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.

Characterization of the APSES-family transcriptional regulators of Histoplasma capsulatum.

The fungal APSES protein family of transcription factors is characterized by a conserved DNA-binding motif facilitating regulation of gene expression in fungal development and other biological processes. However, their functions in the thermally dimorphic fungal pathogen Histoplasma capsulatum are unexplored. Histoplasma capsulatum switches between avirulent hyphae in the environment and virulent yeasts in mammalian hosts. We identified five APSES domain-containing proteins in H. capsulatum homologous to Swi6, Mbp1, Stu1 and Xbp1 proteins and one protein found in related Ascomycetes (APSES-family protein 1; Afp1). Through transcriptional analyses and RNA interference-based functional tests we explored their roles in fungal biology and virulence. Mbp1 serves an essential role and Swi6 contributes to full yeast cell growth. Stu1 is primarily expressed in mycelia and is necessary for aerial hyphae development and conidiation. Xbp1 is the only factor enriched specifically in yeast cells. The APSES proteins do not regulate conversion of conidia into yeast and hyphal morphologies. The APSES-family transcription factors are not individually required for H. capsulatum infection of cultured macrophages or murine infection, nor do any contribute significantly to resistance to cellular stresses including cell wall perturbation, osmotic stress, oxidative stress or antifungal treatment. Further studies of the downstream genes regulated by the individual APSES factors will be helpful in revealing their functional roles in H. capsulatum biology.

A Case of Disseminated Histoplasmosis in a Patient with Rheumatoid Arthritis on Abatacept.

Biologic agents are effective treatments for rheumatoid arthritis but are associated with important risks, including severe infections. Tumor Necrosis Factor (TNF) α inhibitors are known to increase the risk of systemic fungal infections such as disseminated histoplasmosis. Abatacept is a biologic agent with a mechanism different from that of TNFα inhibitors: It suppresses cellular immunity by competing for the costimulatory signal on antigen-presenting cells. The risk of disseminated histoplasmosis for patients on abatacept is not known. We report a case of abatacept-associated disseminated histoplasmosis and review the known infectious complications of abatacept. While the safety of resuming biologic agents following treatment for disseminated histoplasmosis is also not known, abatacept is recommended over TNFα inhibitors for rheumatoid arthritis patients with a prior serious infection. We discuss the evidence supporting this recommendation and discuss alternative treatments for rheumatoid arthritis patients with a history of a serious infection.

L-tyrosine induces the production of a pyomelanin-like pigment by the parasitic yeast-form of Histoplasma capsulatum.

Melanization of Histoplasma capsulatum remains poorly described, particularly in regards to the forms of melanin produced. In the present study, 30 clinical and environmental H. capsulatum strains were grown in culture media with or without L-tyrosine under conditions that produced either mycelial or yeast forms. Mycelial cultures were not melanized under the studied conditions. However, all strains cultivated under yeast conditions produced a brownish to black soluble pigment compatible with pyomelanin when grew in presence of L-tyrosine. Sulcotrione inhibited pigment production in yeast cultures, strengthening the hyphothesis that H. capsulatum yeast forms produce pyomelanin. Since pyomelanin is produced by the fungal parasitic form, this pigment may be involved in H. capsulatum virulence.

Differentiation of the fungus Histoplasma capsulatum into a pathogen of phagocytes.

Mammalian body temperature triggers differentiation of the fungal pathogen Histoplasma capsulatum into yeast cells. The Drk1 regulatory kinase and an interdependent network of Ryp transcription factors establish the yeast state. Beyond morphology, the differentiation-dependent expression program equips yeasts for invasion and survival within phagosomes. Yeast cells produce α-glucan and the Eng1 endoglucanase which hide yeasts from immune detection. Secretion of yeast phase-specific Sod3 and CatB detoxify phagocyte-derived reactive oxygen molecules. Histoplasma cells adapt to iron and zinc limitation in activated macrophages by production of siderophores and the Zrt2 transporter, respectively. Yeasts also respond to inflammation-associated hypoxia. Histoplasma pathogenicity thus relies on factors controlled by yeast differentiation as well as environment-dependent responses.

Investigation of an optimal cell lysis method for the study of the zinc metalloproteome of Histoplasma capsulatum.

