Fernando Leal

The zrfA and zrfB Genes of Aspergillus fumigatus Encode the Zinc Transporter Proteins of a Zinc Uptake System Induced in an Acid, Zinc-Depleted Environment

ABSTRACT 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 zrfAΔ and zrfBΔ mutants and a double zrfAzrfBΔ mutant revealed that the deletion of zrfB causes a greater defect in growth than the single deletion of zrfA. Deletion of both genes has a dramatic effect on growth under acid, zinc-limiting conditions. Interestingly, in neutral or slightly alkaline zinc-depleted medium, the transcriptional expression of both genes is down-regulated to such an extent that even in the absence of a supplement of zinc, the expression of zrfA and zrfB is strongly reduced. This fact correlates with the growth observed in alkaline medium, in which even a zrfAzrfBΔ double mutant was able to grow in a similar way to the wild-type under extremely zinc-limiting conditions. In sum, the zinc transport proteins encoded by zrfA and zrfB are members of a zinc uptake system of A. fumigatus that operates mainly under acid, zinc-limiting conditions.

The ZrfC alkaline zinc transporter is required for Aspergillus fumigatus virulence and its growth in the presence of the Zn/Mn-chelating protein calprotectin

Aspergillus fumigatus can invade the lungs of immunocompromised individuals causing a life‐threatening disease called invasive pulmonary aspergillosis (IPA). To grow in the lungs, A. fumigatus obtains from the host all nutrients, including zinc. In living tissues, however, most zinc is tightly bound to zinc‐binding proteins. Moreover, during infection the bioavailability of zinc can be further decreased by calprotectin, an antimicrobial Zn/Mn‐chelating protein that is released by neutrophils in abscesses. Nevertheless, A. fumigatus manages to uptake zinc from and grow within the lungs of susceptible individuals. Thus, in this study we investigated the role of the zrfA, zrfB and zrfC genes, encoding plasma membrane zinc transporters, in A. fumigatus virulence. We showed that zrfC is essential for virulence in the absence of zrfA and zrfB, which contribute to fungal pathogenesis to a lesser extent than zrfC and are dispensable for virulence in the presence of zrfC. The special ability of ZrfC to scavenge and uptake zinc efficiently from lungtissue depended on its N‐terminus, which is absent in the ZrfA and ZrfB transporters. In addition, under Zn‐ and/or Mn‐limiting conditions zrfC enables A. fumigatus to grow in the presence of calprotectin, which is detected in fungal abscesses of non‐leucopenic animals. This study extends our knowledge about the pathobiology of A. fumigatus and suggests that fungal zinc uptake could be a promising target for new antifungals.

Distinctive properties of the catalase B of Aspergillus nidulans

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‐1,2,4‐triazole.

Targeting zinc homeostasis to combat Aspergillus fumigatus infections.

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/Mn-chelating 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.

Culture conditions for zinc- and pH-regulated gene expression studies in Aspergillus fumigatus

In Aspergillus fumigatus, the regulation of zinc homeostasis is strongly influenced by environmental pH. Thus, the study of zinc-regulated gene expression in A. fumigatus requires controlling variations in culture pH, as this may affect zinc availability. However, depending on the nitrogen source, the pH of the culture can change dramatically over time. In addition, due to the ubiquitous distribution of zinc and that it is an essential micronutrient required in minute amounts for optimal fungal growth, neither buffering of the culture media to prevent pH variations nor the use of chelating agents is advisable if mycelium is to be used for expression analyses. In this work, the growth of A. fumigatus in several culture media was examined in order to determine the conditions yielding mycelia suitable for gene expression analyses in acid and neutral media, regardless of zinc availability. Our results showed that a zinc-limiting synthetic basal medium could be readily converted into a zinc-replete one and subsequently into acid or neutral medium by using, respectively, ammonium or nitrate as nitrogen source.

