Cell wall glucan synthases and GTPases in Paracoccidioides brasiliensis.

In this report we identified orthologues of fungal AGS1, RHO1, RHO2, RAC1 and CDC42 genes in the dimorphic fungus Paracoccidioides brasiliensis. Based on its homology to known fungal sequences, P. brasiliensis Ags1 was identified as an alpha-1,3-glucan synthase, while Rho1, Rho2, Rac1 and Cdc42 proteins were classified into the Rho1, Rho2, Rac1 and Cdc42 subgroups of fungal Rho GTPases, respectively. Of them, Rho1 is one of two subunits of a putative beta-1,3-glucan synthase complex, the other being the synthase itself (Fks1), while Rho2 has been associated to the alpha-1,3-glucan synthase (Ags1). Expression studies showed that mRNAs levels of RHO2 and AGS1 kept a direct relationship but the levels of RHO1 and FKS1 did not. P. brasiliensis RHO1 successfully restored growth of Saccharomyces cerevisiae rho1 mutant under restrictive temperature conditions. Chemical analyses of P. brasiliensis alpha-1,3-glucan, synthesized by Ags1p, indicated that it is essentially a linear polysaccharide, with <3% of alpha-1,4-linked glucose branches, occasionally attached as single units to the alpha-1,3-backbone.

Cell wall polysaccharides isolated from the fungus Neotestudina rosatii, one of the etiologic agents of mycetoma in man.

The alkali-extractable water-soluble polysaccharides F1SS isolated from the cell wall of two isolates of the pathogen Neotestudina rosatii and one of Pseudophaeotrichum sudanense, which is now considered as a synonym of the former, have been studied by methylation analysis, GC-MS and NMR spectroscopy. The three polysaccharides differ mainly in their content in galactofuranose, and have the following idealized repeating unit: with m approximately 19, and p approximately 6 in all cases, but being n approximately 1 for N. rosatii CBS 271.75, n approximately 0.5 for N. rosatii CBS 331.78, and n approximately 0.15 for P. sudanense.

Fungal cell wall polysaccharides isolated from Discula destructiva spp.

The alkali-extractable water-soluble polysaccharides F1SS isolated from the cell wall of four species of Discula destructiva have been studied by methylation analysis and NMR spectroscopy, and their idealized structures established as [structure: see text] where n approximately 2 for strains CBS 109771 and CBS 133.91, n approximately 1 for CBS 132.91, and it has an intermediate value in strain CBS 130.91. The mannan core was obtained by mild hydrolysis of the F1SS polysaccharide and its structure consisted of a skeleton of alpha-(1-->6)-mannopyranan, with around one out of eleven residues substituted at C-2 by short chains (one to six units) of 2-substituted mannopyranoses.

Structural elucidation of fungal polysaccharides isolated from the cell wall of Plectosphaerella cucumerina and Verticillium spp.

The structure of acidic fungal polysaccharides isolated from the cell wall of Plectosphaerella cucumerina, Verticillium dahliae, and V. albo-atrum has been investigated by chemical analysis, methylation analysis, and 1D and 2D 1H and 13C NMR spectroscopy. The polysaccharides have an idealized repeating block of the type: [carbohydrates: see text] linked to a small mannan core (<15%), where n=13, m=13, p=5, and q=8 for P. cucumerina, and n=16, m=16, p=6, and q <1 for both Verticillium species.

DC-SIGN mediates the binding of Aspergillus fumigatus and keratinophylic fungi by human dendritic cells.

Human contact with fungi does not usually lead to pathological consequences, as the immune system manages to defeat the invader pathogens. Nevertheless, under immune suppression, fungi overcome immune defenses and cause diseases that range from nonserious colonizations of keratinizated tissue (Dermatophytosis) to life threatening disseminated infections (Aspergillosis). Host defenses against fungi rely on innate and adaptative responses, with dendritic cell (DC) and macrophage surface receptors having a major role in the recognition of fungal pathogens and in the orchestration of an effective immune response. DC-SIGN is a C-type lectin involved in the recognition of bacterial, viral and parasitic pathogens, as well as in interactions between cells of the immune system. Its expression is restricted to DCs and subsets of macrophages. Here we show that DC-SIGN mediates the binding and capture of Aspergillus fumigatus and keratinophylic fungi, including Chrysosporium tropicum, by human DCs, describe the requirements of these interactions and discuss their potential involvement in the onset and persistence of pulmonary fungal infections.

Off-label use of maraviroc in HIV-1-infected paediatric patients in clinical practice.

