Pathogenic Yeast Form Research Articles

Kinase Inhibitor Library Screening Identifies the Cancer Therapeutic Sorafenib and Structurally Similar Compounds as Strong Inhibitors of the Fungal Pathogen Histoplasma capsulatum.

Histoplasma capsulatum is a dimorphic fungal pathogen endemic to the midwestern and southern United States. It causes mycoses ranging from subclinical respiratory infections to severe systemic disease, and is of particular concern for immunocompromised patients in endemic areas. Clinical management of histoplasmosis relies on protracted regimens of antifungal drugs whose effectiveness can be limited by toxicity. In this study, we hypothesize that conserved biochemical signaling pathways in the eukaryotic domain can be leveraged to repurpose kinase inhibitors as antifungal compounds. We conducted a screen of two kinase inhibitor libraries to identify compounds inhibiting the growth of Histoplasma capsulatum in the pathogenic yeast form. Our approach identified seven compounds with an elongated hydrophobic polyaromatic structure, five of which share a molecular motif including a urea unit linking a halogenated benzene ring and a para-substituted polyaromatic group. The top hits include the cancer therapeutic Sorafenib, which inhibits growth of Histoplasma in vitro and in a macrophage infection model with low host cell cytotoxicity. Our results reveal the possibility of repurposing Sorafenib or derivatives thereof as therapy for histoplasmosis, and suggest that repurposing of libraries developed for human cellular targets may be a fruitful source of antifungal discovery.

Opposing signaling pathways regulate morphology in response to temperature in the fungal pathogen Histoplasma capsulatum.

Phenotypic switching between 2 opposing cellular states is a fundamental aspect of biology, and fungi provide facile systems to analyze the interactions between regulons that control this type of switch. A long-standing mystery in fungal pathogens of humans is how thermally dimorphic fungi switch their developmental form in response to temperature. These fungi, including the subject of this study, Histoplasma capsulatum, are temperature-responsive organisms that utilize unknown regulatory pathways to couple their cell shape and associated attributes to the temperature of their environment. H. capsulatum grows as a multicellular hypha in the soil that switches to a pathogenic yeast form in response to the temperature of a mammalian host. These states can be triggered in the laboratory simply by growing the fungus either at room temperature (RT; which promotes hyphal growth) or at 37 °C (which promotes yeast-phase growth). Prior worked revealed that 15% to 20% of transcripts are differentially expressed in response to temperature, but it is unclear which transcripts are linked to specific phenotypic changes, such as cell morphology or virulence. To elucidate temperature-responsive regulons, we previously identified 4 transcription factors (required for yeast-phase growth [Ryp]1–4) that are required for yeast-phase growth at 37 °C; in each ryp mutant, the fungus grows constitutively as hyphae regardless of temperature, and the cells fail to express genes that are normally induced in response to growth at 37 °C. Here, we perform the first genetic screen to identify genes required for hyphal growth of H. capsulatum at RT and find that disruption of the signaling mucin MSB2 results in a yeast-locked phenotype. RNA sequencing (RNAseq) experiments reveal that MSB2 is not required for the majority of gene expression changes that occur when cells are shifted to RT. However, a small subset of temperature-responsive genes is dependent on MSB2 for its expression, thereby implicating these genes in the process of filamentation. Disruption or knockdown of an Msb2-dependent mitogen-activated protein (MAP) kinase (HOG2) and an APSES transcription factor (STU1) prevents hyphal growth at RT, validating that the Msb2 regulon contains genes that control filamentation. Notably, the Msb2 regulon shows conserved hyphal-specific expression in other dimorphic fungi, suggesting that this work defines a small set of genes that are likely to be conserved regulators and effectors of filamentation in multiple fungi. In contrast, a few yeast-specific transcripts, including virulence factors that are normally expressed only at 37 °C, are inappropriately expressed at RT in the msb2 mutant, suggesting that expression of these genes is coupled to growth in the yeast form rather than to temperature. Finally, we find that the yeast-promoting transcription factor Ryp3 associates with the MSB2 promoter and inhibits MSB2 transcript expression at 37 °C, whereas Msb2 inhibits accumulation of Ryp transcripts and proteins at RT. These findings indicate that the Ryp and Msb2 circuits antagonize each other in a temperature-dependent manner, thereby allowing temperature to govern cell shape and gene expression in this ubiquitous fungal pathogen of humans.

