Agnes Marot-Leblond

Purification and Partial Characterization of a 32-Kilodalton Sialic Acid-Specific Lectin from Aspergillus fumigatus

ABSTRACT 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.

Development and Evaluation of a Rapid Latex Agglutination Test Using a Monoclonal Antibody To Identify Candida dubliniensis Colonies

ABSTRACT Cell components of the dimorphic pathogenic fungus Candida dubliniensis were used to prepare monoclonal antibodies (MAbs). One MAb, designated 12F7-F2, was shown by indirect immunofluorescence to be specific for a surface antigen of Candida dubliniensis yeast cells. No reactivity was observed with other fungal genera or with other Candida species, including Candida albicans, that share many phenotypic features with C. dubliniensis. The use of different chemical and physical treatments for cell component extraction suggested that the specific epitope probably resides on a protein moiety absent from C. albicans. However, we failed to identify the target protein by Western blotting, owing to its sensitivity to heat and sodium dodecyl sulfate. MAb 12F7-F2 was further used to develop a commercial latex agglutination test to identify C. dubliniensis colonies (Bichro-dubli Fumouze test; Fumouze Diagnostics). The test was validated on yeast strains previously identified by PCR and on fresh clinical isolates; these included 46 C. dubliniensis isolates, 45 C. albicans isolates, and other yeast species. The test had 100% sensitivity and specificity for C. dubliniensis isolated on Sabouraud dextrose, CHROMagar Candida, and CandiSelect media and 97.8% sensitivity for C. dubliniensis grown on Candida ID medium. The test is rapid (5 min) and easy to use and may be recommended for routine use in clinical microbiology laboratories and for epidemiological investigations.

Evaluation of a rapid immunochromatographic assay for identification of Candida albicans and Candida dubliniensis.

ABSTRACT Candida dubliniensis was first established as a novel yeast species in 1995. It is particularly associated with recurrent episodes of oral candidosis in human immunodeficiency virus (HIV)-infected patients, but it has also been detected at other anatomical sites and at a low incidence level in non-HIV-infected patients. It shares so many phenotypic characteristics with C. albicans that it is easily misidentified as such. No rapid, simple, and commercial test that allows differentiation between C. dubliniensis and C. albicans has been developed, until now. Accurate species identification requires the use of genotype-based techniques that are not routinely available in most clinical microbiology diagnostic laboratories. The present study was designed to evaluate the efficiency of a new test (the immunochromatographic membrane [ICM] albi-dubli test; SR2B, Avrillé, France) to differentiate between C. albicans and C. dubliniensis. The organisms evaluated were strains whose identities had previously been confirmed by PCR tests and freshly isolated clinical strains and included 58 C. albicans isolates, 60 C. dubliniensis isolates, and 82 isolates belonging to other species of yeast. The ICM albi-dubli test is based on the principle of immunochromatographic analysis and involves the use of two distinct monoclonal antibodies that recognize two unrelated epitopes expressed by both species or specific to only one species. The assay requires no complex instrumentation for analysis and can be recommended for routine use in clinical microbiology laboratories. Results are obtained within 2 h and 30 min and are easy to interpret. This evaluation demonstrated the good performance of this immunochromatographic test for C. albicans and C. dubliniensis isolated on Sabouraud dextrose agar, CHOROMagar Candida, and CandidaSelect, with sensitivities and specificities ranging from 93.1 to 100%. These parameters decreased, however, to 91.4% when the test was performed with yeast isolated with Candida ID.

Adherence of Platelets to Candida Species In Vivo

ABSTRACT The in vivo interactions of platelets with Candidaspecies yeast cells were investigated in a murine model. Mice were injected intravenously via the lateral caudal vein, and blood drawn by periorbital puncture was collected in phosphate-buffered saline–formaldehyde to avoid in vitro platelet activation. The study of the clearance of blastoconidia of Candida albicans andCandida glabrata showed that these cells disappeared quickly from the bloodstream. Microscopic observation of blood samples, stained by Calcofluor white or May Grunwald Giemsa, demonstrated the rapid attachment of platelets to fungal elements of all theCandida spp. tested. The attachment of murine platelets toC. albicans cells, observed by scanning electron microscopy, revealed morphological changes. The platelets lost their discoid shape, generated pseudopodia, and flattened against the yeast cells. The reversibility of platelet binding to C. albicansby chelating agents suggests a cation-dependent link. In contrast, the fixation of C. glabrata and Candida tropicaliswas not modified by chelating agents. The mechanisms involved in the in vivo adherence of platelets to Candida cells may therefore differ according to the species of Candida.

