Aspergillus fumigatus possesses two catalases (described as fast and slow on the basis of their e... more Aspergillus fumigatus possesses two catalases (described as fast and slow on the basis of their electrophoretic mobility). The slow catalase has been recognized as a diagnostic antigen for aspergillosis in immunocompetent patients. The antigenic catalase has been purified. The enzyme is a tetrameric protein composed of 90-kDa subunits. The corresponding cat1 gene was cloned, and sequencing data show that the cat1 gene codes for a 728-amino-acid polypeptide. A recombinant protein expressed in Pichia pastoris is enzymatically active and has biochemical and antigenic properties that are similar to those of the wild-type catalase. Molecular experiments reveal that CAT1 contains a signal peptide and a propeptide of 15 and 12 amino acid residues, respectively. cat1-disrupted mutants that were unable to produce the slow catalase were as sensitive to H2O2 and polymorphonuclear cells as the wild-type strain. In addition, there was no difference in pathogenicity between the cat1 mutant and it...
ASPND1 and ASPF2 are immunodominant antigens from Aspergillus nidulans and A. fumigatus , respect... more ASPND1 and ASPF2 are immunodominant antigens from Aspergillus nidulans and A. fumigatus , respectively, that are readily synthesized in infections in the human host, as demonstrated by their reactivity with more than 80% of sera from patients with aspergilloma or allergic bronchopulmonary aspergillosis. We demonstrate here that both antigens are exclusively produced under situations of low bioavailability of free Zn 2+ . Addition of micromolar concentrations of Zn 2+ to the culture medium strongly stimulated Aspergillus growth but totally inhibited ASPND1 or ASPF2 production. This effect was specific, since other divalent metals had no effect. Removal of endogenous Zn 2+ by a chelator also stimulated ASPND1 production, and the effect was specifically reversed by Zn 2+ . These results suggest a possible role of these antigens in the survival of the fungus in the lungs.
We have identified, purified, and characterized structurally and functionally a 90-kDa immunodomi... more We have identified, purified, and characterized structurally and functionally a 90-kDa immunodominant antigen associated with the water-soluble fraction of Aspergillus fumigatus. This antigen is recognized by 90.3% of serum samples from patients with aspergilloma and should be considered either by itself or better in combination with other purified antigens as a candidate for developing a standardized immunoassay for the detection of aspergilloma. p90 is a glycoprotein containing at least two two N-linked sugar chains of 2 and 5 kDa, respectively, which are not necessary for its reactivity with aspergilloma serum samples. Using specific anti-p90 rabbit serum, we have demonstrated that under native conditions, p90 exists in oligomeric form and has associated catalase activity. This activity is resistant to extreme temperatures (> 60 degrees C), reducing agents (40 mM dithiothreitol), high concentrations of denaturing agents such as 8 M urea and 8% sodium dodecyl sulfate, and treat...
One of the most important features that enables to grow within a susceptible individual and to ca... more One of the most important features that enables to grow within a susceptible individual and to cause disease is its ability to obtain Zn ions from the extremely zinc-limited environment provided by host tissues. Zinc uptake from this source in relies on ZIP transporters encoded by the , and genes. The expression of these genes is tightly regulated by the ZafA transcription factor that regulates zinc homeostasis and is essential for virulence. We combined the use of microarrays, Electrophoretic Mobility Shift Assays (EMSA) analyses, DNase I footprinting assays and in silico tools to better understand the regulation of the homeostatic and adaptive response of to zinc starvation. We found that under zinc-limiting conditions, ZafA functions mainly as a transcriptional activator through binding to a zinc response sequence located in the regulatory regions of its target genes, although it could also function as a repressor of a limited number of genes. In addition to genes involved in the...
The focus of this symposium was to present new information on the morphogenesis of Candida albica... more The focus of this symposium was to present new information on the morphogenesis of Candida albicans, particularly how it relates to signal transduction pathways and other genes involved in the regulation of morphogenesis. In addition, we discuss the role of adherence and colonization of the oral cavity by the organism and discuss the role of mannan as an adhesin that recognizes the human red blood cell. C. albicans utilizes at least two signal pathways to regulate its conversion from a yeast form to lamentous growth (hyphae). One of these two pathways is similar to the Saccharomyces cere×isiae pseudohyphal:mating pathway, which utilizes the regulatory protein, Cph1p. The other pathway is not totally de ned but requires a second regulatory protein, referred to as Efg1p. Other signal pathways may exist, which include a two-component histidine kinase and response regulator proteins. The latter pathway(s) may include proteins such as Chk1p, Ssk1p, Shi1p and Cos1p:Nik1p. Mutations in strains, which speci cally target these proteins, result in morphogenesis defects and avirulence or attenuation of strains. A growth regulatory gene has also been recently de ned whose expression is associated with growth cessation and which appears to be a necessary prerequisite in conversion of the organism to a lamentous growth form. Starvation of yeast cells induces exponentially grown cells (and usually non-germinative) to germinate. This phenomenon is also observed in cells that are transiently treated with metabolic inhibitors. During each of these treatments (starvation, metabolic inhibition), expression of a growth regulatory gene (CGRI) increases. Adherence of C. albicans to host cells and tissues is complex; several proteins, which appear to have host recognition functions, have been de ned. In the oral cavity, C. albicans selectively adheres to salivary proteins, which are absorbed to many oral surfaces. This mechanism enables the cells to colonize surfaces of the oral cavity. An understanding of these interactions may lead to strategies to prevent oral disease. Mannan from C. albicans may provide a host recognition function for C. albicans. Recent experiments indicate that mannan binds to human red blood cells and causes hemolysis. Binding of mannan to the band 3 protein of human red blood cells has been established. This activity may be associated with the ability of the organism to utilize hemoglobin (and iron).
