Diversification of Fungal Chitinases and Their Functional Differentiation in Histoplasma capsulatum.

Chitinases enzymatically hydrolyze chitin, a highly abundant and utilized polymer of N-acetyl-glucosamine. Fungi are a rich source of chitinases; however, the phylogenetic and functional diversity of fungal chitinases are not well understood. We surveyed fungal chitinases from 373 publicly available genomes, characterized domain architecture, and conducted phylogenetic analyses of the glycoside hydrolase (GH18) domain. This large-scale analysis does not support the previous division of fungal chitinases into three major clades (A, B, C) as chitinases previously assigned to the "C" clade are not resolved as distinct from the "A" clade. Fungal chitinase diversity was partly shaped by horizontal gene transfer, and at least one clade of bacterial origin occurs among chitinases previously assigned to the "B" clade. Furthermore, chitin-binding domains (including the LysM domain) do not define specific clades, but instead are found more broadly across clades of chitinases. To gain insight into biological function diversity, we characterized all eight chitinases (Cts) from the thermally dimorphic fungus, Histoplasma capsulatum: six A clade, one B clade, and one formerly classified C clade chitinases. Expression analyses showed variable induction of chitinase genes in the presence of chitin but preferential expression of CTS3 in the mycelial stage. Activity assays demonstrated that Cts1 (B-I), Cts2 (A-V), Cts3 (A-V), Cts4 (A-V) have endochitinase activities with varying degrees of chitobiosidase function. Cts6 (C-I) has activity consistent with N-acetyl-glucosaminidase exochitinase function and Cts8 (A-II) has chitobiase activity. These results suggest chitinase activity is variable even within subclades and that predictions of functionality require more sophisticated models.

Linoleate diol synthase related enzymes of the human pathogens Histoplasma capsulatum and Blastomyces dermatitidis.

Histoplasma capsulatum is an ascomyceteous fungus and a human lung pathogen, which is present in river valleys of the Americas and other continents. H. capsulatum and two related human pathogens, Blasmomyces dermatitidis and Paracoccidioides brasiliensis, belongs to the Ajellomycetaceae family. The genomes of all three species code for three homologous and tentative enzymes of the linoleate diol synthase (LDS) family of fusion enzymes with dioxygenase (DOX) and cytochrome P450 domains. One group aligned closely with 8R-DOX-5,8-LDS of Aspergilli, which oxidizes linoleic acid to 5S,8R-dihydroxylinoleic acid; this group was not further investigated. The second group aligned with 10R-DOX-epoxy alcohol synthase (EAS) of plant pathogens. Expression of this enzyme from B. dermatitidis revealed only 10R-DOX activities, i.e., oxidation of linoleic acid to 10R-hydroperoxy-8E,12Z-octadecadienoic acid. The third group aligned in a separate entity. Expression of these enzymes of H. capsulatum and B. dermatitidis revealed no DOX activities, but both enzymes transformed 13S-hydroperoxy-9Z,11E-octadecadienoic acid efficiently to 12(13S)epoxy-11-hydroperoxy-9Z-octadecenoic acid. Other 13-hydroperoxides of linoleic and α-linolenic acids were transformed with less efficiency and the 9-hydroperoxides of linoleic acid were not transformed. In conclusion, a novel EAS has been found in H. capsulatum and B. dermititidis with 13S-hydroperoxy-9Z,11E-octadecadienoic acid as the likely physiological substrate.

Two­component histidine kinase DRK1 is required for pathogenesis in Sporothrix schenckii.

