Molecular epidemiology of isolates of the Cryptococcus neoformans species complex from Spain

6 p. : il.To study genetic diversity of Cryptococcus neoformans species complex in Spain, 97 isolates of the yeast recovered from human, animal and environmental samples have been analysed using three molecular epidemiological techniques. One of these, URA5 gene fragment length polymorphism (RFLP) analysis, has been previously described as a molecular epidemiology tool. Thus, standard profiles and reference strains have been defined for it. In addition, 5S rDNA/IGS RFLP and [GACA]4 microsatellite PCR fingerprinting were also used. Our results show five of the previously defined URA5 genotypes with a high frequency (33%) of the VNI type, which is in concordance with other studies. The high presence of VNIII pattern (28.9%) among our strains is remarkable and could be a specific feature of the isolates from our country. 5S rDNA/IGS RFLP showed a low intra-species discriminative power. Three different molecular profiles (S1-3), which showed a good correlation with the different species, varieties and genotypes, were obtained. [GACA]4 microsatellite PCR-fingerprinting analysis showed a high variability of patterns among the studied strains. Molecular profiles represented in a dendrogram clustered strains in four main groups related with the source of the yeast and also in concordance with some of the described genotypes (VNI-IV and VGI). ____________________________________________________________________________________.RESUMEN - Para ampliar el conocimiento de la diversidad genética que presenta el complejo Cryptococcus neoformans en España, se analizaron 97 aislamientos de esta levadura obtenidos a partir de muestras ambientales, veterinarias y clínicas, utilizando 3 técnicas epidemiológicas moleculares diferentes. Uno de estos marcadores moleculares, el análisis del URA5 RFLP, se ha descrito previamente como herramienta epidemiológica, por lo que se dispone de perfiles estándar y cepas de referencia. Además, se utilizaron también el análisis del RFLP del 5S rDNA/IGS y la huella digital tras amplificación por PCR de la secuencia microsatélite [GACA]4. Nuestros resultados muestran 5 de los genotipos URA5 con una elevada frecuencia del tipo VNI (33%), lo que concuerda con otros estudios. Es de resaltar la elevada presencia del perfil VNIII entre nuestras cepas (28,9%), lo que podría ser el rasgo específico más destacable de los aislamientos de nuestro país. El marcador 5S rDNA/IGS mostró un bajo poder discriminativo intraespecie. Se obtuvieron 3 perfiles moleculares distintos (S1-3), que presentaron una buena correlación con las especies, variedades y genotipos. La obtención de perfiles de huella digital con [GACA]4, presentó una gran variabilidad entre las cepas estudiadas. La representación en dendrograma agrupó las cepas en 4 agrupamientos principales relacionados con el origen de las levaduras y también con cierta concordancia con los genotipos descritos (VNI-IV y VGI)

The still obscure attributes of cryptococcal glucuronoxylomannan

6 p. : il.Glucuronoxylomannan (GXM) is the major capsular polysaccharide of Cryptococcus neoformans . It is essential for fungal virulence and causes a number of deleterious effects to host cells. During the last decades, most of the experimental models designed to study the roles of GXM during cryptococcal infection were based on the stimulation of animal cells. This most commonly involved macrophages or other effector cells, with polysaccharide fractions obtained by precipitation with cationic detergents. More recently, it has been demonstrated that GXM interferes with the physiological state of other target cells, such as the epithelium. In addition, recent studies indicate that the structure of the polysaccharide and, consequently, its functions vary according with the method used for its purifi cation. This raises questions as to what is native GXM and the signifi cance of prior studies. In this paper, we discuss some of the aspects of GXM that are still poorly explored in the current literature, including the relevance of the polysaccharide in the interaction of cryptococci with non-phagocytic cells and the relationship between its structure and biological activity

Evidence for branching in cryptococcal capsular polysaccharides and consequences on its biological activity