This work sought to assess optimal extraction conditions in the study of the metalloproteome of the dimorphic fungus Histoplasma capsulatum. One of the body's responses to H. capsulatum infection is sequestration of zinc within host macrophage (MØ), as reported by Vignesh et al. (Immunity 39:697-710, 2013) and Vignesh et al. (PLOS Pathog 9:E1003815, 2013). Thus, metalloproteins containing zinc were of greatest interest as it plays a critical role in survival of the fungus. One challenge in metalloproteomics is the preservation of the native structure of proteins to retain non-covalently bound metals. Many of the conventional cell lysis, separation, and identification techniques in proteomics are carried out under conditions that could lead to protein denaturation. Various cell lysis techniques were investigated in an effort to both maintain the metalloproteins during lysis and subsequent analysis while, at the same time, serving to be strong enough to break the cell wall, allowing access to cytosolic metalloproteins. The addition of 1% Triton x-100, a non-ionic detergent, to the lysis buffer was also studied. Seven lysis methods were considered and these included: Glass Homogenizer (H), Bead Beater (BB), Sonication Probe (SP), Vortex with 1% Triton x-100 (V, T), Vortex with no Triton x-100 (V, NT), Sonication Bath, Vortex, and 1% Triton x-100 (SB, V, T) and Sonication Bath, Vortex, and no Triton x-100 (SB, V, NT). A Qubit® Assay was used to compare total protein concentration and inductively coupled plasma-mass spectrometry (ICP-MS) was utilized for total metal analysis of cell lysates. Size exclusion chromatography coupled to ICP-MS (SEC-HPLC-ICP-MS) was used for separation of the metalloproteins in the cell lysate and the concentration of Zn over a wide molecular weight range was examined. Additional factors such as potential contamination sources were also considered. A cell lysis method involving vortexing H. capsulatum yeast cells with 500 µm glass beads in a 1% Triton x-100 lysis buffer (V, T) was found to be most advantageous to extract intact zinc metalloproteins as demonstrated by the highest Zn to protein ratio, 1.030 ng Zn/µg protein, and Zn distribution among high, mid, and low molecular weights suggesting the least amount of protein denaturation. Graphical abstract In this work, several cell lysis techniques and two lysis buffers were investigated to evaluate the preservation of the zinc metalloproteome of H. capsulatum while maintaining compatibility with the analytical techniques employed.

Histoplasmosis mimicking metastatic spinal tumour.

Histoplasmosis is an infection caused by a fungus called Histoplasma. Diagnosis of histoplasmosis is based on the culture of biological samples and detection of fungus in tissues. Histoplasmosis can mimic malignant lesions. We report a 65-year-old, immunocompetent, male patient with back pain. We describe the main clinical and radiological characteristics in our patient who had vertebral histoplasmosis that mimicked cancer. A computed tomography scan showed lytic lesions of the right side of T4, T5, and T6 vertebral bodies. Magnetic resonance imaging displayed abnormal marrow signals in T4, T5, and T6 vertebral bodies (low signal on T1, high on T2 and short time inversion recovery (STIR)). Which was mimicking malignancy, such as haematological malignancy and metastatic bone cancer. Therefore, thoracic spinal surgery using the anterior approach was performed. An intraoperative frozen section examination and routine postoperative pathology showed thoracic histoplasmosis infection. Treatment of histoplasmosis was performed with oral itraconazole. The lesions did not progress and the patient symptomatically improved at a follow-up of 26 months.

The Mould-specific M46 gene is not essential for yeast-mould dimorphism in the pathogenic fungus Histoplasma capsulatum.

Histoplasma capsulatum (Hc) is the causative agent for the respiratory infection histoplasmosis. The fungus exists in the environment as a saprophytic multi-cellular mould. Spores are inhaled by mammals whereupon the organism will convert into the single-celled yeast morphotype resulting in infection. The shift to the yeast morphotype is required for pathogenesis. Most studies on dimorphism have examined yeast-phase-specific genes and few mould-phase-specific genes have been investigated. It is likely, that some mould-phase-specific genes must be downregulated for the yeast to form or upregulated for the mould to form. We isolated a strongly expressed mould-specific gene, M46, from an expression library enriched for mould upregulated genes in Hc strain G186AS. To determine if M46 is involved in dimorphism, M46 was ectopically expressed in yeast phase growing temperature, and an m46 knockout strain was created via allelic replacement. Ectopically expressing M46 in yeast, did not induce filamentous growth. Genomic disruption of M46 by allelic replacement did not alter the morphology of the mould as seen in bright field microscopy, scanning electron microscopy, and transmission electron microscopy. A growth curve study, revealed that M46 is not involved in maintaining the growth rate of cells. These findings indicate that the mould specific M46 gene is not necessary nor essential for dimorphism, maintaining the normal mould morphology, and growth rate of Histoplasma capsulatum.

Revisiting old friends: Developments in understanding Histoplasma capsulatum pathogenesis.

