Cell surface expression of adhesins for fibronectin correlates with virulence in Sporothrix schenckii.

The virulence of four Sporothrix schenckii isolates was compared in a murine model of sporotrichosis, together with the protein pattern of the yeast cell surface and the capacity to bind the extracellular matrix protein fibronectin. Virulence was determined by the mortality rate, fungal burden and histopathology. Two clinical isolates were more virulent for C57BL/6 mice, but no direct correlation was seen between virulence and the clinical or environmental origin of the isolates. The lowest virulence was observed for an isolate recovered from a patient with meningeal sporotrichosis. Although all isolates could effectively disseminate, the dissemination patterns were not similar. Using flow cytometry analysis, we investigated the interaction of all the strains with fibronectin, and showed that the binding capacity correlated with virulence. Western blot analysis of S. schenckii cell wall extracts revealed positive bands for fibronectin in the range of 37-92 kDa. The 70 kDa adhesin was also recognized by a protective monoclonal antibody raised against a gp70 antigen of S. schenckii (mAb P6E7). Confocal microscopy confirmed the co-localization of fibronectin and mAb P6E7 on the yeast cell surface. To our knowledge, this is the first report identifying adhesins for fibronectin on the surface of this human pathogen.

Melanin is an essential component for the integrity of the cell wall of Aspergillus fumigatus conidia.

BACKGROUND: Aspergillus fumigatus is the most common agent of invasive aspergillosis, a feared complication in severely immunocompromised patients. Despite the recent commercialisation of new antifungal drugs, the prognosis for this infection remains uncertain. Thus, there is a real need to discover new targets for therapy. Particular attention has been paid to the biochemical composition and organisation of the fungal cell wall, because it mediates the host-fungus interplay. Conidia, which are responsible for infections, have melanin as one of the cell wall components. Melanin has been established as an important virulence factor, protecting the fungus against the host's immune defences. We suggested that it might also have an indirect role in virulence, because it is required for correct assembly of the cell wall layers of the conidia. RESULTS: We used three A. fumigatus isolates which grew as white or brown powdery colonies, to demonstrate the role of melanin. Firstly, sequencing the genes responsible for biosynthesis of melanin (ALB1, AYG1, ARP1, ARP2, ABR1 and ABR2) showed point mutations (missense mutation, deletion or insertion) in the ALB1 gene for pigmentless isolates or in ARP2 for the brownish isolate. The isolates were then shown by scanning electron microscopy to produce numerous, typical conidial heads, except that the conidia were smooth-walled, as previously observed for laboratory mutants with mutations in the PKSP/ALB1 gene. Flow cytometry showed an increase in the fibronectin binding capacity of conidia from mutant isolates, together with a marked decrease in the binding of laminin to the conidial surface. A marked decrease in the electronegative charge of the conidia and cell surface hydrophobicity was also seen by microelectrophoresis and two-phase partitioning, respectively. Ultrastructural studies of mutant isolates detected considerable changes in the organisation of the conidial wall, with the loss of the outermost electron dense layer responsible for the ornamentations seen on the conidial surface in wild-type strains. Finally, analysis of the conidial surface of mutant isolates by atomic force microscopy demonstrated the absence of the outer cell wall rodlet layer which is composed of hydrophobins. CONCLUSION: These results suggest that, in addition to a protective role against the host's immune defences, melanin is also a structural component of the conidial wall that is required for correct assembly of the cell wall layers and the expression at the conidial surface of adhesins and other virulence factors.

Hypersusceptibility to azole antifungals in a clinical isolate of Candida glabrata with reduced aerobic growth.