Maraviroc (MVC) is not approved for HIV-1-infected paediatric patients. This is the first assessment of the use of MVC-based salvage therapy in vertically HIV-1-infected paediatric patients in clinical settings. The results suggest that MVC-based salvage therapy is useful in children and adolescents with extensive resistance profile leading to maintained virological suppression in up to 88% of the patients with CCR5-tropic virus. The likelihood of treatment success might increase when MVC is combined with other active drugs.

Fungal cell-wall galactomannans isolated from Geotrichum spp. and their teleomorphs, Dipodascus and Galactomyces.

The alkali-extracted water-soluble galactomannan F1SS isolated from the cell wall of two species each of Geotrichum, Galactomyces, and Dipodascus have been studied by methylation analysis and NMR spectroscopy, and their structure is established as the following: [carbohydrate structure: see text]

Structures of wall heterogalactomannans isolated from three genera of entomopathogenic fungi.

O-linked heterogalactomannans with similar structural features have been purified from the fungal walls of the entomopathogenic fungi Lecanicillium muscarium, Beauveria bassiana, Beauveriabrongniartii, and Cordyceps sphingum. Their composition and structure have been determined using acid hydrolysis, methylation analysis, gas-liquid chromatography-mass spectrometry (GC-MS) and Nuclear magnetic resonance spectroscopy (NMR). All structures have an α-(1→6)-mannose backbone, but one of the two strains of L. muscarium included in this study contained an acidic heterogalactomannan instead of the neutral polysaccharide isolated in the rest of the species analyzed. Sequence analysis of the internal transcribed spacer (ITS) region of this strain indicated that it belongs to the related genus Simplicillium, displaying low identity (83%) with the closest Lecanicillium species. This is a new demonstration of the structural diversity of fungal wall heteromannans and validates their interest as chemotaxonomic markers. The production of a pullulan-like extracellular polysaccharide in strain CBS 413.70C of L. muscarium is also reported.

An assessment of fungal wall heteromannans as a phylogenetically informative character in ascomycetes.

The fungal wall contains a small proportion of alkali-extractable water-soluble heteromannans (F1SS). They are the glycidic moiety of glycoproteins that have important roles in the biology of fungi. A considerable number of these polysaccharides has been described, differing in composition or linkage types. Their structure is similar in all species of a well-delimited genus, and teleomorphs and their corresponding anamorphs. Therefore, these polysaccharides have been used as chemotaxonomic markers at the genus level. Here we review cases where they have been found to resolve relationships around the genus level, and assess their phylogenetic informativeness in the delineation of taxa at family and higher ranks in the ascomycetes by comparison with molecular trees. Generally, the correlation is extremely good, from the species to the class level, though there are some divergences. In particular, comparisons suggest that the concept of the Sordariomycetes may eventually require revision as more molecular data become available. An analysis of the different chemical structures of these polysaccharides can lead to the proposal and testing of phylogenetic hypotheses, in a parallel manner to those generated from molecular trees. These molecules serve as an independent character similar to morphological or molecular characters.

Isolation and structural determination of a unique polysaccharide containing mannofuranose from the cell wall of the fungus Acrospermum compressum.

The alkali-extractable and water-soluble fungal polysaccharide F1SS isolated from the cell wall of Acrospermum compressum has been studied by methylation analyses, reductive cleavage and 1D- and 2D-NMR spectroscopy. The polysaccharide consists of a regular disaccharide repeating unit with the structure: [structure: see text]. The mannan core was obtained by mild hydrolysis of the polysaccharide F1SS and its structure was deduced to be composed of a skeleton of alpha-(1-->6)-mannopyranan, with around 1 out of 11 residues substituted at position 2 by short chains (one to six units) of 2-substituted mannopyranoses. DOSY experiments provided molecular sizes of 60 kDa and 2.5 kDa for the polysaccharide F1SS and the Mannan core, respectively. This is the first report of a fungal mannofuranose-containing cell wall polysaccharide.

Dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin mediates binding and internalization of Aspergillus fumigatus conidia by dendritic cells and macrophages.

Aspergillus fumigatus is responsible for a large percentage of nosocomial opportunistic fungal infections in immunocompromised hosts, especially during cytotoxic chemotherapy and after bone marrow transplantation, and is currently a major direct cause of death in leukemia patients. Dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN) is a type II C-type lectin that functions as an adhesion receptor and is used by viral and bacterial pathogens to gain access to human DC. We report that DC-SIGN specifically interacts with clinical isolates of A. fumigatus. DC-SIGN-dependent binding of A. fumigatus conidia can be demonstrated with stable transfectants and monocyte-derived DC and is inhibited by anti-DC-SIGN Abs. Binding and internalization of A. fumigatus conidia correlates with DC-SIGN cell surface expression levels and is abolished in the presence of A. fumigatus-derived cell wall galactomannans. The clinical relevance of this interaction is emphasized by the presence of DC-SIGN in lung DC and alveolar macrophages, and further illustrated by the DC-SIGN-dependent attachment of A. fumigatus conidia to the cell membrane of IL-4-treated monocyte-derived macrophages. Our results suggest the involvement of DC-SIGN in the initial stages of pulmonary infection as well as in fungal spreading during invasive aspergillosis.