Cryptic links between apparently unrelated metabolic systems represent potential new drug targets... more Cryptic links between apparently unrelated metabolic systems represent potential new drug targets in fungi. Evidence of such a link between zinc and gliotoxin (GT) biosynthesis in Aspergillus fumigatus is emerging. Expression of some genes of the GT biosynthetic gene cluster gli is influenced by the zinc-dependent transcription activator ZafA, zinc may relieve GT-mediated fungal growth inhibition and, surprisingly, GT biosynthesis is influenced by zinc availability. In A. fumigatus, dithiol gliotoxin (DTG), which has zinc-chelating properties, is converted to either GT or bis-dethiobis(methylthio)gliotoxin (BmGT) by oxidoreductase GliT and methyltransferase GtmA, respectively. A double deletion mutant lacking both GliT and GtmA was previously observed to be hypersensitive to exogenous GT exposure. Here we show that compared to wild-type exposure, exogenous GT and the zinc chelator N,N,N′,N′-tetrakis(2-pyridinylmethyl)−1,2-ethanediamine (TPEN) inhibit A. fumigatus ΔgliTΔgtmA growth, ...
Aspergillus fumigatus is a saprophyte fungus that typically grows on organic decaying matter but ... more Aspergillus fumigatus is a saprophyte fungus that typically grows on organic decaying matter but can also parasitize immunosuppressed hosts. This is explained, in part, by its great ability to take up Zn 2+ ions from living tissues, which is induced by the ZafA transcription factor. This study shows that the ZafAmediated regulation of fungal growth is also influenced by iron availability and that A. fumigatus is well adapted to grow in zinc-limiting and zincreplete media with Zn:Fe ratios lower in the former than in the latter. Accordingly, this indicates that iron availability appears to be more critical for fungal growth in zinc-replete than in zinc-limiting environments. Interestingly, the cross-regulation of zinc/iron homeostasis under zinc-replete conditions relies on an unprecedented iron-mediated regulation of different zafA transcription units that, along with a limited transcript translation, allows synthesizing the right basal amount of ZafA dependent on iron availability. We posit that this regulatory strategy has evolved in fungi as a mechanism to adjust zinc intake to iron availability under zinc-replete conditions. Thus, fungal growth is enhanced in zinc-and iron-replete media but restricted by reducing zinc intake under iron starvation to prevent the noxious side effects of an intracellular zinc excess during iron deficiency.
can cause pulmonary aspergillosis in immunocompromised patients and is associated with a high mor... more can cause pulmonary aspergillosis in immunocompromised patients and is associated with a high mortality rate due to a lack of reliable treatment options. This opportunistic pathogen requires zinc in order to grow and cause disease. Novel compounds that interfere with fungal zinc metabolism may therefore be of therapeutic interest. We screened chemical libraries containing 59,223 small molecules using a resazurin assay that compared their effects on an wild-type strain grown under zinc-limiting conditions and on a zinc transporter knockout strain grown under zinc-replete conditions to identify compounds affecting zinc metabolism. After a first screen, 116 molecules were selected whose inhibitory effects on fungal growth were further tested by using luminescence assays and hyphal length measurements to confirm their activity, as well as by toxicity assays on HeLa cells and mice. Six compounds were selected following a rescreening, of which two were pyrazolones, two were porphyrins, an...
Antimicrobial agents and chemotherapy, Jan 12, 2018
An increase in the incidence of rare but hard-to-treat invasive fungal pathogens as well as resis... more An increase in the incidence of rare but hard-to-treat invasive fungal pathogens as well as resistance to the currently available antifungal drugs calls for new broad-spectrum antifungals with a novel mechanism of action. Here, we report the identification and characterization of two novel zinc-attenuating compounds ZAC307 and ZAC989, which exhibit broad-spectrumantifungal activity andefficacy in a fungal kidney burden candidiasis model.The compounds were identified serendipitously as part of a drug discovery process aimed at finding novel inhibitors of the fungal plasma membrane proton ATPase, Pma1. Based on their structure, we hypothesized that they might act as zinc chelators. Indeed, both fluorescence-based affinity determination and potentiometric assays revealed these compounds, subsequently termed zinc attenuating compounds (ZACs), to have strong affinity for zinc, and their growth inhibitory effects onandcould be inactivated by the addition of exogenous zinc to fungal growth...
Aspergillus fumigatus can infect immunocompromised patients, leading to high mortality rates due ... more Aspergillus fumigatus can infect immunocompromised patients, leading to high mortality rates due to the lack of reliable treatment options. This pathogen requires uptake of zinc from host tissues in order to successfully grow and cause virulence. Reducing the availability of that micronutrient could help treat A. fumigatus infections. In this study, we examined the in vitro effects of seven chelators using a bioluminescent strain of A. fumigatus . 1,10-Phenanthroline and N , N , N ′, N ′-tetrakis(2-pyridylmethyl)ethane-1,2-diamine (TPEN) proved to be the chelators most effective at inhibiting fungal growth. Intraperitoneal administration of either phenanthroline or TPEN resulted in a significant improvement in survival and decrease of weight loss and fungal burden for immunosuppressed mice intranasally infected with A. fumigatus . In vitro both chelators had an indifferent effect when employed in combination with caspofungin. The use of TPEN in combination with caspofungin also sign...
Journal of immunology (Baltimore, Md. : 1950), Jan 18, 2015
Calprotectin, a heterodimer of S100A8 and S100A9, is an abundant neutrophil protein that possesse... more Calprotectin, a heterodimer of S100A8 and S100A9, is an abundant neutrophil protein that possesses antimicrobial activity primarily because of its ability to chelate zinc and manganese. In the current study, we showed that neutrophils from calprotectin-deficient S100A9(-/-) mice have an impaired ability to inhibit Aspergillus fumigatus hyphal growth in vitro and in infected corneas in a murine model of fungal keratitis; however, the ability to inhibit hyphal growth was restored in S100A9(-/-) mice by injecting recombinant calprotectin. Furthermore, using recombinant calprotectin with mutations in either the Zn and Mn binding sites or the Mn binding site alone, we show that both zinc and manganese binding are necessary for calprotectin's antihyphal activity. In contrast to hyphae, we found no role for neutrophil calprotectin in uptake or killing of intracellular A. fumigatus conidia either in vitro or in a murine model of pulmonary aspergillosis. We also found that an A. fumigatu...