Petite mutations have been described in Saccharomyces cerevisiae and pathogenic yeasts. However, previous studies of the phenotypic traits of these petite mutants reported that they express azole resistance. We describe a clinical isolate of Candida glabrata with a striking association between increased susceptibility to azoles and respiratory deficiency. This isolate was obtained from a urine sample together with a respiration-competent C. glabrata isolate which exhibited azole resistance. The respiratory status of the two isolates was confirmed by cultivation on glycerol-containing agar and oxygraphy. Flow cytometry revealed the normal incorporation of rhodamine 123, and mitochondrial sections with typical cristae were seen by transmission electron microscopy for both isolates. Together, these results suggested a nuclear origin for the reduced respiratory capacity of the hypersusceptible isolate. The sterol contents of these isolates were similar to the sterol content of a reference strain. Sequencing of the ERG11 and PDR1 genes revealed that the sequences were identical in the two isolates, demonstrating their close relatedness. In addition to silent mutations, they carried a nonsense mutation in PDR1 that led to the truncation of transcription factor Pdr1p. They also overexpressed both PDR1 and one of its targets, CDR1, providing a possible explanation for the azole resistance of the respiration-competent isolate. In conclusion, in addition to azole resistance, which is a common feature of C. glabrata mitochondrial petite mutants, the mutation of a nuclear gene affecting aerobic growth may lead to azole hypersusceptibility; however, the mechanisms underlying this phenotype remain to be determined.

A nonsense mutation in the ERG6 gene leads to reduced susceptibility to polyenes in a clinical isolate of Candida glabrata.

Unlike the molecular mechanisms that lead to azole drug resistance, the molecular mechanisms that lead to polyene resistance are poorly documented, especially in pathogenic yeasts. We investigated the molecular mechanisms responsible for the reduced susceptibility to polyenes of a clinical isolate of Candida glabrata. Sterol content was analyzed by gas-phase chromatography, and we determined the sequences and levels of expression of several genes involved in ergosterol biosynthesis. We also investigated the effects of the mutation harbored by this isolate on the morphology and ultrastructure of the cell, cell viability, and vitality and susceptibility to cell wall-perturbing agents. The isolate had a lower ergosterol content in its membranes than the wild type, and the lower ergosterol content was found to be associated with a nonsense mutation in the ERG6 gene and induction of the ergosterol biosynthesis pathway. Modifications of the cell wall were also seen, accompanied by increased susceptibility to cell wall-perturbing agents. Finally, this mutation, which resulted in a marked fitness cost, was associated with a higher rate of cell mortality. Wild-type properties were restored by complementation of the isolate with a centromeric plasmid containing a wild-type copy of the ERG6 gene. In conclusion, we have identified the molecular event responsible for decreased susceptibility to polyenes in a clinical isolate of C. glabrata. The nonsense mutation detected in the ERG6 gene of this isolate led to a decrease in ergosterol content. This isolate may constitute a useful tool for analysis of the relevance of protein trafficking in the phenomena of azole resistance and pseudohyphal growth.

Adherence mechanisms in human pathogenic fungi.

Interactions of human pathogenic fungi with the host tissues are key factors in the pathogenesis of mycoses. Based on the concept that adherence of microorganisms is a prerequisite for initiation of the disease, numerous studies have been conducted to identify the fungal adhesins and their respective receptors. Several adhesins recognizing different host ligands, sometimes with multifunctional properties, have been described. Some of them have been extensively characterized, and their expression analyzed according to morphological changes or culture conditions. For some ligands, the amino acid or carbohydrate motifs participating in these interactions have been identified. Various host proteins or glycoproteins have been suggested as ligands, including components of biological fluids, or extracellular matrix and basement membrane proteins; equally adherence to several cell types, mainly epithelial and endothelial cells, or to biomaterials has been considered. This review synthesizes available information regarding adherence of the most important human fungal pathogens. It is divided into three sections corresponding to the three main groups of pathogenic fungi: Candida yeasts, opportunistic moulds and other filamentous fungal pathogens, and dimorphic fungi.

Reduced susceptibility to polyenes associated with a missense mutation in the ERG6 gene in a clinical isolate of Candida glabrata with pseudohyphal growth.

