Microbiome specificity and fluxes between two distant plant taxa in Iberian forests.

BACKGROUND: Plant-associated microbial communities play important roles in host nutrition, development and defence. In particular, the microbes living within internal plant tissues can affect plant metabolism in a more intimate way. Understanding the factors that shape plant microbial composition and discovering enriched microbes within endophytic compartments would thus be valuable to gain knowledge on potential plant-microbial coevolutions. However, these interactions are usually studied through reductionist approaches (in vitro models or crop controlled systems). Here, we investigate these ecological factors in wild forest niches using proximally located plants from two distant taxa (blueberry and blackberry) as a model. RESULTS: Although the microbial communities were quite similar in both plants, we found that sampling site had a high influence on them; specifically, its impact on the rhizosphere communities was higher than that on the roots. Plant species and sample type (root vs. rhizosphere) affected the bacterial communities more than the fungal communities. For instance, Xanthobacteraceae and Helotiales taxa were more enriched in roots, while the abundance of Gemmatimonadetes was higher in rhizospheres. Acidobacteria abundance within the endosphere of blueberry was similar to that in soil. Several taxa were significantly associated with either blackberry or blueberry samples regardless of the sampling site. For instance, we found a significant endospheric enrichment of Nevskia in blueberry and of Sphingobium, Novosphingobium and Steroidobacter in blackberry. CONCLUSIONS: There are selective enrichment and exclusion processes in the roots of plants that shapes a differential composition between plant species and sample types (root endosphere-rhizosphere). The special enrichment of some microbial taxa in each plant species might suggest the presence of ancient selection and/or speciation processes and might imply specific symbiosis. The selection of fungi by the host is more pronounced when considering the fungal trait rather than the taxonomy. This work helps to understand plant-microbial interactions in natural ecosystems and the microbiome features of plants.

The interplay between zinc and iron homeostasis in Aspergillus fumigatus under zinc-replete conditions relies on the iron-mediated regulation of alternative transcription units of zafA and the basal amount of the ZafA zinc-responsiveness transcription factor.

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 Zn2+ ions from living tissues, which is induced by the ZafA transcription factor. This study shows that the ZafA-mediated regulation of fungal growth is also influenced by iron availability and that A. fumigatus is well adapted to grow in zinc-limiting and zinc-replete 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.

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.

A Novel Polyaminocarboxylate Compound To Treat Murine Pulmonary Aspergillosis by Interfering with Zinc Metabolism.

Aspergillus fumigatus 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 A. fumigatus 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, and two were polyaminocarboxylates. All three groups showed good in vitro activity, but only one of the polyaminocarboxylates was able to significantly improve the survival of immunosuppressed mice suffering from pulmonary aspergillosis. This two-tier screening approach led us to the identification of a novel small molecule with in vivo fungicidal effects and low murine toxicity that may lead to the development of new treatment options for fungal infections by administration of this compound either as a monotherapy or as part of a combination therapy.

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.

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.

Aspergillus fumigatus survival in alkaline and extreme zinc-limiting environments relies on the induction of a zinc homeostasis system encoded by the zrfC and aspf2 genes.

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 PacC. ZafA upregulates the expression of zrfC and aspf2 under zinc-limiting conditions regardless of the ambient pH, whereas PacC represses the expression of these genes under acidic growth conditions. Interestingly, the mode of action of PacC for zrfC-aspf2 transcription contrasts with the more widely accepted model for PacC function, according to which under alkaline growth conditions PacC would activate the transcription of alkaline-expressed genes but would repress the transcription of acid-expressed genes. In sum, this report provides a good framework for investigating several important aspects of the biology of species of Aspergillus, including the repression of alkaline genes by PacC at acidic pH and the interrelationship that must exist between tissue pH, metal availability in the host tissue, and fungal virulence.

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.

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 (AU)

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The regulation of zinc homeostasis by the ZafA transcriptional activator is essential for Aspergillus fumigatus virulence.

We have previously shown that Aspergillus fumigatus is able to grow in zinc-limiting media and that this ability is regulated at transcriptional level by both the availability of zinc and pH. When A. fumigatus grows as a pathogen, it must necessarily obtain zinc from the zinc-limiting environment provided by host tissue. Accordingly, the regulation of zinc homeostasis by some zinc-responsive transcriptional regulator in A. fumigatus must be essential for fungal growth within tissues of an immunocompromised host and, in turn, for pathogenicity. Here we provide evidence of the role of the zafA gene in regulating zinc homeostasis and its relevance in the virulence of A. fumigatus. Thus, we observed that (i) zafA can functionally replace the ZAP1 gene from Saccharomyces cerevisiae that encodes the zinc-responsive transcriptional activator Zap1 protein; (ii) the expression of zafA itself is induced in zinc-limiting media and repressed by zinc; (iii) deletion of zafA impairs the germination and growth capacity of A. fumigatus in zinc-limiting media; and (iv) the deletion of zafA abrogates A. fumigatus virulence in a murine model of invasive aspergillosis. In light of these observations, we concluded that ZafA is a zinc-responsive transcriptional activator that represents an essential attribute for A. fumigatus pathogenicity. Consequently, ZafA may constitute a new target for the development of chemotherapeutic agents against Aspergillus, because no zafA orthologues have been found in mammals.

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.

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 zrfADelta and zrfBDelta mutants and a double zrfAzrfBDelta 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 zrfAzrfBDelta 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.