Non-serum-dependent chemotactic factors produced by Candida albicans stimulate chemotaxis by binding to the formyl peptide receptor on neutrophils and to an unknown receptor on macrophages.
Serum-free culture filtrates of six Candida species and Saccharomyces cerevisiae were found to contain chemoattractants for human polymorphonuclear leukocytes (PMNs) and a mouse macrophage-like cell line, J774. The chemotactic factors differed for the PMN and J774 cells, however, in terms of heat stability, kinetics of liberation by the yeast cells, and divalent cation requirements for production. The chemoattractant in Candida albicans culture filtrates appeared to act through the formyl peptide receptor (FPR) of PMNs, since it was found to induce chemotaxis of Chinese hamster ovary (CHO) cells that were expressing the human FPR but did not induce chemotaxis of wild-type CHO cells. The C. albicans culture filtrates also induced migration of PMNs across confluent monolayers of a human gastrointestinal epithelial cell line, T84; migration occurred in the basolateral-to-apical direction but not the reverse direction, unless the epithelial tight junctions were disrupted. J774 cells did not migrate toward the formylated peptide (fMet-Leu-Phe; fMLF), and chemotaxis toward the C. albicans culture filtrate was not inhibited by an FPR antagonist (t-butoxycarbonyl-Met-Leu-Phe), suggesting that a different receptor mediated J774 cell chemotaxis. In conclusion, we have identified a receptor by which a non-serum-dependent chemotactic factor (NSCF) produced by C. albicans induced chemotaxis of PMNs. Additionally, we have shown that NSCF was active across epithelial monolayers. These findings suggest that NSCFs produced by C. albicans and other yeast species may influence host-pathogen interactions at the gastrointestinal tract mucosal surface by inducing phagocytic-cell infiltration. (+info)
Role of the extracellular signal-regulated protein kinase cascade in human neutrophil killing of Staphylococcus aureus and Candida albicans and in migration.
Killing of Staphylococcus aureus and Candida albicans by neutrophils involves adherence of the microorganisms, phagocytosis, and a collaborative action of oxygen reactive species and components of the granules. While a number of intracellular signalling pathways have been proposed to regulate neutrophil responses, the extent to which each pathway contributes to the killing of S. aureus and C. albicans has not been clearly defined. We have therefore examined the effect of blocking one such pathway, the extracellular signal-regulated protein kinase (ERK) cascade, using the specific inhibitor of the mitogen-activated protein kinase/ERK kinase, PD98059, on the ability of human neutrophils to kill S. aureus and C. albicans. Our data demonstrate the presence of ERK2 and a 43-kDa form of ERK but not ERK1 in human neutrophils. Upon stimulation with formyl methionyl leucyl phenylalanine (fMLP), the activities of both ERK2 and the 43-kDa form were stimulated. Despite abrogating the activity of both ERK forms, PD98059 only slightly reduced the ability of neutrophils to kill S. aureus or C. albicans. This is consistent with our finding that PD98059 had no effect on neutrophil adherence or degranulation, although pretreatment of neutrophils with PD98059 inhibited fMLP-stimulated superoxide production by 50%, suggesting that a change in superoxide production per se is not strictly correlated with microbicidal activity. However, fMLP-stimulated chemokinesis was markedly inhibited, while random migration and fMLP-stimulated chemotaxis were partially inhibited, by PD98059. These data demonstrate, for the first time, that the ERK cascade plays only a minor role in the microbicidal activity of neutrophils and that the ERK cascade is involved primarily in regulating neutrophil migration in response to fMLP. (+info)
BE-31405, a new antifungal antibiotic produced by Penicillium minioluteum. I. Description of producing organism, fermentation, isolation, physico-chemical and biological properties.
A new antifungal antibiotic, BE-31405, was isolated from the culture broth of a fungal strain, Penicillium minioluteum F31405. BE-31405 was isolated by adsorption on high porous polymer resin (Diaion HP-20), followed by solvent extraction, precipitation and crystallization. BE-31405 showed potent growth inhibitory activity against pathogenic fungal strains such as Candida albicans, Candida glabrata and Cryptococcus neoformans, but did not show cytotoxic activity against mammalian cells such as P388 mouse leukemia. The mechanism studies indicated that BE-31405 inhibited the protein synthesis of C. albicans but not of mammalian cells. (+info)
Synergic effects of tactolimus and azole antifungal agents against azole-resistant Candida albican strains.