Efficient Diagnosis of Vulvovaginal Candidiasis by Use of a New Rapid Immunochromatography Test

ABSTRACT The clinical symptoms of vulvovaginal candidiasis (VVC) are nonspecific, and misdiagnosis is common, leading to a delay in the initiation of antifungal treatment. We evaluated a new immunochromatography test (ICT), the CandiVagi assay (SR2B, Avrille, France), for the rapid diagnosis of VVC. This test, which employs an immunoglobulin M antibody directed against the β-1,2-mannopyranosyl epitopes found in the yeast cell wall, was compared with direct microscopic examination and culture of vaginal swabs. Two-hundred five women were investigated, including 130 women with symptomatic vaginitis and 75 asymptomatic controls. Two vaginal swabs were obtained from each woman: one was used to prepare a wet mount and Gram-stained preparations for direct microscopic examination and was also cultured on Sabouraud dextrose agar for the isolation of Candida spp., and the second swab was used for ICT. The sensitivities of microscopic examination, culture, and ICT for the diagnosis of VVC were 61%, 100%, and 96.6%, respectively, while the specificities of the three methods were 100%, 82%, and 98.6%, respectively. ICT had a negative predictive value of 98.6%, a positive predictive value of 96.6%, and an efficiency of 98%. ICT provided a rapid result and a better compromise between sensitivity and specificity than conventional microscopy and culture for the diagnosis of VVC. This easy-to-perform diagnostic test will be useful to practitioners treating women with symptoms of vaginitis.

Purification and Characterization of a Mycelial Catalase from Scedosporium boydii, a Useful Tool for Specific Antibody Detection in Patients with Cystic Fibrosis

ABSTRACT Scedosporium boydii is an opportunistic filamentous fungus which may be responsible for a wide variety of infections in immunocompetent and immunocompromised individuals. This fungus belongs to the Scedosporium apiospermum species complex, which usually ranks second among the filamentous fungi colonizing the airways of patients with cystic fibrosis (CF) and may lead to allergic bronchopulmonary mycoses, sensitization, or respiratory infections. Upon microbial infection, host phagocytic cells release reactive oxygen species (ROS), such as hydrogen peroxide, as part of the antimicrobial response. Catalases are known to protect pathogens against ROS by detoxification of the hydrogen peroxide. Here, we investigated the catalase equipment of Scedosporium boydii, one of the major pathogenic species in the S. apiospermum species complex. Three catalases were identified, and the mycelial catalase A1 was purified to homogeneity by a three-step chromatographic process. This enzyme is a monofunctional tetrameric protein of 460 kDa, consisting of four 82-kDa glycosylated subunits. The potential usefulness of this enzyme in serodiagnosis of S. apiospermum infections was then investigated by an enzyme-linked immunosorbent assay (ELISA), using 64 serum samples from CF patients. Whatever the species involved in the S. apiospermum complex, sera from infected patients were clearly differentiated from sera from patients with an Aspergillus fumigatus infection or those from CF patients without clinical and biological signs of a fungal infection and without any fungus recovered from sputum samples. These results suggest that catalase A1 is a good candidate for the development of an immunoassay for serodiagnosis of infections caused by the S. apiospermum complex in patients with CF.

Recognition of Candida albicans Als3 by the germ tube-specific monoclonal antibody 3D9.3

Monoclonal antibody 3D9.3 (MAb 3D9.3) reacts with the surface of Candida albicans germ tubes and recognizes a protein epitope. We used a two-step chromatography procedure to purify and identify the antigen (3D9) from C. albicans strain 66396 germ tubes. MAb 3D9.3 recognized two intense protein bands at 140 and 180 kDa. A comparative analysis between theoretical and experimental mass spectrum peaks showed that both bands corresponded to Als3. This conclusion was supported by lack of reactivity between MAb 3D9.3 and an als3Delta/als3Delta mutant strain, and the fact that an immunoglobulin preparation enriched for Als3 specificity recognized the purified 3D9 antigen. PCR demonstrated that C. albicans strain 66396 has two different-sized ALS3 alleles that correspond to the two purified protein bands. Strain- and species-specificity of the 3D9 epitope were studied with various C. albicans strains and Candida species, such as closely related Candida dubliniensis. The 3D9 epitope was detected only in C. albicans, demonstrating the utility of MAb 3D9.3 for differentiation between C. albicans and C. dubliniensis. Adhesion assays demonstrated that MAb 3D9.3 blocks adhesion of C. albicans germ tubes to human buccal epithelial cells and vascular endothelial cells.

Susceptibilities of Cryptococcus neoformans strains to platelet binding in vivo and to the fungicidal activity of thrombin-induced platelet microbicidal proteins in vitro.

ABSTRACT In this study we investigated the interactions between capsular and acapsular strains of Cryptococcus neoformans and blood platelets. In vivo microscopic observation of blood samples from mice inoculated with C. neoformans yeast cells demonstrated that encapsulated and nonencapsulated yeast cells disappeared quickly from the bloodstream and that platelets were attached solely to yeast cells of the nonencapsulated strains. In vitro we observed that only the acapsular strains were susceptible to the fungicidal activity of thrombin-induced platelet microbicidal proteins.

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.