Aspergillus fumigatus is able to invade and grow in the lungs of immunosuppressed individuals and... more Aspergillus fumigatus is able to invade and grow in the lungs of immunosuppressed individuals and causes invasive pulmonary aspergillosis. The concentration of free zinc in living tissues is much lower than that required for optimal fungal growth in vitro because most of it is tightly bound to proteins. To obtain efficiently zinc from a living host A. fumigatus uses the zinc transporters ZrfA, ZrfB, and ZrfC. The ZafA transcriptional regulator induces the expression of all these transporters and is essential for virulence. Thus, ZafA could be targeted therapeutically to inhibit fungal growth. The ZrfC transporter plays the major role in zinc acquisition from the host whereas ZrfA and ZrfB rather have a supplementary role to that of ZrfC. In addition, only ZrfC enables A. fumigatus to overcome the inhibitory effect of calprotectin, which is an antimicrobial Zn/Mnchelating protein synthesized and released by neutrophils within the fungal abscesses of immunosuppressed non-leucopenic animals. Hence, fungal survival in these animals would be undermined upon blocking therapeutically the function of ZrfC. Therefore, both ZafA and ZrfC have emerged as promising targets for the discovery of new antifungals to treat Aspergillus infections.
For the first time, an immunodominant Aspergillus nidulans antigen (ASPND1) consistently reactive... more For the first time, an immunodominant Aspergillus nidulans antigen (ASPND1) consistently reactive with serum samples from aspergilloma patients has been purified and characterized, and its coding gene (aspnd1) has been cloned and sequenced. ASPND1 is a glycoprotein with four N-glycosidically-bound sugar chains (around 2.1 kDa each) which are not necessary for reactivity with immune human sera. The polypeptide part is synthesized as a 277-amino-acid precursor of 30.6 kDa that after cleavage of a putative signal peptide of 16 amino acids, affords a mature protein of 261 amino acids with a molecular mass of 29 kDa and a pI of 4.24 (as deduced from the sequence). The ASPND1 protein is 53.1% identical to the AspfII allergen from Aspergillus fumigatus and 48% identical to an unpublished Candida albicans antigen. All of the cysteine residues and most of the glycosylation sites are perfectly conserved in the three proteins, suggesting a similar but yet unknown function. Analysis of the primary structure of the ASPND1 coding gene (aspnd1) has allowed the establishment of a clear relationship between several previously reported A. fumigatus and A. nidulans immunodominant antigens. The increasing incidence of Aspergillus-related diseases (43) has prompted many investigators to search for fungal molecules relevant either for the immunodiagnosis of or for understanding the pathology of the different forms of aspergillosis (ranging from pulmonary aspergilloma to invasive aspergillosis, passing through allergic situations). The role of several putative virulence factors has been investigated without too much success (for a review, see reference 3). In contrast, several antigens of demonstrated value as immunodiagnostic reagents for different Aspergillus infections have been purified, and some of their coding genes have been cloned. The AspfI ribotoxin (27), the AspfII allergen (2), and heat shock protein 1 (HSP-1) (16) from Aspergillus fumigatus have been obtained as recombinant antigens. The CAT1 (the subunit of a catalase) (22) and superoxide dismutase (13) antigens have been purified to homogeneity from A. fumigatus water-soluble extracts. All of these antigens have been shown to be consistently reactive with sera from patients with different forms of aspergillosis but not with sera from control or healthy individuals (2, 10, 24, 26). Certain other purified molecules, such as the 58-kDa (6) and gp55 (40) antigens, or semipurified fractions, such as the CS2 complex (30) or the cytosolic fraction complex (CFC) (24) from A. fumigatus, have been claimed to be useful reagents for immunodiagnosis. In this paper, we attempt to simplify the entangled antigenic array of A. fumigatus antigens by using data obtained from the purification and immunochemical characterization of a previously reported Aspergillus nidulans antigen (4) (now designated ASPND1), cloning of its coding gene, and analysis of its primary structure. The conclusion of this study is that several previously reported antigens or antigenic Aspergillus preparations contain a polypeptide core of common origin, which is the part of the molecule mainly responsible for the immunogenic ability. Different glycosylation levels or light proteolytic digestions could account for the variations in molecular mass, pI, or reactivity with a particular group of patient sera. Consequently, the design of a standard reagent containing all of the antigens of demonstrated immunodiagnostic value, preferably from recombinant origin, is feasible. This should help in accurate diagnosis of the different forms of aspergillosis, mainly allergic bronchopulmonary aspergillosis (ABPA) and pulmonary aspergilloma. The universality of the strong response of the immune system to the family of AspfII and ASPND1 antigens suggests a significant role for these molecules in the pathogenesis of infections caused by Aspergillus spp. MATERIALS AND METHODS Organisms and growth conditions. A. nidulans G1059wt (adF17 pabaA1 yA2) was obtained from A. J. Clutterbuck, Glasgow, Scotland. The organism was maintained on solid YED medium (1% [wt/vol] D-glucose, 1% [wt/vol] Difco yeast extract, 2% [wt/vol] agar). To obtain high yields of conidia, the fungus was grown on solid Aspergillus complete minimal medium (AMM) containing 0.1% glucose, 0.6% NaNO 3 , 0.052% MgSO 4 , 0.052% KCl, 0.15% KH 2 PO 4 , traces of FeSO 4 and ZnSO 4 , and 1.5% (wt/vol) agar (pH 6.5). Plates were incubated at 28ЊC for at least 4 days. For liquid growth cultures, Bacto Czapek-Dox broth-Bacto synthetic broth AOAC (1:1) (CDA) medium was used (obtained from Difco Laboratories, Detroit, Mich.). AMM and CDA media were supplemented with 10 mg of p-aminobenzoic acid and 200 mg of adenine per liter. Aspergillus cells were grown by inoculation of 10 5 conidia per ml in 1-liter Erlenmeyer flasks containing 300 ml of the corresponding liquid medium followed by incubation at 37ЊC in an Adolph Kühner orbital shaker at 280 rpm. Mycelia were harvested