We investigated the effects of combining tacrolimus and azole antifungal agents in azole-resistant strains of Candida albicans by comparing the accumulation of [3H]itraconazole. The CDR1-expressing resistant strain C26 accumulated less itraconazole than the CaMDR-expressing resistant strain C40 or the azole-sensitive strain B2630. A CDR1-expressing Saccharomyces cerevisiae mutant, DSY415, showed a marked reduction in the accumulation of both fluconazole and itraconazole. A CaMDR-expressing S. cerevisiae mutant, DSY416, also showed lower accumulation of fluconazole, but not of itraconazole. The addition of sodium azide, an electron-transport chain inhibitor, increased the intracellular accumulation of itraconazole only in the C26 strain, and not in the C40 or B2630 strains. Addition of tacrolimus, an inhibitor of multidrug resistance proteins, resulted in the highest increase in itraconazole accumulation in the C26 strain. The combination of itraconazole and tacrolimus was synergic in azole-resistant C. albicans strains. In the C26 strain, the MIC of itraconazole decreased from >8 to 0.5 mg/L when combined with tacrolimus. Our results showed that two multidrug resistance phenotypes (encoded by the CDR1 and CaMDR genes) in C. albicans have different substrate specificity for azole antifungal agents and that a combination of tacrolimus and azole antifungal agents is effective against azole-resistant strains of C. albicans. (+info)
In-vivo therapeutic efficacy in experimental murine mycoses of a new formulation of deoxycholate-amphotericin B obtained by mild heating.
Heat-induced 'superaggregation' of deoxycholate-amphotericin B (AmB-DOC, Fungizone) was shown previously to reduce the in-vitro toxicity of this antifungal agent. We compared AmB-DOC with the formulation obtained by heating the commercial form (Fungizone, Bristol Myers Squibb, Paris, France) for 20 min at 70 degrees C, in the treatment of murine infections. An improvement of antifungal activity was obtained with heated AmB-DOC formulations due to a lower toxicity which allowed the administration of higher drug doses than those achievable with the commercial preparation. Single intravenous injections of heated AmB-DOC solutions were demonstrated to be two-fold less toxic than unheated ones to healthy mice. For mice infected with Candida albicans, the maximum tolerated dose was higher with heated than with unheated AmB-DOC solutions. In the model of murine candidiasis, following a single dose of heated AmB-DOC 0.5 mg/kg, 85% of mice survived for 3 weeks, whereas at this dose the immediate toxicity of the standard formulation in infected mice restricted the therapeutic efficacy to 25% survival. Both formulations were equally effective in increasing the survival time for murine cryptococcal pneumonia and meningoencephalitis. Injection of heated AmB-DOC solutions at a dose two-fold higher than the maximal tolerated dose observed with the unheated preparation (1.2 mg/kg) increased the survival time by a factor of 1.4 in cryptococcal meningoencephalitis. These results indicate that mild heat treatment of AmB-DOC solutions could provide a simple and economical method to improve the therapeutic index of this antifungal agent by reducing its toxicity on mammalian cells. (+info)
Adhesive and mammalian transglutaminase substrate properties of Candida albicans Hwp1.
The pathogenesis of candidiasis involves invasion of host tissues by filamentous forms of the opportunistic yeast Candida albicans. Morphology-specific gene products may confer proinvasive properties. A hypha-specific surface protein, Hwp1, with similarities to mammalian small proline-rich proteins was shown to serve as a substrate for mammalian transglutaminases. Candida albicans strains lacking Hwp1 were unable to form stable attachments to human buccal epithelial cells and had a reduced capacity to cause systemic candidiasis in mice. This represents a paradigm for microbial adhesion that implicates essential host enzymes. (+info)
The cellular target of histatin 5 on Candida albicans is the energized mitochondrion.
Histatin 5 is a human basic salivary peptide with strong fungicidal properties in vitro. To elucidate the mechanism of action, the effect of histatin 5 on the viability of Candida albicans cells was studied in relation to its membrane perturbing properties. It was found that both the killing activity and the membrane perturbing activity, studied by the influx of a DNA-specific marker propidium iodide, were inhibited by high salt conditions and by metabolic inhibitors, like sodium azide. In addition, exposure to histatin 5 resulted in a loss of the mitochondrial transmembrane potential in situ, measured by the release of the potential-dependent distributional probe rhodamine 123. Localization studies using tetramethylrhodamine isothiocyanate-labeled histatin 5 or fluorescein isothiocyanate-labeled histatin 5 showed a granular intracellular distribution of the peptide, which co-localized with mitotracker orange, a permeant mitochondria-specific probe. Like the biological effects, uptake of labeled histatin 5 was inhibited by mitochondrial inhibitors and high salt conditions. Our data indicate that histatin 5 is internalized, and targets to the energized mitochondrion. (+info)
Rapid hypothesis testing with Candida albicans through gene disruption with short homology regions.
Disruption of newly identified genes in the pathogen Candida albicans is a vital step in determination of gene function. Several gene disruption methods described previously employ long regions of homology flanking a selectable marker. Here, we describe disruption of C. albicans genes with PCR products that have 50 to 60 bp of homology to a genomic sequence on each end of a selectable marker. We used the method to disrupt two known genes, ARG5 and ADE2, and two sequences newly identified through the Candida genome project, HRM101 and ENX3. HRM101 and ENX3 are homologous to genes in the conserved RIM101 (previously called RIM1) and PacC pathways of Saccharomyces cerevisiae and Aspergillus nidulans. We show that three independent hrm101/hrm101 mutants and two independent enx3/enx3 mutants are defective in filamentation on Spider medium. These observations argue that HRM101 and ENX3 sequences are indeed portions of genes and that the respective gene products have related functions. (+info)