Development and Use of a Polyvalent Conjugate to Differentiate Histoplasma capsulatum and Histoplasma duboisii from Other Pathogens

Previous investigations have demonstrated the existence of five Histoplasma capsulatum serotypes. Available specific fluorescent-antibody reagents stain only four of the five serotypes. Antibodies produced against the most complete H. capsulatum serotype were labeled with fluorescein isothiocyanate to develop a reagent specific for H. capsulatum that was reactive with all the known serotypes. The unadsorbed reagent not only stained all the H. capsulatum serotypes, but it also stained cultures of Blastomyces dermatitidis, H. duboisii, several Candida species, and a variety of other fungi. Adsorption of the conjugate with antigens of C. albicans produced a reagent that intensely stained only H. capsulatum, H. duboisii, and B. dermatitidis. Differentiation of B. dermatitidis from the Histoplasma species was accomplished by application of a B. dermatitidis specific fluorescent antibody to antigens positive with the H. capsulatum reagent. At present, differentiation of H. capsulatum from H. duboisii may be accomplished only by animal inoculation. Our data substantiate the antigenic relationships hypothesized earlier, and they indicate that H. capsulatum shares at least two antigens with the other fungi that were studied.     

Comparative study of the effect of thiabendazole and fungizone on Histoplasma capsulatum in macrophages.

The antifungal properties of Fungizone (amphotericin B intravenous solution) and thiabendazole on Histoplasma capsulatum within guinea pig macrophages were compared using the staining method and a newly developed plating method to determine the viability of intracellular H. capsulatum. The two methods were compared to determine the effectiveness of Fungizone and thiabendazole on H. capsulatum within macrophages. Fungizone was fungicidal for intracellular H. capsulatum, killing 99.9% of the yeasts at a concentration of 0.5 microgram/ml. There was some indication that non-viable intracellular yeasts were stained which could result in misinterpretation of the effectiveness of Fungizone using the staining method unless the yeasts are very closely examined for staining abnormalities. There was a good correlation between the two methods to demonstrate suppression of the multiplication of intracellular H. capsulatum in macrophages treated with 50 microgram/ml of thiabendazole. Thiabendazole was lethal for some intracellular H. capsulatum.     

African histoplasmosis: a review

African histoplasmosis caused by Histoplasma capsulatum var. duboisii is an important deep mycosis endemic in Central and West Africa and in the island of Madagascar. The disease is characterized by presence of granulomatous lesions in the skin, subcutaneous tissues and bones. Lungs and other internal organs are rarely involved. The natural reservoir of the etiological agent has only been recently discovered in a bat cave in Nigeria. The status of asymptomatic infection is not certain. Investigations on skin and serum reactivity have suggested frequent prevalence of asymptomatic infections due to H. capsulatum var. duboisii among the residents in the vicinity of the cave microfocus of the fungus. The exact portal of entry into the body is not known, but inhalation into the lungs and direct inoculation in the skin have been incriminated. Laboratory diagnosis is confirmed by in vitro conversion into large yeast forms (8-15 mum in diameter) and by the demonstration of these forms within giant cells of tissues of experimentally infected animals There are no major clean-cut physiological differences between the two varieties, viz. capsulatum and duboisii. The cell wall of H. capsulatum var duboisii contains a glucan with beta 1-4 linkages in addition to a galactomannan shared with H. capsulatum var. capsulatum. Like the var. capsulatum var. duboisii has marked proteinase and collagenase activities in both mycelial and yeast forms, suggesting a possible pathogenic role for these enzymes. Both varieties have a common exoantigen. The yeast form of H. capsulatum var. duboisii contains the antigen found in the serotype 1,4 of var. capsulatum. A monoclonal antibody test has been developed that can recognize some epitopes in H. capsulatum var. capsulatum but not in the var. duboisii. There is need to develop specific serological diagnosis for the disease. Also there should be greater international awareness about African histoplasmosis. Amphotericin B and several antimycotic azoles like ketoconazole, itraconazole and fluconazole have been successfully employed for treatment.     

