Dimorphism in Histoplasma capsulatum: a model for the study of cell differentiation in pathogenic fungi.

Several fungi can assume either a filamentous or a unicellular morphology in response to changes in environmental conditions. This process, known as dimorphism, is a characteristic of several pathogenic fungi, e.g., Histoplasma capsulatum, Blastomyces dermatitidis, and Paracoccidioides brasiliensis, and appears to be directly related to adaptation from a saprobic to a parasitic existence. H. capsulatum is the most extensively studied of the dimorphic fungi, with a parasitic phase consisting of yeast cells and a saprobic mycelial phase. In culture, the transition of H. capsulatum from one phase to the other can be triggered reversibly by shifting the temperature of incubation between 25 degrees C (mycelia) and 37 degrees C (yeast phase). Mycelia are found in soil and never in infected tissue, in contrast to the yeast phase, which is the only form present in patients. The temperature-induced phase transition and the events in establishment of the disease state are very likely to be intimately related. Furthermore, the temperature-induced phase transition implies that each growth phase is an adaptation to two critically different environments. A fundamental question concerning dimorphism is the nature of the signal(s) that responds to temperature shifts. So far, both the responding cell component(s) and the mechanism(s) remain unclear. This review describes the work done in the last several years at the biochemical and molecular levels on the mechanisms involved in the mycelium to yeast phase transition and speculates on possible models of regulation of morphogenesis in dimorphic pathogenic fungi.     

Regulation of dimorphism in Histoplasma capsulatum by cyclic adenosine 3',5'-monophosphate.

During temperature-induced transition of the dimorphic pathogenic fungus Histoplasma capsulatum from the single yeast cell form to the multicellular mycelial form, there was an increase in intracellular cyclic adenosine 3',5'-monophosphate (cAMP) levels as well as a striking accumulation of cAMP in the medium. cAMP levels also changed during the reverse mycelium-to-yeast transition.     

Insights into the pathogenicity of Penicillium marneffei.

Penicillium marneffei is a significant pathogen of AIDS patients in Southeast Asia. This fungus is unique in that it is the only dimorphic member of the genus. Pathogenesis of P. marneffei requires the saprobic mold form to undergo a morphological change upon tissue invasion. The in vivo form of this fungus reproduces as a fission yeast that capably evades the host immune system. The processes that control these morphological changes, better termed as phase transition, can be replicated in vitro by incubation of the mold form at 37 degrees C. The unidentified molecular mechanisms regulating phase transition in this fungus are now being uncovered using modern methodologies and novel strategies. A better comprehension of these underlying regulatory pathways will provide insight into eukaryotic cellular development as well as the potential factors responsible for infections caused by P. marneffei and other fungi. Such knowledge may lead to better chemotherapeutic interventions of fungal diseases.     

Expression and interstrain variability of the YPS3 gene of Histoplasma capsulatum

The YPS3 locus of the dimorphic fungus Histoplasma capsulatum encodes a secreted and surface-localized protein specific to the pathogenic yeast phase. In this study we examined this locus in 32 H. capsulatum strains and variants. Although protein production is limited to a select group of strains, the North American restriction fragment length polymorphism class 2/NAm 2 isolates, the locus was present in all the strains we examined. The YPS3 gene is well conserved in its 5' and 3' regions but displays an intragenic hypervariable region of tandem repeats that fluctuates in size between strains. This feature is similar to that seen with genes encoding several cell surface proteins in other fungi.     

Purification and characterization of a cysteine dioxygenase from the yeast phase of Histoplasma capsulatum

A cysteine dioxygenase, cysteine oxidase (EC 1.13.11.20), has been purified from the cytosolic fraction of yeast phase cells of the dimorphic fungus Histoplasma capsulatum. The cysteine oxidase is an iron-containing dioxygenase with a molecular weight of 10500 (+/- 1500) and is present only in the yeast phase of the fungus. The enzyme is highly specific for L-cysteine, with a Km of 2 X 10(-5) M in vitro. The product of cysteine oxidation is cysteinesulfinic acid, as analyzed by thin-layer chromatography and mass spectroscopy. To our knowledge, this is the first cysteine oxidase isolated from a fungus, and it probably plays an important role in the mycelial to yeast phase transition of H. capsulatum during which redox potential and cysteine levels are crucial factors.     

Antibiotic sensitivity of the yeast and mycelial phases of Histoplasma capsulatum.

Cycloheximide and chloramphenicol in combination have a greater effect on yeast cells of Histoplasma capsulatum than on the mycelial phase of this fungus. In contrast, clotrimazole was found to be more effective against mycelia. Miconazole produced a pronounced effect against both phases wheras tolnaftate was only slightly active. Sulfadiazine and griseofulvin were completely inactive against both phases. The differential sensitivities of the 2 phases of H. capsulatum to various antibiotics can be useful in studying the transition of the dimorphic fungus from 1 phase to the other.     

Histoplasma capsulatum molecular genetics, pathogenesis, and responsiveness to its environment

Histoplasma capsulatum is a thermally dimorphic ascomycete that is a significant cause of respiratory and systemic disease in mammals including humans, especially immunocompromised individuals such as AIDS patients. As an environmental mold found in the soil, it is a successful member of a competitive polymicrobial ecosystem. Its host-adapted yeast form is a facultative intracellular pathogen of mammalian macrophages. H. capsulatum faces a variety of environmental changes during the course of infection and must survive under harsh conditions or modulate its microenvironment to achieve success as a pathogen. Histoplasmosis may be considered the fungal homolog of the bacterial infection tuberculosis, since both H. capsulatum and Mycobacterium tuberculosis exploit the macrophage as a host cell and can cause acute or persistent pulmonary and disseminated infection and reactivation disease. The identification and functional analysis of biologically or pathogenically important H. capsulatum genes have been greatly facilitated by the development of molecular genetic experimental capabilities in this organism. This review focuses on responsiveness of this fungus to its environment, including differential expression of genes and adaptive phenotypic traits.     

