Peptide derived from plant defensins: A promising 68Ga radiolabelled agent for diagnostic of infection foci in PET.

The development of new radiopharmaceuticals for the detection of hidden infection foci has great relevance for early detection and the selection of the correct treatment, particularly in immunosuppressed patients. In that sense, the labelling of antimicrobial peptides (AMPs) that are capable of binding specifically to the pathogenic microorganism which causes the infection, should provide a sufficiently specific agent, able to distinguish an infection from a sterile inflammation. Defensins are particularly interesting molecules with antimicrobial activity, the EcgDf1 defensin was identified from the genome of a Uruguayan native plant, Erythrina crista-galli, the 'Ceibo' tree. Our group has previously reported a synthetic biologically active short analogue EcgDf21 (ERFTGGHCRGFRRRCFCTKHC) successfully labelled with 99mTc. Herein we present a shorter analogue which also preserves the γ-core domain, as a pharmacophore for a potential infection detection agent. This peptide was derivatized with the bifunctional chelating agent 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) through a lysine linker in the amino-terminal group (NOTA-KGHCRGFRRRC) and radiolabelled with 68Ga ([68Ga]Ga-NOTA-K-EcgDf1(10)). The [68Ga]Ga-NOTA-K-EcgDf1(10) labelling procedure rendered a product with high radiochemical purity and stability in the labelling milieu. The Log P value indicated that the complex has a hydrophilic behaviour, confirmed by the biodistribution profile. The [68Ga]Ga-NOTA-K-EcgDf1(10) complex demonstrated specific binding to cultures of Candida albicans and Aspergillus niger. Its biodistribution showed renal elimination and low accumulation in the rest of the body. It was possible to successfully differentiate sterile inflammation from infection by PET images in nude mice with a target/non-target ratio of 3.3 for C. albicans and 3.7 for A. niger, respectively.

[99m Tc]Tc-HYNIC-EcgDf21: A defensin short analogue with potential application in infection foci imaging.

Opportunistic infections are a problem of great relevance in public health and the precise detection and localization of infection in the early stages of the disease is of great importance for patient management as well as cost containment. Our proposal seeks to contribute to developing a new agent that meets the needs of diagnosis and follow-up of fungal and bacterial infections, focused on the design of a radiotracer with the potential for recognition of hidden infection foci. Defensins are plant antimicrobial peptides that not only show activity against plant pathogens but also against human ones. A short analogue of EcgDf1 defensin, EcgDf21d (NH2 -ERFTGGHCRGFRRRCFCTKHC-COOH), was labelled through the formation of a 99m Tc-HYNIC complex which was assessed for physicochemical and biological behaviour both in vitro and in vivo. The [99m Tc]Tc-HYNIC-EcgDf21 labelling procedure rendered a single product with remarkably high RCP and stability in the labelling milieu. The Log p value indicated that [99m Tc]Tc-HYNIC-EcgDf21 has a hydrophilic behaviour, confirmed by the biodistribution profiles. The optimal uptake value was obtained for Candida albicans infection model reaching a lesion/muscle ratio of 3, this correlates with in vitro binding studies, and the lesion can be definitely observed in the scintigraphic images.

Antimicrobial peptides in the seedling transcriptome of the tree legume Peltophorum dubium.

Antimicrobial peptides (AMPs) play an essential role in plant defense against invading pathogens. Due to their biological properties, these molecules have been considered useful for drug development, as novel agents in disease therapeutics, applicable to both agriculture and medicine. New technologies of massive sequencing open opportunities to discover novel AMP encoding genes in wild plant species. This work aimed to identify cysteine-rich AMPs from Peltophorum dubium, a legume tree from South America. We performed whole-transcriptome sequencing of P. dubium seedlings followed by de novo transcriptome assembly, uncovering 78 AMP transcripts classified into five families: hevein-like, lipid-transfer proteins (LTPs), alpha hairpinins, defensins, and snakin/GASA (Giberellic Acid Stimulated in Arabidopsis) peptides. No transcripts with similarity to cyclotide or thionin genes were identified. Genomic DNA analysis by PCR confirmed the presence of 18 genes encoding six putative defensins and 12 snakin/GASA peptides and allowed the characterization of their exon-intron structure. The present work demonstrates that AMP prediction from a wild species is possible using RNA sequencing and de novo transcriptome assembly, regarding a starting point for studies focused on AMP gene evolution and expression. Moreover, this study allowed the detection of strong AMP candidates for drug development and novel biotechnological products.