Sporothrix schenckii is a pathogenic dimorphic fungus with a global distribution. It grows in a multicellular hyphal form at 25˚C and a unicellular yeast form at 37˚C. The morphological switch from mold to yeast form is obligatory for establishing pathogenicity in S. schenckii. Two­component signaling systems are utilized by eukaryotes to sense and respond to external environmental changes. DRK1is a hybrid histidine kinase, which functions as a global regulator of dimorphism and virulence in Blastomyces dermatitidis and Histoplasma capsulatum. An intracellular soluble hybrid histidine kinase, homologous to DRK1 in B. dermatitidis, has previously been identified in S. schenckii and designated as SsDRK1. In the present study, the function of SsDRK1 was investigated using double stranded RNA interference mediated by Agrobacterium tumefaciens. SsDRK1 was demonstrated to be required for normal asexual development, yeast­phase cell formation, cell wall composition and integrity, melanin synthesis, transcription of the morphogenesis­associated gene Ste20 that is involved in the high osmolarity glycerol/mitogen­activated protein kinase pathway, and pathogenicity of S. schenckii in a murine model of cutaneous infection. Further investigations into the signals SsDRK1 responds to, and the interactions of upstream transmembrane hybrid histidine kinases with SsDRK1, are required to uncover novel targets for anti­fungal therapies.

Eng1 and Exg8 Are the Major ß-Glucanases Secreted by the Fungal Pathogen Histoplasma capsulatum.

Fungal cell walls contain ß-glucan polysaccharides that stimulate immune responses when recognized by host immune cells. The fungal pathogen Histoplasma capsulatum minimizes detection of ß-glucan by host cells through at least two mechanisms: concealment of ß-glucans beneath α-glucans and enzymatic removal of any exposed ß-glucan polysaccharides by the secreted glucanase Eng1. Histoplasma yeasts also secrete the putative glucanase Exg8, which may serve a similar role as Eng1 in removing exposed ß-glucans from the yeast cell surface. Here, we characterize the enzymatic specificity of the Eng1 and Exg8 proteins and show that Exg8 is an exo-ß1,3-glucanase and Eng1 is an endo-ß1,3-glucanase. Together, Eng1 and Exg8 account for nearly all of the total secreted glucanase activity of Histoplasma yeasts. Both Eng1 and Exg8 proteins are secreted through a conventional secretion signal and are modified post-translationally by O-linked glycosylation. Both glucanases have near maximal activity at temperature and pH conditions experienced during infection of host cells, supporting roles in Histoplasma pathogenesis. Exg8 has a higher specific activity than Eng1 for ß1,3-glucans; yet despite this, Exg8 does not reduce detection of yeasts by the host ß-glucan receptor Dectin-1. Exg8 is largely dispensable for virulence in vivo, in contrast to Eng1. These results show that Histoplasma yeasts secrete two ß1,3-glucanases and that Eng1 endoglucanase activity is the predominant factor responsible for removal of exposed cell wall ß-glucans to minimize host detection of Histoplasma yeasts.

The Eng1 ß-Glucanase Enhances Histoplasma Virulence by Reducing ß-Glucan Exposure.

UNLABELLED: The fungal pathogen Histoplasma capsulatum parasitizes host phagocytes. To avoid antimicrobial immune responses, Histoplasma yeasts must minimize their detection by host receptors while simultaneously interacting with the phagocyte. Pathogenic Histoplasma yeast cells, but not avirulent mycelial cells, secrete the Eng1 protein, which is a member of the glycosylhydrolase 81 (GH81) family. We show that Histoplasma Eng1 is a glucanase that hydrolyzes ß-(1,3)-glycosyl linkages but is not required for Histoplasma growth in vitro or for cell separation. However, Histoplasma yeasts lacking Eng1 function have attenuated virulence in vivo, particularly during the cell-mediated immunity stage. Histoplasma yeasts deficient for Eng1 show increased exposure of cell wall ß-glucans, which results in enhanced binding to the Dectin-1 ß-glucan receptor. Consistent with this, Eng1-deficient yeasts trigger increased tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) cytokine production from macrophages and dendritic cells. While not responsible for large-scale cell wall structure and function, the secreted Eng1 reduces levels of exposed ß-glucans at the yeast cell wall, thereby diminishing potential recognition by Dectin-1 and proinflammatory cytokine production by phagocytes. In α-glucan-producing Histoplasma strains, Eng1 acts in concert with α-glucan to minimize ß-glucan exposure: α-glucan provides a masking function by covering the ß-glucan-rich cell wall, while Eng1 removes any remaining exposed ß-glucans. Thus, Histoplasma Eng1 has evolved a specialized pathogenesis function to remove exposed ß-glucans, thereby enhancing the ability of yeasts to escape detection by host phagocytes. IMPORTANCE: The success of Histoplasma capsulatum as an intracellular pathogen results, in part, from an ability to minimize its detection by receptors on phagocytic cells of the immune system. In this study, we showed that Histoplasma pathogenic yeast cells, but not avirulent mycelia, secrete a ß-glucanase, Eng1, which reduces recognition of fungal cell wall ß-glucans. We demonstrated that the Eng1 ß-glucanase promotes Histoplasma virulence by reducing levels of surface-exposed ß-glucans on yeast cells, thereby enabling Histoplasma yeasts to escape detection by the host ß-glucan receptor, Dectin-1. As a consequence, phagocyte recognition of Histoplasma yeasts is reduced, leading to less proinflammatory cytokine production by phagocytes and less control of Histoplasma infection in vivo Thus, Histoplasma yeasts express two mechanisms to avoid phagocyte detection: masking of cell wall ß-glucans by α-glucan and enzymatic removal of exposed ß-glucans by the Eng1 ß-glucanase.