Genetic diversity of Histoplasma capsulatum strains in Brazil

This study establishes the genetic relatedness among Brazilian Histoplasma capsulatum samples obtained from different sources. A PCR-based random amplified polymorphic DNA (RAPD) assay was used to delineate polymorphisms among isolates in geographically diverse regions in Brazil. RAPD fingerprints revealed distinct DNA profiles and provided a high level of discrimination among H. capsulatum strains from different locations. Cluster I was composed of H. capsulatum isolates from the northeast region. The majority of strains from southeast and south were categorized as major cluster II. The strain 84564 from Rio de Janeiro State showed no genetic correlation to any of the isolates from the same state. The RAPD patterns of H. capsulatum isolates from Goias (Cluster III) were unrelated to DNA fingerprints observed among the other H. capsulatum strains (48% similarity). This study is the first report that stratifies the clusters of H. capsulatum strains from Brazil by molecular typing and associates them with the geographical origin.     

VEA1 is required for cleistothecial formation and virulence in Histoplasma capsulatum

Histoplasma capsulatum is a pathogenic fungus dependent on dimorphism for virulence. Among the four described Velvet family genes, two of them, Ryp2 and Ryp3, have been shown to be required for dimorphism. It is known that Velvet A (VeA) is necessary for sexual development and toxin production in Aspergillus nidulans. However, the role of the VeA ortholog in H. capsulatum has not yet been explored. Vea1, H. capsulatum homolog of VeA, was studied to determine its role in cleistothecial formation, dimorphism, and virulence. H. capsulatum Vea1 restores cleistothecial formation and partially restores sterigmatocystin production in an A. nidulans veA deletion strain. Furthermore, silencing VEA1 in an H. capsulatum strain capable of forming cleistothecia abolishes cleistothecial formation. Silenced strains also switch to mycelial phase faster, and show impaired switching to the yeast phase once in mycelial phase. Virulence in mice and macrophages is attenuated in VEA1 silenced strains and silenced strains demonstrate increased sensitivity during growth under acidic conditions. These results indicate that H. capsulatum Vea1 shares a similar role in development as VeA. H. capsulatum is also more susceptible to growth in acidic conditions when VEA1 is silenced, which may contribute to the silenced strains' attenuated virulence in mice and macrophages.     

Geographical distribution of genetic polymorphism of the pathogen Histoplasma capsulatum isolated from infected bats, captured in a central zone of Mexico

Fourteen Histoplasma capsulatum isolates recovered from infected bats captured in Mexican caves and two human H. capsulatum reference strains were analyzed using random amplification of polymorphic DNA PCR-based and partial DNA sequences of four genes. Cluster analysis of random amplification of polymorphic DNA-patterns revealed differences for two H. capsulatum isolates of one migratory bat Tadarida brasiliensis. Three groups were identified by distance and maximum-parsimony analyses of arf, H-anti, ole, and tub1 H. capsulatum genes. Group I included most isolates from infected bats and one clinical strain from central Mexico; group II included the two isolates from T. brasiliensis; the human G-217B reference strain from USA formed an independent group III. Isolates from group II showed diversity in relation to groups I and III, suggesting a different H. capsulatum population.     

Knocking on the right door and making a comfortable home: Histoplasma capsulatum intracellular pathogenesis

Histoplasma capsulatum is a successful intracellular pathogen of mammalian macrophages. As such, this fungus must survive and/or subvert hostile environmental onslaughts in a professionally antimicrobial host cell. H. capsulatum uses different host receptors for binding to macrophages (beta 2 integrins) than it uses for binding to dendritic cells (the fibronectin receptor); the fungus experiences different degrees of success in survival in these two cells. Surface expression of HSP60 as the specific adhesin for macrophage beta 2 integrins represents a novel mechanism for binding. Long considered a resident of the phagolysosome, H. capsulatum may also reside in a modified phagosome without experiencing phagolysosomal fusion. H. capsulatum must compete with the host to acquire the essential nutrient iron, and has several potential mechanisms for accomplishing this necessary feat. Finally, H. capsulatum displays morphotype-specific expression of several genes, and a calcium-binding protein expressed only by the pathogenic yeast phase has been demonstrated as essential for full virulence. An organism's environment is of great importance to its success or failure, and H. capsulatum is good at finding or making the right environment in the host.     