Histoplasma capsulatum is a dimorphic pathogenic fungus and causative agent of histoplasmosis, which is a respiratory and systemic infection that is particularly severe in immunocompromised hosts and represents the fungal homolog of tuberculosis. In highly endemic regions, the majority of individuals have been infected and carry the organism in a persistent latent form that is a danger for reactivation if host defenses are suppressed. H. capsulatum has been a model organism for intracellular pathogenesis and fungal morphogenesis for decades. New genomic information and application of approaches for molecular genetic manipulation are shedding new light on virulence mechanisms.

Sexual variability in Histoplasma capsulatum and its possible distribution: What is going on? / Variabilidad sexual de Histoplasma capsulatum y su posible distribución: fundamento

Histoplasma capsulatum is a dimorphic fungal pathogen naturally found in the soil. Inhalation of conidia can result in pulmonary histoplasmosis and, in some cases, causes severe disseminated disease and death. This fungus is an ascomycete that has an anamorphic or asexual stage and a teleomorphic or sexual stage, known as Ajellomyces capsulatus, which results from (+) and (−) mating types. Sexual reproduction is regulated by a specialized genomic region known as the mating-type (MAT1) locus. The mating process in this heterothallic species is represented by isolates that contain only one of the two different MAT1 locus idiomorphs (MAT1-1 or MAT1-2) that have unrelated sequences encoding different transcription factors. In medically important dimorphic pathogens and in most ascomycete molds, one MAT locus idiomorph encodes a high-mobility-group (HMG) box-domain transcription factor, and the other idiomorph encodes an alpha-box domain transcription factor. There is scarce molecular information about H. capsulatum mating type although recombinant population structures have been reported that could occur in nature and this process has been documented in distinct models such as parasites and other fungi. In this review, we shall focus on published studies on H. capsulatum sexuality, and outline the distribution of the two H. capsulatum mating types in Latin America (AU)

Histoplasma capsulatum es un patógeno fúngico, dimórfico que habita en suelos ricos en materia orgánica. La inhalación de los conidios puede inducir histoplasmosis pulmonar y, en algunos casos, enfermedad diseminada grave y la muerte. Este ascomiceto caracterizado por un estadio anamórfico asexual y un estado teleomórfico o sexual, conocido como Ajellomyces capsulatus, que es consecuencia de los tipos de apareamiento (MAT+ y MAT−) (mating-type, por sus siglas en inglés). La reproducción sexual está regulada por una región genómica especializada, conocida como locus MAT1. El proceso de apareamiento en esta especie heterotálica (o autoincompatible) está representado por aislamientos que solo contienen uno de los 2 diferentes idiomorfos del locus MAT1 (MAT1-1 y MAT1-2), que tienen secuencias muy distintas que codifican diferentes factores de transcripción. En los patógenos dimórficos importantes desde un punto de vista médico y en la mayoría de los ascomicetos filamentosos, un idiomorfo del locus MAT codifica el dominio-caja HMG (high-mobility-group, por sus siglas en inglés) de un factor de transcripción, y el otro idiomorfo codifica el dominio-caja alfa de otro factor de transcripción. Apenas disponemos de información molecular sobre el mating type de H. capsulatum, aunque se ha descrito que en la naturaleza podrían estar presentes estructuras de población recombinante. Este proceso se ha documentado en distintos modelos como parásitos y otros hongos. En esta revisión nos hemos centrado en los estudios publicados sobre la sexualidad de H. capsulatum, y hemos abordado la distribución de los 2 mating type de H. capsulatum en Sudamérica (AU)

Comparison of PCR protocols for detecting Histoplasma capsulatum DNA through a multicenter study

Background. A multicenter study was conducted. A panel containing DNA from Histoplasma capsulatum, as well as negative and cross-reaction controls, was sent to five different laboratories, members of the MICOMOL network from CYTED Program. Aims. The objective was to assess the accuracy of different PCR protocols to detect H. capsulatum DNA. Methods. Seven different PCR protocols were tested. They were based on PCR techniques and used unicopy and multicopy targets. Results. Most of these protocols (4/7) were able to detect the smallest amounts of fungal DNA (102 fg/ml). Overall sensitivity was 86% and specificity was 100%. The protocol based on a unicopy target (SCAR220) presented lower sensitivity (43%) but 100% specificity. The real-time protocols tested were highly reproducible, sensitive, and specific. Neither false positives nor cross-reactions were detected in any protocol. Conclusions. All laboratories were able to amplify H. capsulatum DNA, and real-time PCR seems to be a promising tool to efficiently detect this pathogen in clinical samples (AU)

Use of image cytometry for quantification of pathogenic fungi in association with host cells.