N-acetylated α-linked acidic dipeptidase is identified as an antigen of Histoplasma capsulatum.

Histoplasmosis, one of the most important mycoses, needs to be diagnosed rapidly and accurately. The main method used to diagnose histoplasmosis is serological detection of antibodies to the Histoplasma capsulatum H and M antigens. Several other protein antigens have been reported in H. capsulatum; however, they have not been used for diagnosis. In this study, we explored novel antigens that were detected during H. capsulatum infection. We obtained a protein mixture from H. capsulatum yeast cells after vigorous mixing in a 0.1% Triton X-100 solution. From the resultant pool, we detected nine spots that reacted with sera from patients with histoplasmosis and identified eight seroactive proteins with mass spectrometry. The seroactive proteins were purified, and their antigenicities were tested with an enzyme-linked immunosorbent assay (ELISA). ELISA revealed that the titer of the patients' sera to N-acetylated α-linked acidic dipeptidase was significantly higher than those of healthy volunteers (P < 0.01). These data indicate that N-acetylated α-linked acidic dipeptidase of H. capsulatum is recognized as a major antigen during histoplasmosis.

Isotopic fractionation by a fungal P450 nitric oxide reductase during the production of N2O.

Nitrous oxide (N2O) is a potent greenhouse gas with a 100-year global warming potential approximately 300 times that of CO2. Because microbes account for over 75% of the N2O released in the U.S., understanding the biochemical processes by which N2O is produced is critical to our efforts to mitigate climate change. In the current study, we used gas chromatography-isotope ratio mass spectrometry (GC-IRMS) to measure the δ(15)N, δ(18)O, δ(15)N(α), and δ(15)N(ß) of N2O generated by purified fungal nitric oxide reductase (P450nor) from Histoplasma capsulatum. The isotope values were used to calculate site preference (SP) values (difference in δ(15)N between the central (α) and terminal (ß) N atoms in N2O), enrichment factors (ε), and kinetic isotope effects (KIEs). Both oxygen and N(α) displayed normal isotope effects during enzymatic NO reduction with ε values of -25.7‰ (KIE = 1.0264) and -12.6‰ (KIE = 1.0127), respectively. However, bulk nitrogen (average δ(15)N of N(α) and N(ß)) and N(ß) exhibited inverse isotope effects with ε values of 14.0‰ (KIE = 0.9862) and 36.1‰ (KIE = 0.9651), respectively. The observed inverse isotope effect in δ(15)N(ß) is consistent with reversible binding of the first NO in the P450nor reaction mechanism. In contrast to the constant SP observed during NO reduction in microbial cultures, the site preference measured for purified H. capsulatum P450nor was not constant, increasing from ∼ 15‰ to ∼ 29‰ during the course of the reaction. This indicates that SP for microbial cultures can vary depending on the growth conditions, which may complicate source tracing during microbial denitrification.