Monosaccharide and Chitin Content of Cell Walls of Histoplasma capsulatum and Blastomyces dermatitidis

Cell walls of Histoplasma capsulatum and Blastomyces dermatitidis, obtained by mechanical breakage of yeast- and mycelial-phase cultures, were lipid-extracted and then fractionated with ethylenediamine. Unextracted cell walls, lipid-extracted cell walls, and the three fractions resulting from ethylenediamine treatment were examined for monosaccharide and chitin content. The yeast-phase cell walls of five strains of H. capsulatum fell into two categories, designated chemotypes I and II, one of which, chemotype II, was similar to yeast-phase cell walls derived from three strains of B. dermatitidis. H. capsulatum chemotype I cell walls were characterized by lower content of material soluble in ethylenediamine, higher chitin content, and lower monosaccharide content than H. capsulatum chemotype II or B. dermatitidis cell walls. Approximately 80% of the monosaccharides of chemotype I cell walls was combined in forms susceptible to attack by mild acid hydrolysis, compared with about 50% of the monosaccharides of chemotype II and B. dermatitidis. H. capsulatum and B. dermatitidis yeast-phase cell walls could be distinguished, however, by their susceptibility to attack by a crude enzyme system derived from a Streptomyces sp. incubated with chitin as the only carbon source. Both glucose and acetylglucosamine were released from H. capsulatum cell walls, regardless of chemotype, during enzymatic hydrolysis, whereas only acetylglucosamine was released from B. dermatitidis yeast-phase cell walls. Mycelial-phase cell walls of H. capsulatum and B. dermatitidis were characterized by lower content of material soluble in ethylenediamine, higher proportions of mannose, and lower chitin content than their respective yeast phases. Glucose and acetylglucosamine were both released from all mycelial-phase cell walls, whether H. capsulatum or B. dermatitidis, by the crude enzyme system.     

A medium for inducing conversion of Histoplasma capsulatum var. capsulatum into its yeast-like form.

Histoplasma capsulatum var. capsulatum is a dimorphic fungus that, under special conditions, converts from its more common mycelial form to a yeast-like form. Achieving this conversion, however, has been problematical for researchers. The present study tested conversion rates in ten Histoplasma capsulatum var. capsulatum strains using seven culture media, four of which were conventional and three novel. One of our novel media, MLGema, induced complete conversion of two strains within five days of incubation at 35 degrees C, and of all strains that eventually converted by the time of the second subculturing transfer, under defined experimental conditions. MLGema is also inexpensive and easy to produce.     

Adhesion of Histoplasma capsulatum to pneumocytes and biofilm formation on an abiotic surface

The pathogenic fungus, Histoplasma capsulatum, causes the respiratory and systemic disease 'histoplasmosis'. This disease is primarily acquired via inhalation of aerosolized microconidia or hyphal fragments of H. capsulatum. Evolution of this respiratory disease depends on the ability of H. capsulatum yeasts to survive and replicate within alveolar macrophages. It is known that adhesion to host cells is the first step in colonization and biofilm formation. Some microorganisms become attached to biological and non-biological surfaces due to the formation of biofilms. Based on the importance of biofilms and their persistence on host tissues and cell surfaces, the present study was designed to investigate biofilm formation by H. capsulatum yeasts, as well as their ability to adhere to pneumocyte cells. H. capsulatum biofilm assays were performed in vitro using two different clinical strains of the fungus and biofilms were characterized using scanning electron microscopy. The biofilms were measured using a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium-hydroxide (XTT) reduction assay. The results showed that both the H. capsulatum strains tested were very efficient at adhering to host cells and forming biofilm. Therefore, this is a possible survival strategy adopted by this fungus.     