from liquid medium cultures by filtration through Whatman GF/C paper and were washed thoroughly with double-distilled H 2 O. The wet cake was immediately frozen and kept at Ϫ70ЊC until used. Escherichia coli NM538 was used for propagation of bacteriophage EMBL4. All plasmid subcloning experiments were performed with E. coli DH5␣. To obtain singlestranded DNA for sequencing, E. coli MV1190 was used. All bacterial strains were grown at 37ЊC in Luria broth medium (35). Preparation of cell extracts. Frozen mycelia were thawed and mixed with lysing buffer (100 mM Tris-HCl [pH 7.5], containing 1 mM EDTA, 5 mM dithiothreitol [DTT], 1 mM freshly added phenylmethylsulfonyl fluoride [Sigma Chemical Co.], 5 g of aprotinin per ml, and 5 g of pepstatin A per ml [both obtained from Boehringer Mannheim]) to give a dense suspension. Samples were then disrupted in a French press (SLM Aminco) with the 20,000-lb/in 2 cell, previously refrigerated at Ϫ20ЊC, at a pressure of 16,000 lb/in 2. Complete breakage was monitored by microscopical observation. Broken mycelia were centri
Cytosolic fractions of mycelial extracts from Aspergillus nidulans, A. flaws, and three different... more Cytosolic fractions of mycelial extracts from Aspergillus nidulans, A. flaws, and three different isolates of A. fumigatus, grown to stationary phase in Czapek-Dox-AOAC medium, were tested by immunoblotting for the presence of antigens reactive to 80 serum samples from aspergilloma patients. Fifty control serum samples were used to determine the specificity of the reactions. In the A. fbmigatus cytosolic fraction a group of four main antigenic bands (p90, p60, p40 and p37) was consistently recognized (in total or partial form) by 90% of the serum samples from the aspergilloma patients. This group of antigens was designated as the 'cytosolic fraction complex' (CFC). As confirmed by two-dimensional electrophoresis followed by immunoblotting with aspergilloma serum samples, each of the four antigenic bands is formed of several isoforms of acidic glycopeptides with slightly different pls. All the isoforms are at least N-glycosylated, as demonstrated by endoglycosidase H removal of a considerable amount of sugar residues. The relationship of these antigens with certain other A. fumigatus antigens previously reported in the literature, and their potential use in the immunodiagnosis of aspergilloma, are discussed.
Aspergillus nidulans catalase B (CatB) was purified to homogeneity and characterized as a hydrope... more Aspergillus nidulans catalase B (CatB) was purified to homogeneity and characterized as a hydroperoxidase which resembles typical catalases in some physicochemical characteristics: (1) it has an apparent molecular weight of 360 000 and is composed of four glycosylated subunits, (2) it has hydrophobic properties as revealed by extractability in ethanol/chloroform and binding to phenyl-Superose, and (3) it has an acidic isoelectric point at pH 3.5. Also CatB exhibits some distinctive properties, e.g. it is not inhibited by the presence of 2% sodium dodecyl sulfate, 9 M urea or reducing agents. Furthermore, even though CatB does not exhibit any residual peroxidase activity, it is able to retain up to 38% of its initial catalase activity after incubation with the typical catalase inhibitor 3-amino-1,2,4triazole.
European Journal of Clinical Microbiology & Infectious Diseases, 1996
At present there are no accepted criteria to assess the usefulness of Western blot assays for the... more At present there are no accepted criteria to assess the usefulness of Western blot assays for the serodiagnosis of aspergilloma. An Aspergillus fumigatus cytosolic fraction complex (CFC) composed of four proteins (p90, p60, p40, and p37) has been identified. The usefulness of Western blotting with CFC antigens for the serodiagnosis of aspergilloma was evaluated in 25 patients with well-established diagnoses and in 94 controls. The most consistently reactive antigen was p90 (92% of patients with aspergilloma), followed by p40 (76%) and the entire CFC taken together (76%). With these data, interpretive criteria for positive and negative immunoblots were established, with p90 indicated as a helpful marker of aspergilloma.
Zinc is an essential micronutrient that cells must obtain from the environment in order to develo... more Zinc is an essential micronutrient that cells must obtain from the environment in order to develop their normal growth. Previous work performed at our laboratory showed that the synthesis of immunodominant antigens from Aspergillus spp., including A. fumigatus , was up-regulated by a low environmental concentration of zinc. These results suggested that a tightly regulated system for the fungus to grow under zinc-limiting conditions must underlie the ability of A. fumigatus to acquire zinc in such environments. In this work, we show that zrfA and zrfB are two of the genes that encode membrane zinc transporters from A. fumigatus in this system. Expression of these genes is differentially down-regulated by increasing concentrations of zinc in the medium. Thus, the transcription of zrfB is turned off at a concentration 50-fold higher than that for zrfA transcription. In addition, phenotypic analyses of single zrfA Δ and zrfB Δ mutants and a double zrfAzrfB Δ mutant revealed that the del...
Aspergillus fumigatus has three zinc transporter-encoding genes whose expression is regulated by ... more Aspergillus fumigatus has three zinc transporter-encoding genes whose expression is regulated by both pH and the environmental concentration of zinc. We have previously reported that the zrfA and zrfB genes of A. fumigatus are transcribed at higher levels and are required for fungal growth under acidic zinc-limiting conditions whereas they are dispensable for growth in neutral or alkaline zinc-limiting media. Here we report that the transporter of the zinc uptake system that functions in A. fumigatus growing in neutral or alkaline environments is encoded by zrfC . The transcription of zrfC occurs divergently with respect to the adjacent aspf2 gene, which encodes an immunodominant antigen secreted by A. fumigatus . The two genes— zrfC and aspf2 —are required to different extents for fungal growth in alkaline and extreme zinc-limiting media. Indeed, these environmental conditions induce the simultaneous transcription of both genes mediated by the transcriptional regulators ZafA and Pa...