In Vitro Susceptibility of Berberine Combined with Antifungal Agents Against the Yeast Form of Talaromyces marneffei.

Talaromyces (Penicillium) marneffei can cause fatal disseminated infection in immunocompromised hosts. However, therapeutic strategies for the mycosis are limited. Reports of the other fungi suggest that berberine, a component of traditional herb, inhibitors interact with antifungal agents to improve the treatment outcomes. In the study, we evaluated the in vitro efficacy of berberine in combination with conventional antifungal agents against the pathogenic yeast form of T. marneffei. We demonstrate the synergistic effect of combination of berberine with fluconazole (52.38%), itraconazole (66.67%), voriconazole (71.43%), amphotericin B (71.43%) or caspofungin (52.38%) of T. marneffei strains, respectively. Time-kill curves confirmed the synergistic interaction, and no antagonistic was observed in all of the combinations. In conclusion, berberine could enhance the efficacy of conventional antifungal agents against the yeast form of T. marneffei in vitro. The results indicated berberine might have a potential role in combination therapy for talaromycosis.

Clotrimazole is highly effective in vitro against feline Sporothrix brasiliensis isolates.

Sporothrix brasiliensis, the most virulent species in the Sporothrix schenckii complex, is responsible for the ongoing epidemics of human and animal sporotrichosis in Brazil. Feline outbreaks are usually driven by S. brasiliensis and followed by extensive transmission to humans. Itraconazole is the first-line treatment for both feline and human sporotrichosis; however, reduced sensitivity is an emerging issue. Thus, we investigated the effect of the widely used antifungal clotrimazole - alone or in combination with itraconazole - against the pathogenic (yeast) form of feline and human S. brasiliensis isolates, in vitro. Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) values were determined for treatment with clotrimazole and itraconazole, as monotherapy or in combination. In addition, the effect of the drugs on neutral lipid levels and the yeast ultrastructure were evaluated by flow cytometry and transmission electron microscopy (TEM), respectively. The MIC and MFC values show that clotrimazole was more effective than itraconazole against feline S. brasiliensis isolates, while human isolates were more sensitive to itraconazole. Similarly to itraconazole, treatment with clotrimazole induced statistically significant neutral lipid accumulation in S. brasiliensis yeasts, and treated yeasts displayed irregularities in the cell membrane and a thicker cell wall when observed by TEM. Clotrimazole increased the antifungal activity of itraconazole in combination assays, with a synergistic effect for two feline isolates. The strong activity of clotrimazole against feline S. brasiliensis isolates suggests that this drug is potentially a new alternative for the treatment of feline sporotrichosis, alone or in combination with itraconazole.

Extensive Metabolic Remodeling Differentiates Non-pathogenic and Pathogenic Growth Forms of the Dimorphic Pathogen Talaromyces marneffei.

Fungal infections are an increasing public health problem, particularly in immunocompromised individuals. While these pathogenic fungi show polyphyletic origins with closely related non-pathogenic species, many undergo morphological transitions to produce pathogenic cell types that are associated with increased virulence. However, the characteristics of these pathogenic cells that contribute to virulence are poorly defined. Talaromyces marneffei grows as a non-pathogenic hyphal form at 25°C but undergoes a dimorphic transition to a pathogenic yeast form at 37°C in vitro and following inhalation of asexual conidia by a host. Here we show that this transition is associated with major changes in central carbon metabolism, and that these changes are correlated with increased virulence of the yeast form. Comprehensive metabolite profiling and 13C-labeling studies showed that hyphal cells exhibited very active glycolytic metabolism and contain low levels of internal carbohydrate reserves. In contrast, yeast cells fully catabolized glucose in the mitochondrial TCA cycle, and store excess glucose in large intracellular pools of trehalose and mannitol. Inhibition of the yeast TCA cycle inhibited replication in culture and in host cells. Yeast, but not hyphae, were also able to use myo-inositol and amino acids as secondary carbon sources, which may support their survival in host macrophages. These analyses suggest that T. marneffei yeast cells exhibit a more efficient oxidative metabolism and are capable of utilizing a diverse range of carbon sources, which contributes to their virulence in animal tissues, highlighting the importance of dimorphic switching in pathogenic yeast.