Differences among the cell wall galactomannans from Aspergillus wentii and Chaetosartorya chrysella and that of Aspergillus fumigatus.

The alkali extractable and water-soluble cell wall polysaccharides F1SS from Aspergillus wentii and Chaetosartorya chrysella have been studied by methylation analysis, 1D- and 2D-NMR, and MALDI-TOF analysis. Their structures are almost identical, corresponding to the following repeating unit: [--> 3)-beta-D-Gal f -(1 --> 5)-beta-D-Gal f-(1 -->]n --> mannan core. The structure of this galactofuranose side chain differs from that found in the pathogenic fungus Aspergillus fumigatus, in other Aspergillii and members of Trichocomaceae: [--> 5)-beta-D-Gal f-(1 -->]n --> mannan core. The mannan cores have also been investigated, and are constituted by a (1 --> 6)-alpha-mannan backbone, substituted at positions 2 by chains from 1 to 7 residues of (1 --> 2) linked alpha-mannopyranoses.

Structural differences between the alkali-extracted water-soluble cell wall polysaccharides from mycelial and yeast phases of the pathogenic dimorphic fungus Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis is a pathogenic dimorphic fungus causing paracoccidioidomycosis, the most widespread systemic mycosis in Latin America. We have studied the structure of the alkali-extracted water-soluble cell wall polysaccharides (F1SS) from both mycelial and yeast phases of this fungus by using chemical analysis and NMR spectroscopic techniques. The F1SS polysaccharide from the mycelial phase consists of a trisaccharidic repeating unit of -->6)-[alpha-Galf -(1-->6)-alpha-Manp-(1-->2)]-alpha-Manp-(1-->. The F1SS polysaccharide of the yeast phase maintains 10% of the structure of the mycelium phase, but the main structure contain a disaccharide repeating unit of -->6)-[-alpha-Manp-(1-->2)]-alpha-Manp-(1-->, alternating with a trisaccharide repeating block of -->6)-[beta-Galf -(1-->6)-alpha-Manp-(1-->2)]-alpha-Manp-(1-->.

Variación de las subpoblaciones de células T segun la progresión clínica e inmunológica en niños infectados verticalmente por HIV-1 / T-cell subsets variation during clinical and immunological progression in vertically HIV-infected children

El objetivo de este trabajo es estudiar las subpoblaciones de células T vírgenes, memoria y activadas en sangre periférica de 65 niños infectados verticalmente por el HIV, considerando su categoría clínica, inmunológica y los valores de carga viral (CV). Todos los niños HIV fueron tratados con terapia antirretroviral (AR) (26 niños con terapia AR combinada y 39 en terapia AR altamente agresiva). Las subpoblaciones de linfocitos T se analizaron por citometría de flujo con marcaje triple y se expresaron en porcentaje. Las células T CD4+ vírgenes (CD45RA+CD62L+) estuvieron disminuidas en niños con bajo porcentaje CD4+, pero no en niños en estadios avanzados de la enfermedad o con CV elevadas. Por el contrario, las células T CD8+ vírgenes estuvieron disminuidas en niños con bajo porcentaje CD4+, en estadio avanzado de la enfermedad y con CV elevadas. Las células T CD4+ y CD8+ de memoria (CD45RO+) y activadas (CD38+, HLA-DR+ y CD38+HLA-DR+) estuvieron elevadas en niños con bajo porcentaje CD4+, en estadio avanzado de la enfermedad y con CV elevadas. Sin embargo, las células CD4+CD38+ estuvieron más elevadas en los niños HIV con CD4+>25 por ciento que en el grupo control (p<0,001) y más disminuidas en los niños con bajo porcentaje CD4+. El porcentaje de células T CD4+ y CD8+ vírgenes y memoria depende del porcentaje CD4+ más que de la categoría clínica o el valor de CV. Nuestros datos indican una asociación entre bajo porcentaje CD4+ y CV elevadas con la expresión elevada de marcadores de activación celular, pero no así en estadios clínicos avanzados, posiblemente debido al tratamiento antirretroviral.