Aspergillus fumigatus possesses two catalases (described as fast and slow on the basis of their e... more Aspergillus fumigatus possesses two catalases (described as fast and slow on the basis of their electrophoretic mobility). The slow catalase has been recognized as a diagnostic antigen for aspergillosis in immunocompetent patients. The antigenic catalase has been purified. The enzyme is a tetrameric protein composed of 90-kDa subunits. The corresponding cat1 gene was cloned, and sequencing data show that the cat1 gene codes for a 728-amino-acid polypeptide. A recombinant protein expressed in Pichia pastoris is enzymatically active and has biochemical and antigenic properties that are similar to those of the wild-type catalase. Molecular experiments reveal that CAT1 contains a signal peptide and a propeptide of 15 and 12 amino acid residues, respectively. cat1-disrupted mutants that were unable to produce the slow catalase were as sensitive to H2O2 and polymorphonuclear cells as the wild-type strain. In addition, there was no difference in pathogenicity between the cat1 mutant and it...
ASPND1 and ASPF2 are immunodominant antigens from Aspergillus nidulans and A. fumigatus , respect... more ASPND1 and ASPF2 are immunodominant antigens from Aspergillus nidulans and A. fumigatus , respectively, that are readily synthesized in infections in the human host, as demonstrated by their reactivity with more than 80% of sera from patients with aspergilloma or allergic bronchopulmonary aspergillosis. We demonstrate here that both antigens are exclusively produced under situations of low bioavailability of free Zn 2+ . Addition of micromolar concentrations of Zn 2+ to the culture medium strongly stimulated Aspergillus growth but totally inhibited ASPND1 or ASPF2 production. This effect was specific, since other divalent metals had no effect. Removal of endogenous Zn 2+ by a chelator also stimulated ASPND1 production, and the effect was specifically reversed by Zn 2+ . These results suggest a possible role of these antigens in the survival of the fungus in the lungs.
We have identified, purified, and characterized structurally and functionally a 90-kDa immunodomi... more We have identified, purified, and characterized structurally and functionally a 90-kDa immunodominant antigen associated with the water-soluble fraction of Aspergillus fumigatus. This antigen is recognized by 90.3% of serum samples from patients with aspergilloma and should be considered either by itself or better in combination with other purified antigens as a candidate for developing a standardized immunoassay for the detection of aspergilloma. p90 is a glycoprotein containing at least two two N-linked sugar chains of 2 and 5 kDa, respectively, which are not necessary for its reactivity with aspergilloma serum samples. Using specific anti-p90 rabbit serum, we have demonstrated that under native conditions, p90 exists in oligomeric form and has associated catalase activity. This activity is resistant to extreme temperatures (> 60 degrees C), reducing agents (40 mM dithiothreitol), high concentrations of denaturing agents such as 8 M urea and 8% sodium dodecyl sulfate, and treat...
One of the most important features that enables to grow within a susceptible individual and to ca... more One of the most important features that enables to grow within a susceptible individual and to cause disease is its ability to obtain Zn ions from the extremely zinc-limited environment provided by host tissues. Zinc uptake from this source in relies on ZIP transporters encoded by the , and genes. The expression of these genes is tightly regulated by the ZafA transcription factor that regulates zinc homeostasis and is essential for virulence. We combined the use of microarrays, Electrophoretic Mobility Shift Assays (EMSA) analyses, DNase I footprinting assays and in silico tools to better understand the regulation of the homeostatic and adaptive response of to zinc starvation. We found that under zinc-limiting conditions, ZafA functions mainly as a transcriptional activator through binding to a zinc response sequence located in the regulatory regions of its target genes, although it could also function as a repressor of a limited number of genes. In addition to genes involved in the...
The focus of this symposium was to present new information on the morphogenesis of Candida albica... more The focus of this symposium was to present new information on the morphogenesis of Candida albicans, particularly how it relates to signal transduction pathways and other genes involved in the regulation of morphogenesis. In addition, we discuss the role of adherence and colonization of the oral cavity by the organism and discuss the role of mannan as an adhesin that recognizes the human red blood cell. C. albicans utilizes at least two signal pathways to regulate its conversion from a yeast form to lamentous growth (hyphae). One of these two pathways is similar to the Saccharomyces cere×isiae pseudohyphal:mating pathway, which utilizes the regulatory protein, Cph1p. The other pathway is not totally de ned but requires a second regulatory protein, referred to as Efg1p. Other signal pathways may exist, which include a two-component histidine kinase and response regulator proteins. The latter pathway(s) may include proteins such as Chk1p, Ssk1p, Shi1p and Cos1p:Nik1p. Mutations in strains, which speci cally target these proteins, result in morphogenesis defects and avirulence or attenuation of strains. A growth regulatory gene has also been recently de ned whose expression is associated with growth cessation and which appears to be a necessary prerequisite in conversion of the organism to a lamentous growth form. Starvation of yeast cells induces exponentially grown cells (and usually non-germinative) to germinate. This phenomenon is also observed in cells that are transiently treated with metabolic inhibitors. During each of these treatments (starvation, metabolic inhibition), expression of a growth regulatory gene (CGRI) increases. Adherence of C. albicans to host cells and tissues is complex; several proteins, which appear to have host recognition functions, have been de ned. In the oral cavity, C. albicans selectively adheres to salivary proteins, which are absorbed to many oral surfaces. This mechanism enables the cells to colonize surfaces of the oral cavity. An understanding of these interactions may lead to strategies to prevent oral disease. Mannan from C. albicans may provide a host recognition function for C. albicans. Recent experiments indicate that mannan binds to human red blood cells and causes hemolysis. Binding of mannan to the band 3 protein of human red blood cells has been established. This activity may be associated with the ability of the organism to utilize hemoglobin (and iron).