Miltefosine is active against Sporothrix brasiliensis isolates with in vitro low susceptibility to amphotericin B or itraconazole.

Sporotrichosis is a common mycosis caused by dimorphic fungi from the Sporothrix schenckii complex. In recent years, sporotrichosis incidence rates have increased in the Brazilian state of Rio de Janeiro, where Sporothrix brasiliensis is the species more frequently isolated from patients. The standard antifungals itraconazole and amphotericin B are recommended as first-line therapy for cutaneous/lymphocutaneous and disseminated sporotrichosis, respectively, although decreased sensitivity to these drugs in vitro was reported for clinical isolates of S. brasiliensis. Here, we evaluated the activity of the phospholipid analogue miltefosine - already in clinical use against leishmaniasis - towards the pathogenic yeast form of S. brasiliensis isolates with low sensitivity to itraconazole or amphotericin B in vitro. Miltefosine had fungicidal activity, with minimum inhibitory concentration (MIC) values of 1-2 µg ml(-1). Miltefosine exposure led to loss of plasma membrane integrity, and transmission electron microscopy (TEM) analysis revealed a decrease in cytoplasmic electron density, alterations in the thickness of cell wall layers and accumulation of an electron-dense material in the cell wall. Flow cytometry analysis using an anti-melanin antibody revealed an increase in cell wall melanin in yeasts treated with miltefosine, when compared with control cells. The cytotoxicity of miltefosine was comparable to those of amphotericin B, but miltefosine showed a higher selectivity index towards the fungus. Our results suggest that miltefosine could be an effective alternative for the treatment of S. brasiliensis sporotrichosis, when standard treatment fails. Nevertheless, in vivo studies are required to confirm the antifungal potential of miltefosine for the treatment of sporotrichosis.

In vitro interactions of calcineurin inhibitors with conventional antifungal agents against the yeast form of Penicillium marneffei.

Penicillium marneffei can cause a life-threatening disseminated mycosis in immunocompromised hosts. However, therapeutic strategies for the treatment of this infectious disease are limited. Reports of other fungi suggest that calcineurin inhibitors interact with antifungal agents to improve the treatment outcomes. Here, we evaluated the in vitro interaction of the calcineurin inhibitors cyclosporine A and tacrolimus (FK506) combined with conventional antifungal agents against the pathogenic yeast form of P. marneffei. We demonstrate that the combination of cyclosporine A with amphotericin B, itraconazole, or fluconazole was synergistic for 85, 65, and 30 % of P. marneffei strains, respectively. In contrast, no synergism was observed in all the combinations containing tacrolimus. Furthermore, antagonism was not observed for any combination. In conclusion, the therapeutic potential of a combinatory approach using the calcineurin inhibitor cyclosporine A with conventional antifungal drugs may lead to improved treatment regimens for P. marneffei infections. We propose that mechanism of action studies with cyclosporine A and antifungal agents is needed.

Transcriptomic reprogramming of genus Paracoccidioides in dimorphism and host niches

The thermodimorphic fungi Paracoccidioides brasiliensis and Paracoccidioides lutzii are the etiologic agents of Paracoccidioidomycosis (PCM), the most important endemic systemic mycosis in Latin America. Paracoccidioides grows as saprophytic mycelia that produce infective conidia propagules, which are inhaled into the lungs where the fungus converts to the pathogenic yeast form. From the lungs, Paracoccidioides may disseminate through blood and lymphatics to several other organs and tissues. During the last decade we have witnessed the generation of a large amount of transcriptomic data regarding the events leading to the morphological transition and host niche adaptation. In this review we summarize those findings and discuss the consequence of gene expression plasticity in the persistence and survival of this pathogen. In addition, we discuss the future trends on the host–pathogen studies and how new molecular strategies, such as RNA-seq, dual RNA-seq and Chip-Seq can be powerful tools to improve our understanding on the pathobiology of this systemic mycosis in Latin America.