Temperature- and cyclic nucleotide-induced phase transitions of Histoplasma capsulatum.

The transition from yeast to mycelia of Histoplasma capsulatum could be accomplished by shifting the temperature of incubation from 37 to 25 degrees C. It was accompanied by many changes in cellular metabolism, including changes in respiration, intracellular cyclic adenosine 3',5'-monophosphate (cAMP) levels, and activities of two enzymes specific for the yeast phase, cystine reductase (EC 1.6.4.1) and cysteine oxidase (EC 1.13.11.20). Even at 37 degrees C, the yeast to mycelial transition could be induced by cAMP and agents which raise the intracellular levels of cAMP (theophylline, acetylsalicylic acid, prostaglandin E1, and nerve growth factor). During this morphogenesis the same pattern of changes occurred as in the temperature-induced transition. Therefore, these changes were not simply dependent on a shift in temperature, but rather were part of the process of the phase transition.     

Characterization of cerebrosides from the thermally dimorphic mycopathogen Histoplasma capsulatum: expression of 2-hydroxy fatty N-acyl (E)-Delta(3)-unsaturation correlates with the yeast-mycelium phase transition

Cerebroside (monohexosylceramide) components were identified in neutral lipids extracted from both the yeast and mycelial forms of the thermally dimorphic mycopathogen Histoplasma capsulatum. The components were purified from both forms and their structures elucidated by 1- and 2-dimensional nuclear magnetic resonance (NMR) spectroscopy, electrospray ionization mass spectrometry (ESI-MS), and low energy tandem collision-induced dissociation mass spectrometry (ESI-MS/CID-MS). Both components were characterized as beta-glucopyranosylceramides (GlcCers) containing (4E,8E)-9-methyl-4,8-sphingadienine as the long-chain base, attached to 18-carbon 2-hydroxy fatty N-acyl components. However, while the fatty acid of the yeast form GlcCer was virtually all N-2'-hydroxyoctadecanoate, the mycelium form GlcCer was characterized by almost exclusive expression of N-2'-hydroxy-(E)-delta(3)-octadecenoate. These results suggest that the yeast-mycelium transition is accompanied by up-regulation of an as yet uncharacterized ceramide or cerebroside 2-hydroxy fatty N-acyl (E)-delta(3)-desaturase activity. They also constitute further evidence for the existence of two distinct pathways for ceramide biosynthesis in fungi, since glycosylinositol phosphorylceramides (GIPCs), the other major class of fungal glycosphingolipids, are found with ceramides consisting of 4-hydroxysphinganine (phytosphingosine) and longer chain 2-hydroxy fatty acids. In addition to identification of the major glucocerebroside components, minor components (< 5%) detectable by molecular weight differences in the ESI-MS profiles were also characterized by tandem ESI-MS/CID-MS analysis. These minor components were identified as variants differing in fatty acyl chain length, or the absence of the sphingoid 9-methyl group or (E)-delta(8)-unsaturation, and are hypothesized to be either biosynthetic intermediates or the result of imperfect chemical transformation by the enzymes responsible for these features. Possible implications of these findings with respect to chemotaxonomy, compartmentalization of fungal glycosphingolipid biosynthetic pathways, and regulation of morphological transitions in H.capsulatum and other dimorphic fungi are discussed.     

Protein Profiling of the Dimorphic Pathogenic Fungus, Sporothrix schenckii

Sporotrichosis is a common cutaneous mycosis caused by the dimorphic fungus Sporothrix schenckii, which exhibits a temperature-dependent dimorphic switch. At 25°C, it grows in a mycelial phase, while at 37°C, it forms unicellular yeast cells. The formation of yeast cells was thought to be a requisite for the pathogenicity of S. schenckii. To identify fragments that might be related to morphogenesis, whole-cell proteins from the mold and early yeast stages of S. schenckii were analyzed using 2DE. Among thousands of protein molecules displayed, more than 300 showed a differential expression between the two phases. In particular, 24 yeast-specific proteins were identified using MALDI-TOF/MS. One of the most interesting proteins was a hybrid histidine kinase, DRK1, a global regulator of dimorphism and virulence in Blastomyces dermatitidis and Histoplasma capsulatum that was abundant in the yeast phase. Our study introduced a new approach to study dimorphism in S. schenckii, and the data may help us better understand the molecular mechanisms of phase transition.     

Functional and genetic characterization of calmodulin from the dimorphic and pathogenic fungus Paracoccidioides brasiliensis

Calmodulin (CaM) modulates intracellular calcium signalling and acts on several metabolic pathways and gene expression regulation in many eukaryotic organisms including human fungal pathogens, such as Candida albicans and Histoplasma capsulatum. The temperature-dependent dimorphic fungus Paracoccidioides brasiliensis is the aetiological agent of paracoccidioidomycosis (PCM). The mycelium (M) to yeast (Y) transition has been shown to be essential for establishment of the infection, although the precise molecular mechanisms of dimorphism in P. brasiliensis are still unknown. In this work, several inhibitory drugs of the Ca(2+)/calmodulin signalling pathway were tested to verify the role of this pathway in the cellular differentiation process of P. brasiliensis. EGTA and the drugs calmidazolium (R24571), trifluoperazine (TFP), and W7 were able to inhibit the M-Y transition. We have cloned and characterized the calmodulin gene from P. brasiliensis, which comprises 924 nucleotides and five introns that are in a conserved position among calmodulin genes.