In silico anti-fungal efficacy and the mechanism of binding of some Syzygium aromaticum ingredient compounds to aspartate semialdehyde dehydrogenase, 6C8W and 6C85, enzymes from Blastomyces dermatitidis.

This in silico work was carried out to reveal the proposed anti-fungal efficacy of some clove ingredient compounds against aspartate semialdehyde dehydrogenase, 6C8W and 6C85, enzymes from Blastomyces dermatitidis. The molecular docking simulation was implemented utilizing the Auto Dock 4.2. software. A set of 17 compounds were selected for this study, which is known to be active ingredients of Syzygium aromaticum crude and oil. The best docking scores associated with the Blastomyces dermatitidis enzymes 6C85 and 6C8W were for Maslinic acid and Oleanolic acid, followed by Stigmasterol and Campesterol. It was found that these compounds possess inhibitory potential against 6C85 and 6C8W and hence have anti-fungal efficacy. Maslinic acid and Oleanolic acid produced the strongest binding to 6C85 and 6C8W over the remaining bioactive compounds by forming H-bonds with some amino acids in these enzymes.

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.

Structural insights into inhibitor binding to a fungal ortholog of aspartate semialdehyde dehydrogenase.

The aspartate pathway, uniquely found in plants and microorganisms, offers novel potential targets for the development of new antimicrobial drugs. Aspartate semialdehyde dehydrogenase (ASADH) catalyzes production of a key intermediate at the first branch point in this pathway. Several fungal ASADH structures have been determined, but the prior crystallization conditions had precluded complex formation with enzyme inhibitors. The first inhibitor-bound and cofactor-bound structures of ASADH from the pathogenic fungi Blastomyces dermatitidis have now been determined, along with a structural and functional comparison to other ASADH family members. The structure of this new ASADH is similar to the other fungal orthologs, but with some critical differences in the orientation of some active site functional groups and in the subunit interface region. The presence of this bound inhibitor reveals the first details about inhibitor binding interactions, and the flexible orientation of its aromatic ring provides helpful insights into the design of potentially more potent and selective antifungal compounds.

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.

Blastomyces dermatitidis serine protease dipeptidyl peptidase IVA (DppIVA) cleaves ELR+ CXC chemokines altering their effects on neutrophils.

Blastomycosis elicits a pyogranulomatous inflammatory response that involves a prominent recruitment of neutrophils to the site of infection. Although neutrophils are efficiently recruited to the site of infection, this event is paradoxically coupled with the host's inability to control infection by Blastomyces dermatitidis, the causative agent. The mechanisms underlying this characteristic pyogranulomatous response and inability of neutrophils to kill the yeast are poorly understood. We recently reported that the fungal protease dipeptidyl peptidase IVA (DppIVA) promotes B. dermatitidis virulence by cleaving a dipeptide from the N-terminus of C-C chemokines and granulocyte/macrophage-colony stimulating factor, thereby inactivating them. Herein, we present evidence that DppIVA can also truncate the N-terminus of members of the ELR+ CXC chemokine family, which are known to modulate neutrophil function. We show that the DppIVA cleaved form of human (h) CXCL-2, for example, hCXCL-2 (3-73), is a more potent neutrophil chemoattractant than its intact counterpart, but hCXCL-2 (3-73) is conversely impaired in its ability to prime the reactive oxygen species response of neutrophils. Thus, DppIVA action on ELR+ CXC chemokines may promote the pyogranulomatous response that is typical of blastomycosis, while also explaining the inability of neutrophils to control infection.

Fludioxonil Induces Drk1, a Fungal Group III Hybrid Histidine Kinase, To Dephosphorylate Its Downstream Target, Ypd1.

Novel antifungal drugs and targets are urgently needed. Group III hybrid histidine kinases (HHKs) represent an appealing new therapeutic drug target because they are widely expressed in fungi but absent from humans. We investigated the mode of action of the widely utilized, effective fungicide fludioxonil. The drug acts in an HHK-dependent manner by constitutive activation of the HOG (high-osmolarity glycerol) pathway, but its mechanism of action is poorly understood. Here, we report a new mode of drug action that entails conversion of the HHK from a kinase into a phosphatase. We expressed Drk1 (dimorphism-regulating kinase), which is an intracellular group III HHK from the fungal pathogen Blastomyces dermatitidis, in Saccharomyces cerevisiae Drk1 engendered drug sensitivity in B. dermatitidis and conferred sensitivity upon S. cerevisiae In response to fludioxonil, Drk1 behaved as a phosphatase rather than as a kinase, leading to dephosphorylation of its downstream target, Ypd1, constitutive activation of the HOG pathway, and yeast cell death. Aspartic acid residue 1140 in the Drk1 receiver domain was required for in vivo phosphatase activity on Ypd1, and Hog1 was required for drug effect, indicating fidelity in HHK-dependent drug action. In in vitro assays with purified protein, intact Drk1 demonstrated intrinsic kinase activity, and the Drk1 receiver domain exhibited intrinsic phosphatase activity. However, fludioxonil failed to induce intact Drk1 to dephosphorylate Ypd1. We conclude that fludioxonil treatment in vivo likely acts on an upstream target that triggers HHK to become a phosphatase, which dephosphorylates its downstream target, Ypd1.