N-acetylglucosamine (GlcNAc) Triggers a Rapid, Temperature-Responsive Morphogenetic Program in Thermally Dimorphic Fungi

The monosaccharide N-acetylglucosamine (GlcNAc) is a major component of microbial cell walls and is ubiquitous in the environment. GlcNAc stimulates developmental pathways in the fungal pathogen Candida albicans, which is a commensal organism that colonizes the mammalian gut and causes disease in the setting of host immunodeficiency. Here we investigate GlcNAc signaling in thermally dimorphic human fungal pathogens, a group of fungi that are highly evolutionarily diverged from C. albicans and cause disease even in healthy individuals. These soil organisms grow as polarized, multicellular hyphal filaments that transition into a unicellular, pathogenic yeast form when inhaled by a human host. Temperature is the primary environmental cue that promotes reversible cellular differentiation into either yeast or filaments; however, a shift to a lower temperature in vitro induces filamentous growth in an inefficient and asynchronous manner. We found GlcNAc to be a potent and specific inducer of the yeast-to-filament transition in two thermally dimorphic fungi, Histoplasma capsulatum and Blastomyces dermatitidis. In addition to increasing the rate of filamentous growth, micromolar concentrations of GlcNAc induced a robust morphological transition of H. capsulatum after temperature shift that was independent of GlcNAc catabolism, indicating that fungal cells sense GlcNAc to promote filamentation. Whole-genome expression profiling to identify candidate genes involved in establishing the filamentous growth program uncovered two genes encoding GlcNAc transporters, NGT1 and NGT2, that were necessary for H. capsulatum cells to robustly filament in response to GlcNAc. Unexpectedly, NGT1 and NGT2 were important for efficient H. capsulatum yeast-to-filament conversion in standard glucose medium, suggesting that Ngt1 and Ngt2 monitor endogenous levels of GlcNAc to control multicellular filamentous growth in response to temperature. Overall, our work indicates that GlcNAc functions as a highly conserved cue of morphogenesis in fungi, which further enhances the significance of this ubiquitous sugar in cellular signaling in eukaryotes.

A Temperature-Responsive Network Links Cell Shape and Virulence Traits in a Primary Fungal Pathogen

Author SummaryMicrobial pathogens of humans display the ability to thrive at host temperature. So-called “thermally dimorphic” fungal pathogens, which include Histoplasma capsulatum, are a class of soil fungi that upon being inhaled into the human lung, undergo dramatic changes in cell shape and virulence gene expression in response to host temperature. The ability of these pathogens to cause disease is exquisitely coupled to temperature response. Here we elucidate the regulatory network that governs the ability of H. capsulatum to switch from a filamentous form in the soil environment to a pathogenic yeast form at body temperature. The circuit is driven by three transcription regulators (Ryp1, Ryp2, and Ryp3) that control yeast-phase growth. We show that these factors, which include two highly conserved proteins of the Velvet family of unknown function, bind to specific regulatory DNA elements and directly regulate expression of virulence genes. We identify and characterize Ryp4, a fourth regulator of this pathway, and define DNA motifs that recruit these transcription factors to their temperature-responsive target genes. Our results provide a molecular understanding of how changes in cell shape are linked to expression of virulence genes in thermally dimorphic fungi.

Characterization of anMPLP6, a gene coding for a yeast phase specific, antigenic mannoprotein inPenicillium marneffei

A gene encoding an antigenic mannoprotein of Penicillium marneffei, MPLP6, was isolated by an antibody screening approach and characterized. The polypeptide chain containing deduced 220 amino acids has a predicted molecular mass of 24 kDa. It has high similarity to Mp1p, the first mannoprotein antigen isolated from P. marneffei. The polypeptide sequence presents the property of cell wall mannoproteins by containing a putative N-terminal signal peptide and potential O-linked glycosylation sites. However, absence of a GPI-anchored signal sequence suggested that this protein is secreted. The MPLP6 transcript was present specifically in the pathogenic yeast form. The transcript was completely absent in the mold phase and conidia. The fusion protein produced in E. coli was Western immunoblotted with P. marneffei-infected human sera and 95% of the patients' sera were positive in the assay. None of the sera obtained from patients with aspergillosis, tuberculosis, histoplasmosis or cryptococcosis tested positive. These results suggest that Mplp6 can be used as a marker in a serodiagnostic assay.