Studies of the cellular pathogenesis mechanisms of pathogenic yeasts such as Candida albicans, Histoplasma capsulatum, and Cryptococcus neoformans commonly employ infection of mammalian hosts or host cells (i.e. macrophages) followed by yeast quantification using colony forming unit analysis or flow cytometry. While colony forming unit enumeration has been the most commonly used method in the field, this technique has disadvantages and limitations, including slow growth of some fungal species on solid media and low and/or variable plating efficiencies, which is of particular concern when comparing growth of wild-type and mutant strains. Flow cytometry can provide rapid quantitative information regarding yeast viability, however, adoption of flow cytometric detection for pathogenic yeasts has been limited for a number of practical reasons including its high cost and biosafety considerations. Here, we demonstrate an image-based cytometric methodology using the Cellometer Vision (Nexcelom Bioscience, LLC) for the quantification of viable pathogenic yeasts in co-culture with macrophages. Our studies focus on detection of two human fungal pathogens: Histoplasma capsulatum and Candida albicans. H. capsulatum colonizes alveolar macrophages by replicating within the macrophage phagosome, and here, we quantitatively assess the growth of H. capsulatum yeasts in RAW 264.7 macrophages using acridine orange/propidium iodide staining in combination with image cytometry. Our method faithfully recapitulates growth trends as measured by traditional colony forming unit enumeration, but with significantly increased sensitivity. Additionally, we directly assess infection of live macrophages with a GFP-expressing strain of C. albicans. Our methodology offers a rapid, accurate, and economical means for detection and quantification of important human fungal pathogens in association with host cells.

Rapid quantification of pathogenic fungi by Cellometer image-based cytometry.

The objective of this study was to develop an image-based cytometric methodology for the quantification of viable pathogenic yeasts, which can offer increased sensitivity and efficiency when compared to the traditional colony forming unit (CFU) assay. Live/dead yeast quantification by flow cytometry has been previously demonstrated, however, adoption of flow cytometric detection of pathogenic yeasts has been limited for a number of practical reasons including its high cost and biosafety considerations. Our studies focus on detection of two human fungal pathogens: Histoplasma capsulatum and Candida albicans. H. capsulatum colonizes alveolar macrophages by replicating within the macrophage phagosome. Here, we quantitatively assess the growth of H. capsulatum yeasts within RAW 264.7 macrophages using acridine orange/propidium iodide staining in combination with Cellometer image-based cytometry; this method faithfully recapitulates growth trends as measured by traditional CFU enumeration, but with significantly increased sensitivity. Additionally, we directly assess infection of bone marrow-derived macrophages with a GFP-expressing strain of C. albicans. To demonstrate that image-based cytometry can be used as a tool to assess the susceptibility of fungi to antifungal drugs, we perform dose response experiments with the antifungal drugs amphotericin B and itraconazole and show that image-based cytometry allows rapid assessment of the kinetics of cytotoxicity induced by these antifungals. Our methodology offers a rapid, accurate, and economical means for detection and quantification of important human fungal pathogens, either alone or in association with host cells.

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.

Actividad antifúngica in vitro del ajoeno en cinco aislamientos clínicos de Histoplasma capsulatum var. capsulatum / In vitro antifungal activity of ajoene on five clinical isolates of Histoplasma capsulatum var. capsulatum

Antecedentes. Con el advenimiento del sida, la histoplasmosis ha aumentado considerablemente y su tratamiento continúa siendo relativamente eficaz, si bien provoca efectos adversos. Objetivo. Evaluar el efecto inhibitorio del ajoeno sobre Histoplasma capsulatum, en fase micelial, utilizando como medios de cultivo caldo glucosado de Sabouraud y RPMI-1640. Métodos. Las curvas de crecimiento y el efecto inhibitorio del ajoeno (1,25-20mg/ml) se realizaron a temperatura ambiente, en agitación y se registraron turbidimétricamente (540nm) cada 48h, durante 14 días. Se construyeron gráficas para estimar el tiempo de generación (Tg), concentración mínima inhibitoria (CMI) y concentración inhibitoria 50% (CI50). La concentración fungicida mínima (CFM) se determinó por el método de cultivo en placas. Se usaron las pruebas de Mann-Whitney y T-test para evaluar la significación estadística entre los medios de cultivo y los parámetros Tg, CIM, CI50, CFM y efecto fungistático (EF) con un nivel de significación de 0,05. Resultados. Para los dos medios de cultivo, en todos los aislamientos se obtuvieron curvas de crecimiento con Tg de 43 a 67h, EF a concentraciones de 1,25 y 2,5mg/ml y CFM de 5 y 10mg/ml. Los valores de la CIM, en CSD fueron de 2,5 y 5 y en RPMI, de 1,25 a 5mg/ml. La CI50 en CSD fue de 1,9 a 2,6 y en RPMI, de 3,8 a 4,3mg/ml de ajoeno. No hubo diferencias significativas entre los medios de cultivo para los parámetros estudiados (p>0,05). Conclusiones. Los hallazgos del presente trabajo corroboran que el ajoeno inhibe el desarrollo de H. capsulatum en fase micelial(AU)