Cryptic links between apparently unrelated metabolic systems represent potential new drug targets... more Cryptic links between apparently unrelated metabolic systems represent potential new drug targets in fungi. Evidence of such a link between zinc and gliotoxin (GT) biosynthesis in Aspergillus fumigatus is emerging. Expression of some genes of the GT biosynthetic gene cluster gli is influenced by the zinc-dependent transcription activator ZafA, zinc may relieve GT-mediated fungal growth inhibition and, surprisingly, GT biosynthesis is influenced by zinc availability. In A. fumigatus, dithiol gliotoxin (DTG), which has zinc-chelating properties, is converted to either GT or bis-dethiobis(methylthio)gliotoxin (BmGT) by oxidoreductase GliT and methyltransferase GtmA, respectively. A double deletion mutant lacking both GliT and GtmA was previously observed to be hypersensitive to exogenous GT exposure. Here we show that compared to wild-type exposure, exogenous GT and the zinc chelator N,N,N′,N′-tetrakis(2-pyridinylmethyl)−1,2-ethanediamine (TPEN) inhibit A. fumigatus ΔgliTΔgtmA growth, ...
Aspergillus fumigatus is a saprophyte fungus that typically grows on organic decaying matter but ... more Aspergillus fumigatus is a saprophyte fungus that typically grows on organic decaying matter but can also parasitize immunosuppressed hosts. This is explained, in part, by its great ability to take up Zn 2+ ions from living tissues, which is induced by the ZafA transcription factor. This study shows that the ZafAmediated regulation of fungal growth is also influenced by iron availability and that A. fumigatus is well adapted to grow in zinc-limiting and zincreplete media with Zn:Fe ratios lower in the former than in the latter. Accordingly, this indicates that iron availability appears to be more critical for fungal growth in zinc-replete than in zinc-limiting environments. Interestingly, the cross-regulation of zinc/iron homeostasis under zinc-replete conditions relies on an unprecedented iron-mediated regulation of different zafA transcription units that, along with a limited transcript translation, allows synthesizing the right basal amount of ZafA dependent on iron availability. We posit that this regulatory strategy has evolved in fungi as a mechanism to adjust zinc intake to iron availability under zinc-replete conditions. Thus, fungal growth is enhanced in zinc-and iron-replete media but restricted by reducing zinc intake under iron starvation to prevent the noxious side effects of an intracellular zinc excess during iron deficiency.
can cause pulmonary aspergillosis in immunocompromised patients and is associated with a high mor... more can cause pulmonary aspergillosis in immunocompromised patients and is associated with a high mortality rate due to a lack of reliable treatment options. This opportunistic pathogen requires zinc in order to grow and cause disease. Novel compounds that interfere with fungal zinc metabolism may therefore be of therapeutic interest. We screened chemical libraries containing 59,223 small molecules using a resazurin assay that compared their effects on an wild-type strain grown under zinc-limiting conditions and on a zinc transporter knockout strain grown under zinc-replete conditions to identify compounds affecting zinc metabolism. After a first screen, 116 molecules were selected whose inhibitory effects on fungal growth were further tested by using luminescence assays and hyphal length measurements to confirm their activity, as well as by toxicity assays on HeLa cells and mice. Six compounds were selected following a rescreening, of which two were pyrazolones, two were porphyrins, an...
Antimicrobial agents and chemotherapy, Jan 12, 2018
An increase in the incidence of rare but hard-to-treat invasive fungal pathogens as well as resis... more An increase in the incidence of rare but hard-to-treat invasive fungal pathogens as well as resistance to the currently available antifungal drugs calls for new broad-spectrum antifungals with a novel mechanism of action. Here, we report the identification and characterization of two novel zinc-attenuating compounds ZAC307 and ZAC989, which exhibit broad-spectrumantifungal activity andefficacy in a fungal kidney burden candidiasis model.The compounds were identified serendipitously as part of a drug discovery process aimed at finding novel inhibitors of the fungal plasma membrane proton ATPase, Pma1. Based on their structure, we hypothesized that they might act as zinc chelators. Indeed, both fluorescence-based affinity determination and potentiometric assays revealed these compounds, subsequently termed zinc attenuating compounds (ZACs), to have strong affinity for zinc, and their growth inhibitory effects onandcould be inactivated by the addition of exogenous zinc to fungal growth...
Aspergillus fumigatus can infect immunocompromised patients, leading to high mortality rates due ... more Aspergillus fumigatus can infect immunocompromised patients, leading to high mortality rates due to the lack of reliable treatment options. This pathogen requires uptake of zinc from host tissues in order to successfully grow and cause virulence. Reducing the availability of that micronutrient could help treat A. fumigatus infections. In this study, we examined the in vitro effects of seven chelators using a bioluminescent strain of A. fumigatus . 1,10-Phenanthroline and N , N , N ′, N ′-tetrakis(2-pyridylmethyl)ethane-1,2-diamine (TPEN) proved to be the chelators most effective at inhibiting fungal growth. Intraperitoneal administration of either phenanthroline or TPEN resulted in a significant improvement in survival and decrease of weight loss and fungal burden for immunosuppressed mice intranasally infected with A. fumigatus . In vitro both chelators had an indifferent effect when employed in combination with caspofungin. The use of TPEN in combination with caspofungin also sign...
Journal of immunology (Baltimore, Md. : 1950), Jan 18, 2015
Calprotectin, a heterodimer of S100A8 and S100A9, is an abundant neutrophil protein that possesse... more Calprotectin, a heterodimer of S100A8 and S100A9, is an abundant neutrophil protein that possesses antimicrobial activity primarily because of its ability to chelate zinc and manganese. In the current study, we showed that neutrophils from calprotectin-deficient S100A9(-/-) mice have an impaired ability to inhibit Aspergillus fumigatus hyphal growth in vitro and in infected corneas in a murine model of fungal keratitis; however, the ability to inhibit hyphal growth was restored in S100A9(-/-) mice by injecting recombinant calprotectin. Furthermore, using recombinant calprotectin with mutations in either the Zn and Mn binding sites or the Mn binding site alone, we show that both zinc and manganese binding are necessary for calprotectin's antihyphal activity. In contrast to hyphae, we found no role for neutrophil calprotectin in uptake or killing of intracellular A. fumigatus conidia either in vitro or in a murine model of pulmonary aspergillosis. We also found that an A. fumigatu...