Redundant catalases detoxify phagocyte reactive oxygen and facilitate Histoplasma capsulatum pathogenesis.

Histoplasma capsulatum is a respiratory pathogen that infects phagocytic cells. The mechanisms allowing Histoplasma to overcome toxic reactive oxygen molecules produced by the innate immune system are an integral part of Histoplasma's ability to survive during infection. To probe the contribution of Histoplasma catalases in oxidative stress defense, we created and analyzed the virulence defects of mutants lacking CatB and CatP, which are responsible for extracellular and intracellular catalase activities, respectively. Both CatB and CatP protected Histoplasma from peroxide challenge in vitro and from antimicrobial reactive oxygen produced by human neutrophils and activated macrophages. Optimal protection required both catalases, as the survival of a double mutant lacking both CatB and CatP was lower than that of single-catalase-deficient cells. Although CatB contributed to reactive oxygen species defenses in vitro, CatB was dispensable for lung infection and extrapulmonary dissemination in vivo. Loss of CatB from a strain also lacking superoxide dismutase (Sod3) did not further reduce the survival of Histoplasma yeasts. Nevertheless, some catalase function was required for pathogenesis since simultaneous loss of both CatB and CatP attenuated Histoplasma virulence in vivo. These results demonstrate that Histoplasma's dual catalases comprise a system that enables Histoplasma to efficiently overcome the reactive oxygen produced by the innate immune system.

Expression of Paracoccidioides brasiliensis AMY1 in a Histoplasma capsulatum amy1 mutant, relates an α-(1,4)-amylase to cell wall α-(1,3)-glucan synthesis.

In the cell walls of the pathogenic yeast phases of Paracoccidioides brasiliensis, Blastomyces dermatitidis and Histoplasma capsulatum, the outer α-(1,3)-glucan layer behaves as a virulence factor. In H. capsulatum, an α-(1,4)-amylase gene (AMY1) is essential for the synthesis of this polysaccharide, hence related to virulence. An orthologous gene to H. capsulatum AMY1 was identified in P. brasiliensis and also labeled AMY1. P. brasiliensis AMY1 transcriptional levels were increased during the yeast phase, which correlates with the presence of α-(1,3)-glucan as the major yeast cell wall polysaccharide. Complementation of a H. capsulatum amy1 mutant strain with P. brasiliensis AMY1, suggests that P. brasiliensis Amy1p may play a role in the synthesis of cell wall α-(1,3)-glucan. To study some biochemical properties of P. brasiliensis Amy1p, the enzyme was overexpressed, purified and studied its activity profile with starch and amylopeptin. It showed a relatively higher hydrolyzing activity on amylopeptin than starch, producing oligosaccharides from 4 to 5 glucose residues. Our findings show that P. brasiliensis Amy1p produces maltooligosaccharides which may act as a primer molecule for the fungal cell wall α-(1,3)-glucan biosynthesis by Ags1p.

Extracellular superoxide dismutase protects Histoplasma yeast cells from host-derived oxidative stress.

In order to establish infections within the mammalian host, pathogens must protect themselves against toxic reactive oxygen species produced by phagocytes of the immune system. The fungal pathogen Histoplasma capsulatum infects both neutrophils and macrophages but the mechanisms enabling Histoplasma yeasts to survive in these phagocytes have not been fully elucidated. We show that Histoplasma yeasts produce a superoxide dismutase (Sod3) and direct it to the extracellular environment via N-terminal and C-terminal signals which promote its secretion and association with the yeast cell surface. This localization permits Sod3 to protect yeasts specifically from exogenous superoxide whereas amelioration of endogenous reactive oxygen depends on intracellular dismutases such as Sod1. While infection of resting macrophages by Histoplasma does not stimulate the phagocyte oxidative burst, interaction with polymorphonuclear leukocytes (PMNs) and cytokine-activated macrophages triggers production of reactive oxygen species (ROS). Histoplasma yeasts producing Sod3 survive co-incubation with these phagocytes but yeasts lacking Sod3 are rapidly eliminated through oxidative killing similar to the effect of phagocytes on Candida albicans yeasts. The protection provided by Sod3 against host-derived ROS extends in vivo. Without Sod3, Histoplasma yeasts are attenuated in their ability to establish respiratory infections and are rapidly cleared with the onset of adaptive immunity. The virulence of Sod3-deficient yeasts is restored in murine hosts unable to produce superoxide due to loss of the NADPH-oxidase function. These results demonstrate that phagocyte-produced ROS contributes to the immune response to Histoplasma and that Sod3 facilitates Histoplasma pathogenesis by detoxifying host-derived reactive oxygen thereby enabling Histoplasma survival.