17 p. : il., tab.The encapsulated fungus Cryptococcus neoformans is a common cause of life-threatening disease in immunocompromised individuals. Its major virulence determinant is the polysaccharide (PS) capsule. An unsolved problem in cryptococcal biology is whether the PSs composing the capsule are linear or complex branched polymers, as well as the implications of this structural composition in pathogenesis. In this study we approached the problem by combining static and dynamic light scattering, viscosity analysis, and high-resolution microscopy and correlated the findings with biological properties. Analysis of the dependence of capsular PS molecular mass and the radius of gyration provided strong evidence against a simple linear PS configuration. Shape factors calculated from light scattering measurements in solution revealed values consistent with polymer branching. Furthermore, viscosity measurements provided complementary evidence for structural branching. Electron microscopy showed PS spherical-like structures similar to other branched PS. Finally, we show that the capacity of capsular PS to interfere in complement mediated phagocytosis, inhibit nitric oxide production by macrophage-like cells, protect against reactive oxygen species, antibody reactivity and half-life in serum were influenced by the degree of branching, providing evidence for the notion that PS branching is an important parameter in determining the biological activity of C. neoformans PS

The capsule of the fungal pathogen Cryptococcus neoformans

64 p. : il.The capsule of the fungal pathogen Cryptococcus neoformans has been studied extensively in recent decades, and a large body of information is now available to the scientific community. Well-known aspects of the capsule include its structure, antigenic properties and its function as a virulence factor. The capsule is composed primarily of two polysaccharides, glucuronoxylomannan (GXM) and galactoxylomannan (GalXM), in addition to a smaller proportion of mannoproteins (MP). Most of the studies on the composition of the capsule have focused on GXM, which comprises more than 90% of the capsule's polysaccharide mass. It is GalXM, however, that is of particular scientific interest because of its immunological properties. The molecular structure of these polysaccharides is very complex and has not yet been fully elucidated. Both GXM and GalXM are high molecular mass polymers with the mass of GXM equaling roughly 10 times that of GalXM. Recent findings suggest, however, that the actual Mw might be different to what it has traditionally been thought to be. In addition to their structural roles in the polysaccharide capsule, these molecules have been associated with many deleterious effects on the immune response. Capsular components are therefore considered key virulence determinants in Cryptococcus neoformans, which has motivated their use in vaccines and made them targets for monoclonal antibody treatments. In this review we will provide an update on the current knowledge of the C. neoformans capsule, covering aspects related to its structure, synthesis, and particularly, its role as a virulence factor

Galactoxylomannan-mediated immunological paralysis results from specific B cell depletion in the context of widespread immune system damage

16 p. : il.The mechanisms responsible for polysaccharide-induced immunological paralysis have remained unexplained almost a century after this phenomenon was first described. Cryptococcus neoformans capsular polysaccharides glucuronoxylomannan (GXM) and galactoxylomannan (GalXM) elicit little or no antibody responses. This study investigates the immunological and biological effects of GalXM in mice. GalXM immunization elicits a state of immunological paralysis in mice characterized by the disappearance of antibody-producing cells in the spleen. Immunological paralysis and lack of immunogenicity could not be overcome by immunization with GalXM conjugated to a protein carrier, Bacillus anthracis protective antigen. Additionally, immunization with GalXM in either complete or incomplete Freund's adjuvant was associated with spleen enlargement in Balb/c mice. Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling (TUNEL) and flow cytometry revealed widespread apoptosis in the spleen after GalXM administration. Administration of a cocktail of Caspase-3 Inhibitor Z-DEVD-FMK and General Caspase Inhibitor Z-VAD-FMK or Fas-deficient mice abrogated the complete disappearance of antibody producing cells. Analysis of spleen cytokine expression in response to GalXM systemic injection revealed that GalXM down-regulated the production of inflammatory cytokines. Hence, we conclude that GalXM-induced immune paralysis is a result of specific B-cell depletion mediated by its pro-apoptotic properties in the context of widespread dysregulation of immune function

Cryptococcus neoformans responds to mannitol by increasing capsule size in vitro and in vivo

22 p. : il., tab.The polysaccharide capsule of the fungus Cryptococcus neoformans is its main virulence factor. In this study, we determined the effects of mannitol and glucose on the capsule and exopolysaccharide production. Growth in mannitol significantly increased capsular volume compared to cultivation in glucose. However, cells grown in glucose concentrations higher than 62.5mM produced more exopolysaccharide than cells grown in mannitol. The fiber lengths and glycosyl composition of capsular polysaccharide from yeast grown in mannitol was structurally different from that of yeast grown in glucose. Furthermore, mannitol treatment of mice infected intratracheally with C. neoformans resulted in fungal cells with significantly larger capsules and the mice had reduced fungal dissemination to the brain. Our results demonstrate the capacity of carbohydrate source and concentration to modify the expression of a major virulence factor of C. neoformans. These findings may impact the clinical management of cryptococcosis