Fungal Mimicry of a Mammalian Aminopeptidase Disables Innate Immunity and Promotes Pathogenicity.

Systemic fungal infections trigger marked immune-regulatory disturbances, but the mechanisms are poorly understood. We report that the pathogenic yeast of Blastomyces dermatitidis elaborates dipeptidyl-peptidase IVA (DppIVA), a close mimic of the mammalian ectopeptidase CD26, which modulates critical aspects of hematopoiesis. We show that, like the mammalian enzyme, fungal DppIVA cleaved C-C chemokines and GM-CSF. Yeast producing DppIVA crippled the recruitment and differentiation of monocytes and prevented phagocyte activation and ROS production. Silencing fungal DppIVA gene expression curtailed virulence and restored recruitment of CCR2(+) monocytes, generation of TipDC, and phagocyte killing of yeast. Pharmacological blockade of DppIVA restored leukocyte effector functions and stemmed infection, while addition of recombinant DppIVA to gene-silenced yeast enabled them to evade leukocyte defense. Thus, fungal DppIVA mediates immune-regulatory disturbances that underlie invasive fungal disease. These findings reveal a form of molecular piracy by a broadly conserved aminopeptidase during disease pathogenesis.

PRP8 intein in Ajellomycetaceae family pathogens: sequence analysis, splicing evaluation and homing endonuclease activity.

Inteins are intervening sequences that are transcribed and translated with flanking host protein sequences and then self-excised by protein splicing. Bi-functional inteins also contain a homing endonuclease responsible for their genetic mobility. The PRP8 intein, the most widespread among fungi, occurs in important pathogens such as Histoplasma capsulatum and Paracoccidioides brasiliensis, from the Ajellomycetaceae family. Herein, we describe the bi-functional PRP8 intein in two other Ajellomycetacean pathogens, Blastomyces dermatitidis and Emmonsia parva. Sequence analysis and experimental evidence suggest that the homing endonuclease from PbrPRP8 is inactive. The splicing activity of the PRP8 intein from the B. dermatitidis, E. parva and P. brasiliensis species complex was demonstrated in a non-native protein context in Escherichia coli. Since the PRP8 intein is located in a functionally essential nuclear protein, it can be considered a promising therapeutic target for anti-fungal drugs, because inhibition of intein splicing should inhibit proliferation of intein-containing pathogens.

Global control of dimorphism and virulence in fungi.

Microbial pathogens that normally inhabit our environment can adapt to thrive inside mammalian hosts. There are six dimorphic fungi that cause disease worldwide, which switch from nonpathogenic molds in soil to pathogenic yeast after spores are inhaled and exposed to elevated temperature. Mechanisms that regulate this switch remain obscure. We show that a hybrid histidine kinase senses host signals and triggers the transition from mold to yeast. The kinase also regulates cell-wall integrity, sporulation, and expression of virulence genes in vivo. This global regulator shapes how dimorphic fungal pathogens adapt to the mammalian host, which has broad implications for treating and preventing systemic fungal disease.

Mechanisms of fungal pathogenicity: correlation of virulence in vivo, susceptibility to killing by polymorphonuclear neutrophils in vitro, and neutrophil superoxide anion induction among Blastomyces dermatitidis isolates.

Seven Blastomyces dermatitidis isolates varying in virulence for mice were compared for susceptibility to polymorphonuclear neutrophil (PMN) killing and the ability to induce superoxide anion (O2-) production by PMNs in vitro. In vitro killing of six B. dermatitidis isolates by murine peripheral blood PMNs or by PMNs elicited from the peritoneal cavity by a local immune reaction (B. dermatitidis-immune mice given killed B. dermatitidis intraperitoneally 24 h earlier) inversely correlated with in vivo virulence (most to least virulent) isolates: VV, V, V40, KL-1, A2, and GA-1). The capacity of isolates to induce O2- production by PMNs also inversely correlated with in vivo virulence. Isolate A, of intermediate in vivo virulence, was a good inducer of O2- production in vitro but was no more susceptible to in vitro killing by PMNs than isolate V, VV, or V40. Fungal intracellular superoxide dismutase or catalase content did not correlate with in vivo virulence or in vitro killing by PMNs. Isolate A, however, had two to four times the intracellular catalase activity as did other B. dermatitidis isolates, suggesting a possible mechanism for its enhanced resistance to in vitro killing by PMNs. Therefore, while in vitro killing by PMNs and the capacity to induce O2- production by PMNs inversely correlated with virulence for six B. dermatitidis isolates, isolate A was an exception: its resistance to killing by PMN-generated oxygen metabolites in vitro but its susceptibility to killing in vivo suggest that its in vivo killing occurs by other, perhaps nonoxidative, mechanisms.

Chitin synthetase from the yeast and mycelial phases of Blastomyces dermatitidis.