Selección y optimización de los métodos basados en PCR para la detección de Histoplasma capsulatum var. capsulatum / Selection and optimization of PCR-based methods for the detection of Histoplasma capsulatum var. capsulatum

Antecedentes. Los métodos actuales para el diagnóstico de laboratorio de la histoplasmosis son problemáticos si consideramos los valores de sensibilidad, especificidad y tiempo de ejecución. Objetivos. En este estudio hemos tratado de seleccionar y optimizar los métodos para la detección de Histoplasma capsulatum var. capsulatum mediante la reacción en cadena de la polimerasa (PCR). Métodos. Se evaluaron tres métodos de extracción de ADN y tres métodos de PCR. Se optimizó la concentración de los componentes de esta reacción de PCR y se determinó su co-positividad (sensibilidad) y co-negatividad (especificidad), utilizando muestras de sangre a las que se había añadido H. capsulatum. Resultados. El método de extracción que dio el ADN de más alta calidad utiliza membranas de sílice (DNeasy Blood & Tissue Kit, Qiagen, Hilden, Germany), y el método de PCR con mayor capacidad de detección es el que incluye un gen diana que codifica una proteína de 100 kDa. Nuestra optimización de las condiciones de PCR indicaron que la reacción trabaja en un rango significativo de las concentraciones de componentes y, además, fue capaz de detectar H. capsulatum mejor que las técnicas de cultivo tradicional, con un límite de detección de solo 10 pg de ADN. Conclusiones. En nuestras condiciones experimentales, el método PCR seleccionado en este trabajo (en lugar de PCR anidada) es una herramienta lo suficientemente sensible para el diagnóstico de la histoplasmosis

Background. Current methods for the laboratory diagnosis of histoplasmosis are problematic in terms of their sensitivity, specificity and runtime. Objectives. Thus, in this study, we sought to select and optimize methods for the detection of Histoplasma capsulatum var. capsulatum by polymerase chain reaction (PCR). Methods. Three DNA extraction methods and three PCR methods were evaluated. We optimised the concentration of the components of this PCR reaction and determined its sensitivity and specificity using blood samples to which H. capsulatum had been added. Results. The DNA extraction method that yielded the highest-quality DNA used silica membranes (DNeasy Blood & Tissue Kit, Qiagen, Hilden, Germany), and the amplification method with the best detection capacity used a target gene encoding a 100-kDa protein. Our optimisation of the PCR conditions indicated that the reaction works over a significant range of component concentrations; in addition, it was able to detect H. capsulatum better than traditional culture techniques, with a detection limit of only 10 pg of DNA. Conclusions. In our experimental conditions, the PCR method selected in this work (instead of nested-PCR) is a tool sensitive enough for the diagnosis of histoplasmosis(AU)

The yeast-phase virulence requirement for α-glucan synthase differs among Histoplasma capsulatum chemotypes.