The recombinant antigen ASPND1r from Aspergillus nidulans is specifically recognized by sera from patients with aspergilloma

A 996 bp Aspergillus nidulans cDNA encoding the ASPND1 immunodominant antigen was cloned and expressed in Escherichia coli as a fusion protein with the enzyme glutathione S-transferase (GST) from Schistosoma japonicum. The GST-ASPND1 fusion protein was purified from isolated bacterial inclusion bodies by preparative SDS-PAGE. After cleavage with thrombin, the ASPND1 recombinant antigen (ASPND1r) and the GST protein were separated by SDS-PAGE and immunoblotted with a number of different human sera. The sera from 22 (88%) of 25 patients with an aspergilloma recognized the ASPND1r recombinant antigen on immunoblots. Forty-nine normal human sera and 14 sera from patients with other infections were unreactive. The ASPND1r expressed in E. coli could therefore be used, in combination with previously reported recombinant antigens, as a standardized antigen for serological and clinical diagnosis of Aspergillus-associated diseases.

Protein involvement in thermally induced structural transitions of pig erythrocyte ghosts

Thermal transitions in pig erythrocyte ghosts were studied by differential scanning calorimetry and thermal gel analysis (TGA). Heating of the suspension of pig erythrocyte ghosts induced at least six thermodynamically irreversible transitions. Each of these transitions is believed to be due to a localized structural transition induced by thermal stress. Using TGA and covalent attachment of the anionic transport inhibitor regions in the thermograms corresponding to the heat sorption of some proteins of the pig erythrocyte ghosts were identified.

The plasma membrane of Saccharomyces cerevisiae Molecular structure and asymmetry

The molecular structure of the plasma membrane of the haploid strain Saccharomyces cerevisiae X-2180 1 A has been studied by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis. Protein and glycoprotein components have been identified and their apparent Mr determined. A glycoprotein showing an apparent Mr of 27500 has been shown to be the main structural component. Treatment of the cells with cycloheximide prior to plasma membrane isolation resulted in a redistribution of the relative amounts of each protein band and a drastic reduction in the number of Schiff positive bands. It is postulated that treatment with this drug rids the plasma membrane of glycoprotein secretory components which are in the process of being secreted to the periplasmic space, thus allowing the study of the basic structural components of the organelle. The electrophoretic pattern of the internal membranes revealed close similarities with that of the plasma membrane and though two-dimensional electrophoresis might disclose greater differences, these similarities suggest a common origin for most of the components of both membranous systems. Finally, radioiodination techniques, have been used in studying the asymmetric disposition of some of the components of the plasma membrane. At least five polypeptides were identified as located to the outer layer of the plasma membrane and two more glycopeptides were shown to span across the bilayer.

The Transcription Factor ZafA Regulates the Homeostatic and Adaptive Response to Zinc Starvation in Aspergillus fumigatus

One of the most important features that enables Aspergillus fumigatus to grow within a susceptible individual and to cause disease is its ability to obtain Zn2+ ions from the extremely zinc-limited environment provided by host tissues. Zinc uptake from this source in A. fumigatus relies on ZIP transporters encoded by the zrfA, zrfB and zrfC genes. The expression of these genes is tightly regulated by the ZafA transcription factor that regulates zinc homeostasis and is essential for A. fumigatus 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 A. fumigatus 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 homeostatic response to zinc deficiency, ZafA also influenced, either directly or indirectly, the expression of many other genes. It is remarkable that the expression of many genes involved in iron uptake and ergosterol biosynthesis is strongly reduced under zinc starvation, even though only the expression of some of these genes appeared to be influenced directly or indirectly by ZafA. In addition, it appears to exist in A. fumigatus a zinc/iron cross-homeostatic network to allow the adaptation of the fungus to grow in media containing unbalanced Zn:Fe ratios. The adaptive response to oxidative stress typically linked to zinc starvation was also mediated by ZafA, as was the strong induction of genes involved in gliotoxin biosynthesis and self-protection against endogenous gliotoxin. This study has expanded our knowledge about the regulatory and metabolic changes displayed by A. fumigatus in response to zinc starvation and has helped us to pinpoint new ZafA target genes that could be important for fungal pathogens to survive and grow within host tissues and, hence, for virulence.