In vitro interactions of micafungin with amphotericin B, itraconazole or fluconazole against the pathogenic phase of Penicillium marneffei

Penicillium marneffei infection is an important disease among human immunodeficiency virus patients in south-east Asia, including southern China. However, therapeutic strategies are limited. Combination regimens with synergistic drugs could provide additional options for treating penicilliosis. We evaluated the in vitro efficacy of combining micafungin with amphotericin B, itraconazole or fluconazole against the pathogenic yeast form of P. marneffei. Twenty isolates of P. marneffei were assayed. Drug interactions were assessed with the chequerboard technique using the CLSI (formerly the NCCLS) microdilution method (M27-A2) with minor modifications. The fractional inhibitory concentration index (FICI) was used to classify drug interactions. Results were interpreted as follows: synergy, FICI < or =0.5; no interaction, FICI >0.5 and < or =4.0; or antagonism, FICI >4.0. The in vitro interactions of micafungin combined with itraconazole showed the highest percentage of synergic interaction (65%); for the micafungin/amphotericin B combination, 50% of the isolates had synergic interaction. Micafungin significantly enhanced the antifungal activity of amphotericin B and itraconazole against P. marneffei. Micafungin, however, did not enhance the activity of fluconazole and no synergism was observed with this combination. Antagonism was not detected for any of the antifungal combinations assayed. The results of this study suggest that micafungin enhances the efficacy of itraconazole or amphotericin B in vitro and indicate that an echinocandin, such as micafungin, might have a potential role in combination therapy among patients infected with P. marneffei.

Conserved factors Ryp2 and Ryp3 control cell morphology and infectious spore formation in the fungal pathogen Histoplasma capsulatum

The human fungal pathogen Histoplasma capsulatum grows in a sporulating filamentous form in the soil and, after inhalation of infectious spores, converts to a pathogenic yeast form inside host macrophages in response to temperature. Here we report the identification of two genes (RYP2 and RYP3) required for yeast-phase growth. Ryp2 and Ryp3 are homologous to each other and to the Velvet A family of regulatory proteins in Aspergillus species and other filamentous fungi. Wild-type H. capsulatum grows as filaments at room temperature and as yeast cells at 37 degrees C, but ryp2 and ryp3 mutants constitutively grow as filaments independent of temperature. RYP2 and RYP3 transcripts accumulate to higher levels at 37 degrees C than at room temperature. This differential expression is similar to the previously identified RYP1 transcript, which encodes a transcriptional regulator required for the yeast-phase expression program. Ryp1 associates with the upstream region of RYP2, and each of the three RYP genes is required for the differential expression of the others at 37 degrees C. In addition to responding to the elevated temperature of the mammalian host, RYP2 and RYP3 are essential for viable spore production and regulation of sporulation at room temperature. This regulatory function is strikingly similar to the role of the Aspergillus Velvet A protein family in spore development in response to light, with the notable distinction that the H. capsulatum circuit responds to temperature.

Temperature-induced switch to the pathogenic yeast form of Histoplasma capsulatum requires Ryp1, a conserved transcriptional regulator

Histoplasma capsulatum, a fungal pathogen of humans, switches from a filamentous spore-forming mold in the soil to a pathogenic budding-yeast form in the human host. This morphologic switch, which is exhibited by H. capsulatum and a group of evolutionarily related fungal pathogens, is regulated by temperature. Using insertional mutagenesis, we identified a gene, RYP1 (required for yeast phase growth), which is required for yeast-form growth at 37 degrees C. ryp1 mutants are constitutively filamentous irrespective of temperature. Ryp1 is a member of a family of fungal proteins that includes Wor1, a master transcriptional regulator of the white-opaque transition required for mating in Candida albicans. Ryp1 associates with its own upstream regulatory region, consistent with a direct role in transcriptional control, and both the protein and its transcript accumulate to high levels in wild-type yeast-phase cells. Microarray analysis demonstrated that Ryp1 is required for the expression of the vast majority of yeast-specific genes, including two genes linked to virulence. Thus, Ryp1 appears to be a critical transcriptional regulator of a temperature-regulated morphologic switch in H. capsulatum.