Chitin synthetase (E.C.2.4.1.16) from mixed membrane fractions of the yeast and mycelial phases of Blastomyces dermatitidis were compared. The behavior of the enzyme from both phases was very similar: N-acetylglucosamine was stimulatory (Km 8.5 mM for yeast and 3.9 mM for mycelium); substrate Michaelis-Menten kinetics were sigmoidal; substrate Km of enzyme from yeast decreased from 3.0 mM at low N-acetylglucosamine (5 mM) levels to 1.4 mM at high (100 mM) levels; substrate Km of enzyme from mycelium was essentially unchanged at 1.4 mM; temperature optimum was 28 degrees C; pH optimum was 7-7.5; Mg+2 optimum was 5-10 mM. The greatest difference was that enzyme from yeast was extracted in a mostly latent form that required trypsin treatment for maximal in vitro activity while enzyme from mycelium was extracted in an active form which was rapidly deactivated by trypsin treatment.

Cell wall studies of Histoplasma capsulatum and Blastomyces dermatitidis using autologous and heterologous enzymes.

Enzymes capable of hydrolyzing cell walls of Blastomyces dermatitidis and chemotypes I and II of Histoplasma capsulatum were prepared in the laboratory or obtained from commercial sources. They included chitinases, beta-1,3-glucanases, beta-1,6-glucanase, and Pronase. Monosaccharides and disaccharides of glucose released from the cell walls by the enzymes were determined qualitatively by paper and gas-liquid chromatography, and monosaccharides were quantitated by the latter technique as well. An enzyme system isolated from Streptomyces sp. containing both chitinase and glucanase released maximum amounts of glucose and N-acetylglucosamine from the cell walls of H. capsulatum chemotype I. A chitinase preparation, free of glucanase, from Serratia marcescens released only chitobiose and N-acetylglucosamine from chemotype I cell walls, but the total quantity of N-acetylglucosamine released was about 60% less than that released by the Streptomyces system. A beta-1,3-glucanase from Bacillus circulans hydrolyzed the cell walls of H. capsulatum chemotype I, but a beta-1,6-glucanase failed to release glucose from the same walls. Autolytic enzymes, viz., beta-1,3-glucanases and several glycosidases were detected as constitutive enzymes in both yeast and mycelial phases of B. dermatitidis and H. capsulatum chemotypes I and II. No difference in the amount of activity was found between cell sap and culture filtrate preparations. The beta-glucanases prepared from the Histoplasma and Blastomyces strains were active on the cell walls of the yeast phases of H. capsulatum chemotypes I and II, releasing laminaribiose and glucose, but were essentially inactive on the cell walls of B. dermatitidis. Chitinase, beta-1,6-glucanase, alpha-glucanase, and alpha-glucosidase activities were absent from these fungal enzyme preparations.

Amino acid synthetic media for fungal pathogens based on aminopeptidase specificities: Histoplasma capsulatum, Blastomyces dermititidis, Paracoccidioides brasiliensis and Cryptococcus neoformans.

The development of simple and chemically defined liquid media for Histoplasma capsulatum, Blastomyces dermatitidis, Paracoccidioides brasiliensis and Crypto-occus neoformans according to their aminopeptidases profiles as amino acid requirement was described. When 1.5% purified agar was added, these media also supported excellent mycelial growth and sporulation of the deep mycoses. H. capsulatum was converted to and maintained in yeast phase when 0.1% L-cystine was added to the solid medium incubated at 37 degrees C.

Identification of yeast phase of pathogenic fungi by the specificity of their aminopeptidase(s).

Specificity of aminopeptidase(s) was fluorimetrically determined in the yeast phase of Histoplasma capsulatum, H. duboisii, H. farciminosum, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Candida albicans and Crytococcus neoformans. After individually incubating each of 26 amino acid-beta-naphthylamides with each yeast, the amount of each amino acid-beta-naphthylamide hydrolyzed was determined by measuring the free naphythylamine. This resulted in a reproducible profile of the aminopeptidase(s) for each fungus when medium, growth time, size of inoculum and incubation period were standardized. This technique provided a rapid and specific means of identification and differentiation among these yeasts. Specific amino acids were identified from the profile of each yeast as those rapidly liberated by the aminopeptidase(s) of that yeast. These amino acids delineated the amino acid requirement for the normal growth of each yeast.

Specific inhibitors of the three RNA polymerases from the aquatic fungus Blastocladiella emersonii.

Specific inhibitors of each of the three RNA polymerases of Blastocladiella emersonii have been found. Cycloheximide specifically inhibited the in vitro activity of the DEAE-fraction I enzyme, alpha-amanitin specifically inhibited the DEAE-fraction II enzyme, and rifampicin specifically inhibited the fraction III enzyme. DNA stimulation and dependency on the four riboside triphosphates were shown to be characteristic of each of the three fractions. Optimum concentrations of magnesium ions required were shown to differ among the three fractions and to be somewhat higher than optimum concentrations of manganese ions. The effect of pH on activity was essentially identical for each of the three fractions. Kinetic experiments and nuclease assays indicated the presence of some interfering substances in the partially purified RNA polymerase fractions.