Structural and functional properties of the trichosporon asahii glucuronoxylomannan

10 p. : il.The virulence attributes of Trichosporon asahii are virtually unknown, despite its growing relevance as causative agent of superficial and invasive diseases in humans. Glucuronoxylomannan (GXM) is a well described virulence factor of pathogenic species in the Cryptococcus genus. GXM is also produced by species of the Trichosporon genus, and both polysaccharides share antigenic determinants, but unlike cryptococcal GXM, relatively little work has been done on trichosporal GXMs. In this study, we analyzed structural and functional aspects of GXM produced by T. asahii and compared them to the properties of the cryptococcal polysaccharide. Trichosporal and cryptococcal GXM shared antigenic reactivity, but the former polysaccharide had smaller effective diameter and negative charge. GXM anchoring to the cell wall was perturbed by dimethylsulfoxide and required interactions of chitin-derived oligomers with the polysaccharide. GXM from T. asahii supernatants are incorporated by acapsular mutants of Cryptococcus neoformans, which renders these cells more resistant to phagocytosis by mouse macrophages. In summary, our results establish that despite similarities in cell wall anchoring, antigenic and antiphagocytic properties, trichosporal and cryptococcal GXMs manifest major structural differences that may directly affect polysaccharide assembly at the fungal surface

Sec6-dependent sorting of fungal extracellular exosomes and laccase of Cryptococcus neoformans

12 p. : il.The cell wall of pathogenic fungi such as Cryptococcus neoformans, provides a formidable barrier to secrete virulence factors that produce host cell damage. To study secretion of virulence factors to the cell periphery, sec6 RNAi mutant strains of C. neoformans were tested for virulence factor expression. The studies reported here show that SEC6 RNAi mutant strains were defective in a number of virulence factors including laccase, urease as well as soluble polysaccharide and demonstrated attenuated virulence in mice. Further analysis by transmission electron microscopy detected the production of abundant extracellular exosomes in wild-type strains containing empty plasmid, but a complete absence in the iSEC6 strain. In addition, a green fluorescent protein–laccase fusion protein demonstrated aberrant localization within cytoplasmic vesicles in iSEC6 strains. In contrast, iSEC6 strains retained normal growth at 37°C, as well as substantially normal capsule formation, phospholipase activity and total secreted protein. These results provide the first molecular evidence for the existence of fungal exosomes and associate these vesicles with the virulence of C. neoformans

Agglutination of histoplasma capsulatum by IgG monoclonal antibodies against Hsp60 impacts macrophage effector functions

11 p. : il., tab.Histoplasma capsulatum can efficiently survive within macrophages, facilitating H. capsulatum translocation from the lung into the lymphatics and bloodstream. We have recently generated monoclonal antibodies (MAbs) to an H. capsulatum surface-expressed heat shock protein of 60 kDa (Hsp60) that modify disease in a murine histoplasmosis model. Interestingly, the MAbs induced different degrees of yeast cell agglutination in vitro. In the present study, we characterized the agglutination effects of the antibodies to Hsp60 on H. capsulatum yeast cells by light microscopy, flow cytometry, dynamic light scattering, measuring zeta potential, and using optical tweezers. We found that immunoglobulin Gs (IgGs) to Hsp60 cause H. capsulatum aggregation dependent on the (i) concentration of MAbs, (ii) MAb binding constant, and (iii) IgG subclass. Furthermore, infection of macrophages using agglutinates of various sizes after incubation with different Hsp60-binding MAbs induced association to macrophages through distinct cellular receptors and differentially affected macrophage antifungal functions. Hence, the capacity of IgG MAbs to agglutinate H. capsulatum significantly impacted pathogenic mechanisms of H. capsulatum during macrophage infection, and the effect was dependent on the antibody subclass and antigen epitope