Histoplasma capsulatum strains can be classified into two chemotypes based on cell wall composition. The cell wall of chemotype II yeast contains a layer of α-(1,3)-glucan that masks immunostimulatory ß-(1,3)-glucans from detection by the Dectin-1 receptor on host phagocytes. This α-(1,3)-glucan cell wall component is essential for chemotype II Histoplasma virulence. In contrast, chemotype I yeast cells lack α-(1,3)-glucan in vitro, yet they remain fully virulent in vivo. Analysis of the chemotype I α-glucan synthase (AGS1) locus revealed a 2.7-kb insertion in the promoter region that diminishes AGS1 expression. Nonetheless, AGS1 mRNA can be detected during respiratory infection with chemotype I yeast, suggesting that α-(1,3)-glucan could be produced during in vivo growth despite its absence in vitro. To directly test whether AGS1 contributes to chemotype I strain virulence, we prevented AGS1 function by RNA interference and by insertional mutation. Loss of AGS1 function in chemotype I does not impair the cytotoxicity of ags1(-) mutant yeast to cultured macrophages, nor does it affect the intracellular growth of yeast. In a murine model of histoplasmosis, the ags1(-) chemotype I mutant strains show no defect in lung infection or in extrapulmonary dissemination. Together, these studies demonstrate that AGS1 expression is dispensable for chemotype I yeast virulence, in contrast to the case for chemotype II yeast. Despite the absence of cell wall α-(1,3)-glucan, chemotype I yeast can avoid detection by Dectin-1 in a growth stage-dependent manner. This suggests the production of a unique Histoplasma chemotype I factor that, at least partially, circumvents the α-(1,3)-glucan requirement for yeast virulence.

Secreted dipeptidyl peptidase IV activity in the dimorphic fungal pathogen Histoplasma capsulatum.

Dipeptidyl peptidase type IV (DppIV) enzymes are broadly distributed phylogenetically and display diverse functions, including intercellular signaling, immunomodulation, protein maturation and processing, metabolism, and nutrient acquisition. We identified a secreted proteolytic activity in Histoplasma capsulatum effective toward DppIV-specific substrates. In order to determine the gene(s) that encodes this activity, we identified two putative DPPIV homologs (HcDPPIVA and HcDPPIVB) in H. capsulatum based on a homology search with Aspergillus fumigatus DppIV. Comparative sequence analysis revealed that HcDppIVA is similar to secreted DppIV enzymes, while HcDppIVB clusters with intracellular DapB-like enzymes. Unexpectedly, silencing of HcDPPIVA by RNA interference (RNAi) had no effect on secreted DppIV activity and an HcDPPIVA-null deletion mutant also showed no abrogation of secreted DppIV activity. In contrast, RNAi silencing of HcDPPIVB significantly reduced the level of secreted DppIV activity. RNAi silencing of HcDPPIVB in the HcDPPIVA-null mutant had no additional effect on secreted DppIV activity, indicating that HcDPPIVA does not contribute to secreted activity. RNAi silencing of HcDPPIVB did not affect the ability to kill a murine macrophage-like cell line, RAW 264.7, indicating that this gene is not required for infection of macrophages.

Histoplasma capsulatum encodes a dipeptidyl peptidase active against the mammalian immunoregulatory peptide, substance P.

The pathogenic fungus Histoplasma capsulatum secretes dipeptidyl peptidase (Dpp) IV enzyme activity and has two putative DPPIV homologs (HcDPPIVA and HcDPPIVB). We previously showed that HcDPPIVB is the gene responsible for the majority of secreted DppIV activity in H. capsulatum culture supernatant, while we could not detect any functional contribution from HcDPPIVA. In order to determine whether HcDPPIVA encodes a functional DppIV enzyme, we expressed HcDPPIVA in Pichia pastoris and purified the recombinant protein. The recombinant enzyme cleaved synthetic DppIV substrates and had similar biochemical properties to other described DppIV enzymes, with temperature and pH optima of 42 degrees C and 8, respectively. Recombinant HcDppIVA cleaved the host immunoregulatory peptide substance P, indicating the enzyme has the potential to affect the immune response during infection. Expression of HcDPPIVA under heterologous regulatory sequences in H. capsulatum resulted in increased secreted DppIV activity, indicating that the encoded protein can be expressed and secreted by its native organism. However, HcDPPIVA was not required for virulence in a murine model of histoplasmosis. This work reports a fungal enzyme that can function to cleave the immunomodulatory host peptide substance P.