Little information is available about the molecular mechanisms responsible for polyene resistance in pathogenic yeasts. A clinical isolate of Candida glabrata with a poor susceptibility to polyenes, as determined by disk diffusion method and confirmed by determination of MIC, was recovered from a patient treated with amphotericin B. Quantitative analysis of sterols revealed a lack of ergosterol and an accumulation of late sterol intermediates, suggesting a defect in the final steps of the ergosterol pathway. Sequencing of CgERG11, CgERG6, CgERG5, and CgERG4 genes revealed exclusively a unique missense mutation in CgERG6 leading to the substitution of a cysteine by a phenylalanine in the corresponding protein. In addition, real-time reverse transcription-PCR demonstrated an overexpression of genes encoding enzymes involved in late steps of the ergosterol pathway. Moreover, this isolate exhibited a pseudohyphal growth whatever the culture medium used, and ultrastructural changes of the cell wall of blastoconidia were seen consisting in a thinner inner layer. Cell wall alterations were also suggested by the higher susceptibility of growing cells to Calcofluor white. Additionally, complementation of this isolate with a wild-type copy of the CgERG6 gene restored susceptibility to polyenes and a classical morphology. Together, these results demonstrated that mutation in the CgERG6 gene may lead to a reduced susceptibility to polyenes and to a pseudohyphal growth due to the subsequent changes in sterol content of the plasma membrane.

Investigation of the antifungal activity of caledonixanthone E and other xanthones against Aspergillus fumigatus.

Among the different xanthones previously isolated from the stem bark of Calophyllum caledonicum, caledonixanthone E presented the strongest activity (MIC (80) = 8 microg/mL) in acidic conditions (pH 3) against the human pathogenic fungus Aspergillus fumigatus. Phase-contrast microscopy studies suggested the assembly or synthesis of cell wall components as the target of the drug. Moreover, the use of fluorescent lectins further supported an impact of caledonixanthone E on the synthesis of chitin, the major structural polysaccharide of the fungal wall. These results suggest that caledonixanthone E may be an interesting model for the design of new antifungal drugs.

Ultrastructural and Immunochemical Changes During the In Vitro Development of Plasmopara halstedii.

ABSTRACT The asexual phase of the life cycle of Plasmopara halstedii, the causal agent of downy mildew of sunflower, plays a key role in the propagation of the disease. We investigated the morphological and ultrastructural changes that occur during the asexual development of the pathogen. Direct examination of infected cotyledons confirmed the presence of sporangiophores. In contact with water, important ultrastructural changes occurred, affecting the surface of zoosporangia, which became smoother, and their cytoplasm, which differentiated into flagellate zoospores. The subsequent encystment of zoospores was characterized by the synthesis of a cell wall and the loss of the flagella. In addition, two monoclonal antibodies (MAbs) specific for P. halstedii were used to analyze the immunochemical changes associated with these modifications. MAb 16A6, which bound to a 48-kDa glycoprotein, mainly labeled the surface of mobile or encysted zoospores and of mother cells of germ tubes. Conversely, MAb 2F9, which recognized highly glycosylated antigens, labeled the surface of zoosporangia and of flagellate zoospores, but not the encysted zoospores. These results provide new insights into the morphological and ultrastructural changes associated with the release and the encystment of zoospores which may be interesting targets for the development of new antimicrobial products.

Purification and partial characterization of a 32-kilodalton sialic acid-specific lectin from Aspergillus fumigatus.

Adherence of the opportunistic fungus Aspergillus fumigatus to the extracellular matrix components is considered a crucial step in the establishment of the infection. Given the high carbohydrate content of these glycoproteins and the role of carbohydrate-protein interactions in numerous adherence processes, the presence of a lectin in A. fumigatus was investigated. Different fungal extracts obtained by sonication or grinding in liquid nitrogen from resting or swollen conidia, as well as from germ tubes and mycelium, were tested by hemagglutination assays using rabbit erythrocytes. A lectin activity was recovered in all the extracts tested. However, sonication of resting conidia resulted in the highest specific activity. Purification of the lectin was achieved by gel filtration followed by ion-exchange and hydrophobic-interaction chromatographies. Analysis of the purified lectin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed an apparent molecular mass of 32 kDa, which is similar to that of the alkaline protease already identified from different strains of A. fumigatus. However, as evidenced by the use of an alkaline protease-deficient mutant, the two activities were supported by distinct proteins. In addition, hemagglutination inhibition experiments using different saccharides and glycoproteins demonstrated the specificity of the lectin for sialic acid residues. Together these results suggest that this lectin may contribute to the attachment of conidia to the extracellular matrix components through the recognition of the numerous terminal sialic acid residues of their carbohydrate chains.