The cAMP pathway is important for controlling the morphological switch to the pathogenic yeast form of Paracoccidioides brasiliensis

Paracoccidioides brasiliensis is a human pathogenic fungus that switches from a saprobic mycelium to a pathogenic yeast. Consistent with the morphological transition being regulated by the cAMP-signalling pathway, there is an increase in cellular cAMP levels both transiently at the onset (< 24 h) and progressively in the later stages (> 120 h) of the transition to the yeast form, and this transition can be modulated by exogenous cAMP. We have cloned the cyr1 gene encoding adenylate cyclase (AC) and established that its transcript levels correlate with cAMP levels. In addition, we have cloned the genes encoding three Gα (Gpa1–3), Gβ (Gpb1) and Gγ (Gpg1) G proteins. Gpa1 and Gpb1 interact with one another and the N-terminus of AC, but neither Gpa2 nor Gpa3 interacted with Gpb1 or AC. The interaction of Gpa1 with Gpb1 was blocked by GTP, but its interaction with AC was independent of bound nucleotide. The transcript levels for gpa1, gpb1 and gpg1 were similar in mycelium, but there was a transient excess of gpb1 during the transition, and an excess of gpa1 in yeast. We have interpreted our findings in terms of a novel signalling mechanism in which the activity of AC is differentially modulated by Gpa1 and Gpb1 to maintain the signal over the 10 days needed for the morphological switch.

Developmental regulation of the glyoxylate cycle in the human pathogen Penicillium marneffei

Penicillium marneffei is a thermally dimorphic opportunistic human pathogen with a saprophytic filamentous hyphal form at 25 degrees C and a pathogenic unicellular yeast form at 37 degrees C. During infection. P. marneffei yeast cells exist intracellularly in macrophages. To cope with nutrient deprivation during the infection process, a number of pathogens employ the glyoxylate cycle to utilize fatty acids as carbon sources. The genes which constitute this pathway have been implicated in pathogenesis. To investigate acetate and fatty acid utilization, the acuD gene encoding a key glyoxylate cycle enzyme (isocitrate lyase) was cloned. The acuD gene is regulated by both carbon source and temperature in P. marneffei, being strongly induced at 37 degrees C even in the presence of a repressing carbon source such as glucose. When introduced into the non-pathogenic monomorphic fungus Aspergillus nidulans, the P. marneffei acuD promoter only responds to carbon source. Similarly, when the A. nidulans acuD promoter is introduced into P. marneffei it only responds to carbon source suggesting that P. marneffei possesses both cis elements and trans-acting factors to control acuD by temperature. The Zn(II)2Cys6 DNA binding motif transcriptional activator FacB was cloned and is responsible for carbon source-, but not temperature-, dependent induction of acuD. The expression of acuD at 37 degrees C is induced by AbaA, a key regulator of morphogenesis in P. marneffei, but deletion of abaA does not completely eliminate temperature-dependent induction, suggesting that acuD and the glyoxylate cycle are regulated by a complex network of factors in P. marneffei which may contribute to its pathogenicity.

Calcium Binding by the Essential Virulence Factor BAD-1 of Blastomyces dermatitidis