Identification and characterization of the gene encoding the Acidobacterium capsulatum major sigma factor

Acidobacterium capsulatum is an acid-tolerant, encapsulated, Gram-negative member of the ubiquitous, but poorly understood Acidobacteria phylum. Little is known about the genetics and regulatory mechanisms of A. capsulatum. To begin to address this gap, we identified the gene encoding the A. capsulatum major sigma factor, rpoD, which encodes a 597-amino acid protein with a predicted sequence highly similar to the major sigma factors of Solibacter usitatus Ellin6076 and Geobacter sulfurreducens PCA. Purified hexahistidine-tagged RpoD migrates at approximately 70 kDa under SDS-PAGE conditions, which is consistent with the predicted MW of 69.2 kDa, and the gene product is immunoreactive with monoclonal antibodies specific for either bacterial RpoD proteins or the N-terminal histidine tag. A. capsulatum RpoD restored normal growth to E. coli strain CAG20153 under conditions that prevent expression of the endogenous rpoD. These results indicate we have cloned the gene encoding the A. capsulatum major sigma factor and the gene product is active in E. coli.     

Autonomous replication of foreign DNA in Histoplasma capsulatum: role of native telomeric sequences.

Genetic transformation of the dimorphic pathogenic fungus Histoplasma capsulatum can result in chromosomal integration of the transforming DNA or the generation of multicopy linear plasmids carrying the transforming DNA. We showed previously that Escherichia coli plasmids do not replicate autonomously in H. capsulatum without significant modifications, one of which is the in vivo addition of Histoplasma telomeres at the termini of linear DNA. To address the requirements for autonomous replication in H. capsulatum, we constructed a circular E. coli plasmid containing adjacent inverted stretches of Histoplasma telomeric repeats separated by a unique restriction site. The linearized plasmid bearing telomeric termini was maintained in H. capsulatum without modification other than the addition of more telomeric sequence. We recovered the original plasmid in E. coli after removal of the telomeric termini by using engineered restriction sites. Thus, no special Histoplasma modification or sequence other than the telomeres was needed for autonomous replication in H. capsulatum. Additionally, this plasmid provides a shuttle vector that replicates autonomously in E. coli (as a circular plasmid) and in H. capsulatum (as a linear plasmid).     

A monoclonal antibody to Histoplasma capsulatum alters the intracellular fate of the fungus in murine macrophages

Monoclonal antibodies (MAbs) to a cell surface histone on Histoplasma capsulatum modify murine infection and decrease the growth of H. capsulatum within macrophages. Without the MAbs, H. capsulatum survives within macrophages by modifying the intraphagosomal environment. In the present study, we aimed to analyze the affects of a MAb on macrophage phagosomes. Using transmission electron and fluorescence microscopy, we showed that phagosome activation and maturation are significantly greater when H. capsulatum yeast are opsonized with MAb. The MAb reduced the ability of the organism to regulate the phagosomal pH. Additionally, increased antigen processing and reduced negative costimulation occur in macrophages that phagocytose yeast cells opsonized with MAb, resulting in more-efficient T-cell activation. The MAb alters the intracellular fate of H. capsulatum by affecting the ability of the fungus to regulate the milieu of the phagosome.     

Protein phosphorylation in submerged spores and vegetative mycelium of Streptomyces granaticolor

Abstract The phylogenetic position of an acidophilic chemo-organotrophic menaquinone-containing bacterium, Acidobacterium capsulatum , was studied on the basis of 16S rRNA gene sequence information. A. capsulatum showed the highest level of sequence similarity to Heliobacterium chlorum , a member of the Gram-positive group, yet this level was only 81%. Distance matrix tree analysis suggested that A. capsulatum belongs to a unique lineage deeply branching from the Chlamydia-Planctomyces group or from the Gram-positive line.     