[Significantly increase of glycolytic flux and pyruvate productivity in Torulopsis glabrata by heterologous expression of NADH alternative oxidase].

OBJECTIVE: This study aimed at increasing the glycolytic flux and pyruvate productivity in Torulopsis glabrata with glucose as carbon source. METHODS: For this target, we introduced a mitochondrial located alternative oxidase encoded by Histoplasma capsulatum AOX1 gene into T. glabrata, and a mutant strain named as AOX with total NADH oxidase activity was 1.8-fold higher than that of the parent stain, was achieved. RESULTS: The heterologous expression of NADH alternative oxidase resulted in decrease of the dry cell weight and fermentation time by 20.3% and 10.7%, but the specific rate of glucose consumption and pyruvate production increased 34.7% and 54.1% higher, respectively. The reasons for high glycolytic flux were the intracellular NADH/NAD+ ratio and ATP concentration decreased 74.7% and 52.9% respectively, the specific activity of phosphofructokinase, pyruvate kinase increased 185.0% and 28.1%. CONCLUSION: Introduction of a novel NADH oxidation pathway by alternative oxidase can efficiently increase the rate of glucose consumption and the target metabolite productivity.

Histoplasma capsulatum secreted gamma-glutamyltransferase reduces iron by generating an efficient ferric reductant.

The intracellular fungal pathogen Histoplasma capsulatum (Hc) resides in mammalian macrophages and causes respiratory and systemic disease. Iron limitation is an important host antimicrobial defence, and iron acquisition is critical for microbial pathogenesis. Hc displays several iron acquisition mechanisms, including secreted glutathione-dependent ferric reductase activity (GSH-FeR). We purified this enzyme from culture supernatant and identified a novel extracellular iron reduction strategy involving gamma-glutamyltransferase (Ggt1) activity. The 320 kDa complex was composed of glycosylated protein subunits of about 50 and 37 kDa. The purified enzyme exhibited gamma-glutamyl transfer activity as well as iron reduction activity in the presence of glutathione. We cloned and manipulated expression of the encoding gene. Overexpression or RNAi silencing affected both GGT and GSH-FeR activities concurrently. Enzyme inhibition experiments showed that the activity is complex and involves two reactions. First, Ggt1 initiates enzymatic breakdown of GSH by cleavage of the gamma-glutamyl bond and release of cysteinylglycine. Second, the thiol group of the released dipeptide reduces ferric to ferrous iron. A combination of kinetic properties of both reactions resulted in efficient iron reduction over a broad pH range. Our findings provide novel insight into Hc iron acquisition strategies and reveal a unique aspect of Ggt1 function in this dimorphic mycopathogen.

Spectroscopic and kinetic studies of Nor1, a cytochrome P450 nitric oxide reductase from the fungal pathogen Histoplasma capsulatum.

The fungal respiratory pathogen Histoplasma capsulatum evades the innate immune response and colonizes macrophages during infection. Although macrophage production of the antimicrobial effector nitric oxide (NO) restricts H. capsulatum growth, the pathogen is able to establish a persistent infection. H. capsulatum contains a P450 nitric oxide reductase homologue (NOR1) that may be important for detoxifying NO during infection. To characterize the activity of this putative P450 enzyme, a 404 amino acid fragment of Nor1p was expressed in Escherichia coli and purified to homogeneity. Spectral characterization of Nor1p indicated that it was similar to other fungal P450 nitric oxide reductases. Nor1p catalyzed the reduction of NO to N2O using NADH as the direct reductant. The K(M) for NO was determined to be 20 microM and the k(cat) to be 5000 min(-1). Together, these results provide evidence for a protective role of a P450 nitric oxide reductase against macrophage-derived NO.

The Histoplasma capsulatum vacuolar ATPase is required for iron homeostasis, intracellular replication in macrophages and virulence in a murine model of histoplasmosis.