BAD-1 (Blastomyces adhesin 1), a 120-kDa protein of Blastomyces dermatitidis, functions as an adhesin, immune modulator, and essential virulence factor. Structurally, BAD-1 is composed of a short N-terminal region, a core of 30 tandem repeats critical for virulence, and a C-terminal epidermal growth factor domain that binds the protein to yeast cell surface chitin. Each of the 30 acidic residue-rich tandem repeats contains a sequence that resembles the calcium-binding loop of the EF-hand domain found in many calcium-binding proteins. Here, we investigated the binding of calcium by BAD-1 and its biological significance. Yeast washed with double distilled H2O released surface-bound BAD-1, but EGTA washes were an order of magnitude more efficient, suggesting an interaction between BAD-1 and calcium. Immobilized BAD-1 was stained with ruthenium red dye, an indicator of calcium-binding proteins. In equilibrium dialysis, BAD-1 bound 45Ca2+ with an affinity of 0.41 x 10(-5) m and a capacity of 27 calcium/mol. Mass spectrometry confirmed this capacity. Elevated [Ca2+] diminished BAD-1 solubility. Upon deletion of its C-terminal epidermal growth factor-like domain, BAD-1 resisted aggregation by elevated [Ca2+] but retained its affinity and capacity for calcium. Removing 20 copies of the tandem repeat, however, sharply reduced the capacity of BAD-1 for calcium. Growth of the bad-1 null yeast was inhibited by 5 mm EGTA, and re-expression of BAD-1 in trans or the addition of exogenous purified BAD-1 restored growth. Thus, BAD-1 is a high capacity calcium-binding protein. This property contributes to the structure and function of BAD-1, as well as to B. dermatitidis acquisition of calcium from the environment.

Transcriptional Profiles of the Human Pathogenic Fungus Paracoccidioides brasiliensis in Mycelium and Yeast Cells

Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis, a disease that affects 10 million individuals in Latin America. This report depicts the results of the analysis of 6,022 assembled groups from mycelium and yeast phase expressed sequence tags, covering about 80% of the estimated genome of this dimorphic, thermo-regulated fungus. The data provide a comprehensive view of the fungal metabolism, including overexpressed transcripts, stage-specific genes, and also those that are up- or down-regulated as assessed by in silico electronic subtraction and cDNA microarrays. Also, a significant differential expression pattern in mycelium and yeast cells was detected, which was confirmed by Northern blot analysis, providing insights into differential metabolic adaptations. The overall transcriptome analysis provided information about sequences related to the cell cycle, stress response, drug resistance, and signal transduction pathways of the pathogen. Novel P. brasiliensis genes have been identified, probably corresponding to proteins that should be addressed as virulence factor candidates and potential new drug targets.

Selective expression of the virulence factor BAD1 upon morphogenesis to the pathogenic yeast form of Blastomyces dermatitidis: evidence for transcriptional regulation by a conserved mechanism.

Most dimorphic fungal pathogens grow as non-pathogenic moulds in soil and convert to pathogenic yeast in the host, suggesting that virulence factors are upregulated during phase transition. Such factors have been difficult to identify. We analysed BAD1 (formerly WI-1), a virulence factor in the dimorphic fungus Blastomyces dermatitidis, for expression in yeast and mycelial morphotypes. BAD1 was expressed in yeast but not in mycelia of North American strains of B. dermatitidis, and this expression pattern was confirmed for BAD1 transcript. BAD1 under the control of its promoter was transferred into African B. dermatitidis lacking a native BAD1 locus, and phase-specific expression was conserved. Sequence similarity was identified between the BAD1 promoter and the promoters of two yeast phase-specific genes in Histoplasma capsulatum. In H. capsulatum BAD1 transformants, yeast phase-specific expression of BAD1 was conserved, and no transcript was detected in mycelia. BAD1 beta-galactosidase reporter fusions analysed in B. dermatitidis and H. capsulatum confirmed that BAD1 is transcriptionally regulated in both fungi. BAD1 promoter activity and surface BAD1 expression were detected 6 h after shifting mycelia to 37 degrees C. Thus, BAD1 is expressed after transition to the pathogenic yeast morphotype and is regulated by a mechanism for phase-specific gene expression that appears to be conserved.

Host organism defense by a peptide-polysaccharide extracted from the fungus Sporothrix schenckii.

A peptide-polysaccharide, a peptide-rhamnomannan, was isolated from the pathogenic yeast form of the fungus Sporothrix schenckii. This substance, which may play a role in fungal virulence, was tested in an animal model of systemic disease, and depression of the immune response was observed in the animals between the 4th and 6th week of infection. Concomitantly, this compound showed mitogenic activity when challenged with normal lymphocytes and was also found to be involved in the inflammatory response. These results provide further information for the understanding of fungal implantation in tissues and of the pathogenicity of this systemic mycosis.