Aspergillus fumigatus is able to invade and grow in the lungs of immunosuppressed individuals and... more Aspergillus fumigatus is able to invade and grow in the lungs of immunosuppressed individuals and causes invasive pulmonary aspergillosis. The concentration of free zinc in living tissues is much lower than that required for optimal fungal growth in vitro because most of it is tightly bound to proteins. To obtain efficiently zinc from a living host A. fumigatus uses the zinc transporters ZrfA, ZrfB, and ZrfC. The ZafA transcriptional regulator induces the expression of all these transporters and is essential for virulence. Thus, ZafA could be targeted therapeutically to inhibit fungal growth. The ZrfC transporter plays the major role in zinc acquisition from the host whereas ZrfA and ZrfB rather have a supplementary role to that of ZrfC. In addition, only ZrfC enables A. fumigatus to overcome the inhibitory effect of calprotectin, which is an antimicrobial Zn/Mnchelating protein synthesized and released by neutrophils within the fungal abscesses of immunosuppressed non-leucopenic animals. Hence, fungal survival in these animals would be undermined upon blocking therapeutically the function of ZrfC. Therefore, both ZafA and ZrfC have emerged as promising targets for the discovery of new antifungals to treat Aspergillus infections.
For the first time, an immunodominant Aspergillus nidulans antigen (ASPND1) consistently reactive... more For the first time, an immunodominant Aspergillus nidulans antigen (ASPND1) consistently reactive with serum samples from aspergilloma patients has been purified and characterized, and its coding gene (aspnd1) has been cloned and sequenced. ASPND1 is a glycoprotein with four N-glycosidically-bound sugar chains (around 2.1 kDa each) which are not necessary for reactivity with immune human sera. The polypeptide part is synthesized as a 277-amino-acid precursor of 30.6 kDa that after cleavage of a putative signal peptide of 16 amino acids, affords a mature protein of 261 amino acids with a molecular mass of 29 kDa and a pI of 4.24 (as deduced from the sequence). The ASPND1 protein is 53.1% identical to the AspfII allergen from Aspergillus fumigatus and 48% identical to an unpublished Candida albicans antigen. All of the cysteine residues and most of the glycosylation sites are perfectly conserved in the three proteins, suggesting a similar but yet unknown function. Analysis of the primary structure of the ASPND1 coding gene (aspnd1) has allowed the establishment of a clear relationship between several previously reported A. fumigatus and A. nidulans immunodominant antigens. The increasing incidence of Aspergillus-related diseases (43) has prompted many investigators to search for fungal molecules relevant either for the immunodiagnosis of or for understanding the pathology of the different forms of aspergillosis (ranging from pulmonary aspergilloma to invasive aspergillosis, passing through allergic situations). The role of several putative virulence factors has been investigated without too much success (for a review, see reference 3). In contrast, several antigens of demonstrated value as immunodiagnostic reagents for different Aspergillus infections have been purified, and some of their coding genes have been cloned. The AspfI ribotoxin (27), the AspfII allergen (2), and heat shock protein 1 (HSP-1) (16) from Aspergillus fumigatus have been obtained as recombinant antigens. The CAT1 (the subunit of a catalase) (22) and superoxide dismutase (13) antigens have been purified to homogeneity from A. fumigatus water-soluble extracts. All of these antigens have been shown to be consistently reactive with sera from patients with different forms of aspergillosis but not with sera from control or healthy individuals (2, 10, 24, 26). Certain other purified molecules, such as the 58-kDa (6) and gp55 (40) antigens, or semipurified fractions, such as the CS2 complex (30) or the cytosolic fraction complex (CFC) (24) from A. fumigatus, have been claimed to be useful reagents for immunodiagnosis. In this paper, we attempt to simplify the entangled antigenic array of A. fumigatus antigens by using data obtained from the purification and immunochemical characterization of a previously reported Aspergillus nidulans antigen (4) (now designated ASPND1), cloning of its coding gene, and analysis of its primary structure. The conclusion of this study is that several previously reported antigens or antigenic Aspergillus preparations contain a polypeptide core of common origin, which is the part of the molecule mainly responsible for the immunogenic ability. Different glycosylation levels or light proteolytic digestions could account for the variations in molecular mass, pI, or reactivity with a particular group of patient sera. Consequently, the design of a standard reagent containing all of the antigens of demonstrated immunodiagnostic value, preferably from recombinant origin, is feasible. This should help in accurate diagnosis of the different forms of aspergillosis, mainly allergic bronchopulmonary aspergillosis (ABPA) and pulmonary aspergilloma. The universality of the strong response of the immune system to the family of AspfII and ASPND1 antigens suggests a significant role for these molecules in the pathogenesis of infections caused by Aspergillus spp. MATERIALS AND METHODS Organisms and growth conditions. A. nidulans G1059wt (adF17 pabaA1 yA2) was obtained from A. J. Clutterbuck, Glasgow, Scotland. The organism was maintained on solid YED medium (1% [wt/vol] D-glucose, 1% [wt/vol] Difco yeast extract, 2% [wt/vol] agar). To obtain high yields of conidia, the fungus was grown on solid Aspergillus complete minimal medium (AMM) containing 0.1% glucose, 0.6% NaNO 3 , 0.052% MgSO 4 , 0.052% KCl, 0.15% KH 2 PO 4 , traces of FeSO 4 and ZnSO 4 , and 1.5% (wt/vol) agar (pH 6.5). Plates were incubated at 28ЊC for at least 4 days. For liquid growth cultures, Bacto Czapek-Dox broth-Bacto synthetic broth AOAC (1:1) (CDA) medium was used (obtained from Difco Laboratories, Detroit, Mich.). AMM and CDA media were supplemented with 10 mg of p-aminobenzoic acid and 200 mg of adenine per liter. Aspergillus cells were grown by inoculation of 10 5 conidia per ml in 1-liter Erlenmeyer flasks containing 300 ml of the corresponding liquid medium followed by incubation at 37ЊC in an Adolph Kühner orbital shaker at 280 rpm. Mycelia were harvested from liquid medium cultures by