Histoplasma capsulatum is a dimorphic fungal pathogen that survives and replicates within macrophages (Mphi). To identify specific genes required for intracellular survival, we utilized Agrobacterium tumefaciens-mediated mutagenesis, and screened for H. capsulatum insertional mutants that were unable to survive in human Mphi. One colony was identified that had an insertion within VMA1, the catalytic subunit A of the vacuolar ATPase (V-ATPase). The vma1 mutant (vma1::HPH) grew normally on iron-replete medium, but not on iron-deficient media. On iron-deficient medium, the growth of the vma1 mutant was restored in the presence of wild-type (WT) H. capsulatum yeasts, or the hydroxamate siderophore, rhodotorulic acid. However, the inability to replicate within Mphi was only partially restored by the addition of exogenous iron. The vma1::HPH mutant also did not grow as a mold at 28 degrees C. Complementation of the mutant (vma/VMA1) restored its ability to replicate in Mphi, grow on iron-poor medium and grow as a mold at 28 degrees C. The vma1::HPH mutant was avirulent in a mouse model of histoplasmosis, whereas the vma1/VMA1 strain was as pathogenic as WT yeasts. These studies demonstrate the importance of V-ATPase function in the pathogenicity of H. capsulatum, in iron homeostasis and in fungal dimorphism.

Histoplasma requires SID1, a member of an iron-regulated siderophore gene cluster, for host colonization.

The macrophage is the primary host cell for the fungal pathogen Histoplasma capsulatum during mammalian infections, yet little is known about fungal genes required for intracellular replication in the host. Since the ability to scavenge iron from the host is important for the virulence of most pathogens, we investigated the role of iron acquisition in H. capsulatum pathogenesis. H. capsulatum acquires iron through the action of ferric reductases and the production of siderophores, but the genes responsible for these activities and their role in virulence have not been determined. We identified a discrete set of co-regulated genes whose transcription is induced under low iron conditions. These genes all appeared to be involved in the synthesis, secretion, and utilization of siderophores. Surprisingly, the majority of these transcriptionally co-regulated genes were found clustered adjacent to each other in the genome of the three sequenced strains of H. capsulatum, suggesting that their proximity might foster coordinate gene regulation. Additionally, we identified a consensus sequence in the promoters of all of these genes that may contribute to iron-regulated gene expression. The gene set included L-ornithine monooxygenase (SID1), the enzyme that catalyzes the first committed step in siderophore production in other fungi. Disruption of SID1 by allelic replacement resulted in poor growth under low iron conditions, as well as a loss of siderophore production. Strains deficient in SID1 showed a significant growth defect in murine bone-marrow-derived macrophages and attenuation in the mouse model of infection. These data indicated that H. capsulatum utilizes siderophores in addition to other iron acquisition mechanisms for optimal growth during infection.

Identification and evolution of fungal mitochondrial tyrosyl-tRNA synthetases with group I intron splicing activity.

The bifunctional Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) both aminoacylates mitochondrial tRNA(Tyr) and acts as a structure-stabilizing splicing cofactor for group I introns. Previous studies showed that CYT-18 has distinct tRNA(Tyr) and group I intron-binding sites, with the latter formed by three small "insertions" in the nucleotide-binding fold and other structural adaptations compared with nonsplicing bacterial tyrosyl-tRNA synthetases. Here, analysis of genomic sequences shows that mitochondrial tyrosyl-tRNA synthetases with structural adaptations similar to CYT-18's are uniquely characteristic of fungi belonging to the subphylum Pezizomycotina, and biochemical assays confirm group I intron splicing activity for the enzymes from several of these organisms, including Aspergillus nidulans and the human pathogens Coccidioides posadasii and Histoplasma capsulatum. By combining multiple sequence alignments with a previously determined cocrystal structure of a CYT-18/group I intron RNA complex, we identify conserved features of the Pezizomycotina enzymes related to group I intron and tRNA interactions. Our results suggest that mitochondrial tyrosyl-tRNA synthetases with group I intron splicing activity evolved during or after the divergence of the fungal subphyla Pezizomycotina and Saccharomycotina by a mechanism involving the concerted differentiation of preexisting protein loop regions. The unique group I intron splicing activity of these fungal enzymes may provide a new target for antifungal drugs.