagnostic and/or therapeutic challenges. Compared with fungal infections in general, AIDS-associated mycoses are more likely to have nonspecific clinical manifestations; moreover, treatments effective in other settings seldom are curative. These problems have led to new vigilance regarding mycoses in the differential diagnosis of complications of infection due to the human immunodeficiency virus (HIV) and have necessitated a redefinition of goals: the aim is now to suppress rather than cure infection in most cases. This change has stimulated trials of new antifungal agents and regimens particularly designed to facilitate long-term outpatient management of mycoses without interfering with treatment of either HIV infection itself or other concomitant complications.

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.

Successful treatment of Candida osteomyelitis with fluconazole. A noncomparative study of two patients.

We describe two patients with osteomyelitis due to Candida spp. treated with fluconazole, a new triazole antifungal. One patient had extensive involvement of ribs and costochondral regions of the anterior chest, and the other had vertebral infection. Both were cured with courses of 10 and 14 months, with greater than or equal to 1 year of follow-up after fluconazole was discontinued. Fluconazole is an attractive agent for the treatment of Candida osteomyelitis and deserves to be studied more extensively for this indication.

Recognition of binding sites on Candida albicans by monoclonal antibodies to human leukocyte antigens.

Candida albicans exhibits examples of human molecular mimicry, including receptors resembling human steroid receptors and human complement receptor (CR)-like receptors that bind the complement fragments C3d and iC3b. To further characterize the epitopes of the Candida human CR-like molecules, a panel of monoclonal antibodies (MAbs) against epitopes within the human CR3 was used. MAbs Mo1, M1/70HL, and 7C3 bound to the germ tube, as assessed by immunofluorescence. MAbs Leu15, 60.1, and 95G8 did not bind. Miscellaneous MAbs against other antigens on human leukocytes (B2, 3D9, and OKT4) did not bind. However, MY9, which recognizes a monocyte antigen, bound extensively to the germ tube. The binding of certain anti-CR3 MAbs suggested limited identity between the Candida CR3-like receptor and the human CR3. The binding of MY9 identified an antigen recognized by a MAb to a human cell antigen not previously known to exist on C. albicans.

Inhibition and killing of fungi by the polyamine oxidase-polyamine system. Antifungal activity of the PAO-polyamine system.

Both components of the polyamine oxidase (PAO)-polyamine system are known to be present in phagocytes and have thus been postulated to contribute to the antimicrobial activity of these cells. Therefore, the effects of the PAO-polyamine system on three medically important opportunistic fungi were examined. Yeasts of Cryptococcus neoformans, but not Candida albicans blastoconidia or Aspergillus fumigatus conidia, were efficiently killed by the system. Two putative end products of the system, hydrogen peroxide and acrolein, both killed C. neoformans at concentrations attainable with the whole system. However, catalase failed to inhibit activity of the whole system, making hydrogen peroxide an unlikely mediator of killing. Although C. albicans blastoconidia and A. fumigatus conidia were not killed by the PAO-polyamine system, germ tube formation by the former, and hyphal growth by the latter, were markedly inhibited. These data establish that the PAO-polyamine system possesses antifungal activity.

Patterns of guanine nucleotide exchange reflecting disparate neutrophil activation pathways by opsonized and unopsonized Candida albicans hyphae.

Through guanosine triphosphate (GTP) regulatory proteins are crucial components in signal transduction by most soluble and opsonized particulate stimuli, previous data suggest that neutrophil (PMNL) activation by unopsonized hyphae differs. Most of the PMNL superoxide response evoked by unopsonized hyphae was independent of both Ca++ ions and pertussis toxin-sensitive guanine nucleotide regulatory proteins. To determine whether related regulatory proteins were involved in PMNL activation by unopsonized hyphae, separated PMNL plasma membranes were incubated with GTP and a poorly hydrolyzed, radiolabeled GTP analogue, 5'-guanylylimido-diphosphate, then stimulated. Particulate Candida albicans hyphae and soluble chemotactic peptide induced comparable guanine nucleotide release. In contrast, while unopsonized hyphae caused release, it was considerably delayed, though opsonization discernibly affected neither PMNL attachment nor spreading over hyphal surfaces. This paralleled earlier observations of other delayed responses by intact PMNL to unopsonized hyphae: phospholipase C activation, the rise in cytosolic free Ca++ ions, and actin polymerization.

Effects of neutrophils and in vitro oxidants on survival and phenotypic switching of Candida albicans WO-1.

The relationship to pathogenesis of the spontaneous phenotypic switching of Candida albicans is uncertain. Since neutrophils are critical in containment of disseminated candidiasis, we used these cells and some of their potentially microbicidal oxidative products to define effects on a C. albicans strain (WO-1) that exhibits characteristic, easily recognized switching between the white and opaque phenotypes. Blastoconidia of the opaque phenotypes were more susceptible than those of the white to killing by either intact neutrophils or cell-free oxidants, including reagent hydrogen peroxide or the myeloperoxidase-H2O2-Cl- system. Paralleling these findings, opaque blastoconidia were 2.8- to 3.6-fold more potent stimuli of neutrophil superoxide generation than were the white cells. In addition, both neutrophils and oxidants (reagent H2O2 or hypochlorous acid as well as the myeloperoxidase-H2O2-Cl- system) induced unidirectional increases in spontaneous rates of switching from white to opaque phenotypes. Differences in expression of C. albicans phenotypes therefore may determine relative susceptibility to neutrophil fungicidal mechanisms, and neutrophils themselves appear to be capable of selectively augmenting the switching process.