filtration through Whatman GF/C paper and were washed thoroughly with double-distilled H 2 O. The wet cake was immediately frozen and kept at Ϫ70ЊC until used. Escherichia coli NM538 was used for propagation of bacteriophage EMBL4. All plasmid subcloning experiments were performed with E. coli DH5␣. To obtain singlestranded DNA for sequencing, E. coli MV1190 was used. All bacterial strains were grown at 37ЊC in Luria broth medium (35). Preparation of cell extracts. Frozen mycelia were thawed and mixed with lysing buffer (100 mM Tris-HCl [pH 7.5], containing 1 mM EDTA, 5 mM dithiothreitol [DTT], 1 mM freshly added phenylmethylsulfonyl fluoride [Sigma Chemical Co.], 5 g of aprotinin per ml, and 5 g of pepstatin A per ml [both obtained from Boehringer Mannheim]) to give a dense suspension. Samples were then disrupted in a French press (SLM Aminco) with the 20,000-lb/in 2 cell, previously refrigerated at Ϫ20ЊC, at a pressure of 16,000 lb/in 2. Complete breakage was monitored by microscopical observation. Broken mycelia were centri
Cytosolic fractions of mycelial extracts from Aspergillus nidulans, A. flaws, and three different... more Cytosolic fractions of mycelial extracts from Aspergillus nidulans, A. flaws, and three different isolates of A. fumigatus, grown to stationary phase in Czapek-Dox-AOAC medium, were tested by immunoblotting for the presence of antigens reactive to 80 serum samples from aspergilloma patients. Fifty control serum samples were used to determine the specificity of the reactions. In the A. fbmigatus cytosolic fraction a group of four main antigenic bands (p90, p60, p40 and p37) was consistently recognized (in total or partial form) by 90% of the serum samples from the aspergilloma patients. This group of antigens was designated as the 'cytosolic fraction complex' (CFC). As confirmed by two-dimensional electrophoresis followed by immunoblotting with aspergilloma serum samples, each of the four antigenic bands is formed of several isoforms of acidic glycopeptides with slightly different pls. All the isoforms are at least N-glycosylated, as demonstrated by endoglycosidase H removal of a considerable amount of sugar residues. The relationship of these antigens with certain other A. fumigatus antigens previously reported in the literature, and their potential use in the immunodiagnosis of aspergilloma, are discussed.
Aspergillus nidulans catalase B (CatB) was purified to homogeneity and characterized as a hydrope... more Aspergillus nidulans catalase B (CatB) was purified to homogeneity and characterized as a hydroperoxidase which resembles typical catalases in some physicochemical characteristics: (1) it has an apparent molecular weight of 360 000 and is composed of four glycosylated subunits, (2) it has hydrophobic properties as revealed by extractability in ethanol/chloroform and binding to phenyl-Superose, and (3) it has an acidic isoelectric point at pH 3.5. Also CatB exhibits some distinctive properties, e.g. it is not inhibited by the presence of 2% sodium dodecyl sulfate, 9 M urea or reducing agents. Furthermore, even though CatB does not exhibit any residual peroxidase activity, it is able to retain up to 38% of its initial catalase activity after incubation with the typical catalase inhibitor 3-amino-1,2,4triazole.
European Journal of Clinical Microbiology & Infectious Diseases, 1996
At present there are no accepted criteria to assess the usefulness of Western blot assays for the... more At present there are no accepted criteria to assess the usefulness of Western blot assays for the serodiagnosis of aspergilloma. An Aspergillus fumigatus cytosolic fraction complex (CFC) composed of four proteins (p90, p60, p40, and p37) has been identified. The usefulness of Western blotting with CFC antigens for the serodiagnosis of aspergilloma was evaluated in 25 patients with well-established diagnoses and in 94 controls. The most consistently reactive antigen was p90 (92% of patients with aspergilloma), followed by p40 (76%) and the entire CFC taken together (76%). With these data, interpretive criteria for positive and negative immunoblots were established, with p90 indicated as a helpful marker of aspergilloma.
Zinc is an essential micronutrient that cells must obtain from the environment in order to develo... more Zinc is an essential micronutrient that cells must obtain from the environment in order to develop their normal growth. Previous work performed at our laboratory showed that the synthesis of immunodominant antigens from Aspergillus spp., including A. fumigatus , was up-regulated by a low environmental concentration of zinc. These results suggested that a tightly regulated system for the fungus to grow under zinc-limiting conditions must underlie the ability of A. fumigatus to acquire zinc in such environments. In this work, we show that zrfA and zrfB are two of the genes that encode membrane zinc transporters from A. fumigatus in this system. Expression of these genes is differentially down-regulated by increasing concentrations of zinc in the medium. Thus, the transcription of zrfB is turned off at a concentration 50-fold higher than that for zrfA transcription. In addition, phenotypic analyses of single zrfA Δ and zrfB Δ mutants and a double zrfAzrfB Δ mutant revealed that the del...
Aspergillus fumigatus has three zinc transporter-encoding genes whose expression is regulated by ... more Aspergillus fumigatus has three zinc transporter-encoding genes whose expression is regulated by both pH and the environmental concentration of zinc. We have previously reported that the zrfA and zrfB genes of A. fumigatus are transcribed at higher levels and are required for fungal growth under acidic zinc-limiting conditions whereas they are dispensable for growth in neutral or alkaline zinc-limiting media. Here we report that the transporter of the zinc uptake system that functions in A. fumigatus growing in neutral or alkaline environments is encoded by zrfC . The transcription of zrfC occurs divergently with respect to the adjacent aspf2 gene, which encodes an immunodominant antigen secreted by A. fumigatus . The two genes— zrfC and aspf2 —are required to different extents for fungal growth in alkaline and extreme zinc-limiting media. Indeed, these environmental conditions induce the simultaneous transcription of both genes mediated by the transcriptional regulators ZafA and Pa...
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Papers by Jose Calera