Background. The number of histoplasmosis cases have considerably increased since the advent of AIDS, and the therapy for this mycosis is not always effective, as well as having adverse effects. Aims. To evaluate the inhibitory effect of ajoene on five clinical isolates of Histoplasma capsulatum, on the mycelial form, using Sabouraud dextrose broth (SDB) and RPMI-1640 culture media. Methods. Growth curves and inhibitory activity of the drug (at concentrations of 1.25 mg/ml to 20mg/ml) were performed at room temperature, under mechanical agitation, and the turbidimetric readings (540nm) were recorded every 48h for 14 days, in both culture media. Generation times (GT) were calculated and graphs were constructed to estimate Minimal Inhibitory Concentrations (MIC) and Inhibitory Concentration 50% (IC50). The fungicidal minimal concentrations (FMC) were determined by plate cultures. The U-Mann-Whitney and t-test with a significance level of 0.05 were used to evaluate the statistical significance between culture media and GT, MIC, IC50 MFC and fungistatic effect (FE). Results. In both media and for all isolates, growth curves showed a GT of 43 to 67hrs, an FE at 1.25-2.5mg/ml, and a MFC at 5-10mg/ml of ajoene. Values of MIC were 2.5-5 in SDB and in RPMI medium these values were 1.25-5mg/ml of ajoene. For IC50, in SDB, the values were 1.9-2.6 mg/ml and in RPMI medium, they were of 3.8-4.3mg/ml of ajoene. There were no significance differences between culture media for GT, FE, MIC, IC50 and MFC (p>0.05). Conclusions. These findings corroborate that ajoene inhibits the growth of the mycelial form of H. capsulatum(AU)

Histoplasma capsulatum heat-shock 60 orchestrates the adaptation of the fungus to temperature stress.

Heat shock proteins (Hsps) are among the most widely distributed and evolutionary conserved proteins. Hsps are essential regulators of diverse constitutive metabolic processes and are markedly upregulated during stress. A 62 kDa Hsp (Hsp60) of Histoplasma capsulatum (Hc) is an immunodominant antigen and the major surface ligand to CR3 receptors on macrophages. However little is known about the function of this protein within the fungus. We characterized Hc Hsp60-protein interactions under different temperature to gain insights of its additional functions oncell wall dynamism, heat stress and pathogenesis. We conducted co-immunoprecipitations with antibodies to Hc Hsp60 using cytoplasmic and cell wall extracts. Interacting proteins were identified by shotgun proteomics. For the cell wall, 84 common interactions were identified among the 3 growth conditions, including proteins involved in heat-shock response, sugar and amino acid/protein metabolism and cell signaling. Unique interactions were found at each temperature [30°C (81 proteins), 37°C (14) and 37/40°C (47)]. There were fewer unique interactions in cytoplasm [30°C (6), 37°C (25) and 37/40°C (39)] and four common interactions, including additional Hsps and other known virulence factors. These results show the complexity of Hsp60 function and provide insights into Hc biology, which may lead to new avenues for the management of histoplasmosis.

Histoplasma capsulatum prosthetic valve endocarditis with negative fungal blood cultures and negative histoplasma antigen assay in an immunocompetent patient.

We report a case of Histoplasma capsulatum endocarditis in which Histoplasma antigen assay and fungal blood cultures were negative. The diagnosis was made by microscopic examination and culture of the excised valve. Histoplasma capsulatum should be considered in the differential diagnosis of culture-negative endocarditis in regions where it is endemic and in travelers.

Indigenous case of disseminated histoplasmosis from the Penicillium marneffei endemic area of China.

This is the first indigenous case of disseminated histoplasmosis reported from the Penicillium marneffei endemic area in southern China. It was diagnosed by histopathology of tissue, gross and microscopic morphology of the culture and PCR assay of the isolated fungus. Successful antifungal treatment was with itraconazole 400 mg/day for 5 months. This case suggests that histoplasmosis should be an important differential diagnosis in immunocompromised patients in southern China and South East Asia (the only endemic area for P. marneffei).