Utilization and cell-surface binding of hemin by Histoplasma capsulatum

Histoplasma capsulatum, a dimorphic fungus capable of causing severe respiratory illness in immunocompromised individuals, resides in macrophages during mammalian infection. Previous studies suggest that siderophore-mediated iron transport may be important for the acquisition of iron from transferrin while the organism resides in macrophages. However, iron is also present as hemin in the intracellular environment of the macrophage and may serve as a major source of iron during infection. Thus the ability of H. capsulatum to use hemin and heme-containing compounds was examined. Histoplasma capsulatum G217B was iron-starved by adding the iron chelator deferoxamine mesylate to the culture. The addition of 10 microM hemin in the presence of deferoxamine mesylate restored growth to the levels seen in the absence of the chelator. Histoplasma capsulatum was also cultivated in an iron-limited, chemically defined medium without the addition of chelators and it was determined that the organism could also use hemoglobin as a sole source of iron. The method of iron internalization from heme was examined by measuring hemin binding to the yeast-cell surface. The ability of H. capsulatum to bind hemin was related to the nutritional status of the cells. Cells grown under iron-limited conditions bound more heme to the cell surface than did cells grown in medium without chelator. Pretreatment of iron-starved cells with proteinase K eliminated the ability of the organism to bind hemin. Additionally, the pre-incubation of iron-starved H. capsulatum with hemin eliminated the ability of these cells to remove hemin from the solution, although pre-incubation of cells with the iron-free form of hemin, protoporphyrin IX, only modestly affected the ability of the organism to bind hemin. These results suggest that H. capsulatum uses hemin as a sole source of iron and that one mechanism of iron acquisition involves a cell-surface receptor for hemin.     

Modulation of the macrophage oxidative burst by Histoplasma capsulatum

The production of reactive oxygen species by phagocytic cells is an important host defense against invading microorganisms. Because pathogens that achieve intracellular survival escape destruction by reactive oxidants, we investigated the relationship between the intracellular survival of H. capsulatum and the macrophage oxidative burst. H. capsulatum yeast failed to stimulate the release of reactive oxygen metabolites in unprimed murine macrophages despite extensive phagocytosis of the microorganisms. This effect was observed with live as well as heat-killed fungi over a wide range of yeast-to-macrophage ratios. Preincubation of murine macrophages with heat-killed H. capsulatum (but not with latex spheres), followed by incubation with unopsonized zymosan, resulted in inhibition of oxidative burst triggering without inhibition of zymosan phagocytosis. Ingestion of H. capsulatum yeast opsonized with the cognate mouse antibody resulted in significant oxidant release, suggesting that suppression of the respiratory burst may be circumvented through Fc-mediated phagocytosis.     

Intraspecific chromosomal variability in human pathogenic fungi, especially in Histoplasma capsulatum

The ploidy, karyotype, and chromosome length polymorphism (CLP) of human pathogenic fungi were revised with emphasis on Histoplasma capsulatum, the causative agent of the systemic mycosis, histoplasmosis. Currently, different systems of gel electrophoresis are being used to determine fungal electrokaryotypes (EK). By renaturation kinetic and genomic reconstruction in H. capsulatum strains (G-186AS and Downs), estimated genome sizes of 23 and 32 Mb were determined for both strains, respectively. The haploid state was proposed for both strains, although aneuploidy was suggested for the Downs strain. Contour-clamped homogeneous electric field (CHEF), field inversion gel electrophoresis (FIGE), and Southern blot using different probes showed the presence of six to seven chromosomes in the Downs strain (low virulence), whereas four chromosomes were identified in the G-186B strain (high virulence). The use of these methods in the three major H. capsulatum reference strains (G-217B and Downs from the United States of America, G-186B from Panama) revealed distinct chromosome sizes, from 0.5 to 5.7 Mb, with CLP associated with chromosomes size and mobility. Recently, by CHEF, using 19 H. capsulatum isolates from Latin-America and the G-186B strain, five to seven chromosomes with 1.1 to 11.2 Mb molecular sizes were revealed, which again suggested CLP in H. capsulatum. However, to elucidate the EKs polymorphism in H. capsulatum and its relationship with the isolates phenotype more studies are needed to understand the mechanisms controlling ploidy variability.     

Molecular epidemiology, pathogenesis, and genetics of the dimorphic fungus Histoplasma capsulatum

Histoplasma capsulatum, the causative agent of the most common systemic fungal infection, histoplasmosis, has become subject to increasing study in parallel with rising prevalence of human immunodeficiency. This review presents a summary of the advances made in the investigation of H. capsulatum genomics, molecular epidemiology, pathogenesis, and molecular genetics.