Gene isolation and structural characterization of a legume tree defensin with a broad spectrum of antimicrobial activity.

MAIN CONCLUSION: The recombinant EcgDf1 defensin has an antimicrobial effect against both plant and human pathogens. In silico analyses predict that EcgDf1 is prone to form dimers capable of interacting with the membranes of microorganisms. Plant defensins comprise a large family of antimicrobial peptides (AMP) with a wide range of biological functions. They are cysteine-rich molecules, highly sequence diverse but with a conserved and stable structure. In this work, a defensin gene (EcgDf1) was isolated from Erythrina crista-galli, a legume tree native from South America. The predicted peptide presents eight cysteines, with a γ-core motif GXCX3-9C and six cysteines distributed like the typical defensin αß motif. The mature EcgDf1 coding sequence was heterologously expressed in Escherichia coli strains and purified by affinity chromatography. Possible dimer and oligomers of EcgDf1 were visible in SDS electrophoresis. Moreover, its 3D structure, determined by homology modeling, docking, and molecular dynamics simulations, was found to be compatible with the formation of homodimers between the ß3 and ß1-loop-α1, leaving the ß2-loop-ß3 free to interact with lipid membranes. The purified recombinant peptide inhibited the growth of several critical plant and human pathogens, like the opportunistic fungi Candida albicans and Aspergillus niger and the plant pathogens Clavibacter michiganensis ssp. michiganensis, Penicillium expansum, Botrytis cinerea, and Alternaria alternata. EcgDf1 is a promising candidate for the development of antimicrobial products for use in agriculture and medicine.

Evaluation of different hurdles on Penicillium crustosum growth in sponge cakes by means of a specific real time PCR.

Limited shelf life of bakery products, caused by microbial deterioration, is a concern for industries due to economic losses. Fungal spoilage of sponge cakes industrially produced in Montevideo was caused mainly by Penicillium species, in particular by Penicillium crustosum. The combination of different hurdles was studied to inhibit P. crustosum growth in sponge cakes. A full factorial design was performed to study the effect of the concentration of potassium sorbate, pH, packaging atmosphere and storage time. The results showed that packaging atmosphere and storage time were the significant factors in the ranges tested. No growth was detected in cakes stored in modified atmosphere packaging (MAP) (N2:CO2 50:50) at room temperature (25 °C) for 15 days. The effect of MAP on P. crustosum growth in cakes at room temperature was compared with the effect of air-packaging and storage at low temperature (4 °C) for 30 days. P. crustosum growth was not detected in cakes packaged in MAP, whereas it was detected after 20 days in cakes packaged in air and stored at 4 °C. This growth was quantified by a specific real time PCR developed in this work. Specific primers were designed using the sequence of ß-tubulin gene of P. crustosum as a target and PCR conditions were adjusted to ensure specificity. PCR efficiency was 107%, with a detection limit of 0.0014 ng of DNA. The qPCR method presented here, resulted specific and sensitive enough to detect the growth of P. crustosum even before biodeterioration signs were visible.

Antimicrobial and structural insights of a new snakin-like peptide isolated from Peltophorum dubium (Fabaceae).

Snakins are antimicrobial peptides (AMPs) found, so far, exclusively in plants, and known to be important in the defense against a wide range of pathogens. Like other plant AMPs, they contain several positively charged amino acids, and an even number of cysteine residues forming disulfide bridges which are considered important for their usual function. Despite its importance, studies on snakin tertiary structure and mode of action are still scarce. In this study, a new snakin-like gene was isolated from the native plant Peltophorum dubium, and its expression was verified in seedlings and adult leaves. The deduced peptide (PdSN1) shows 84% sequence identity with potato snakin-1 mature peptide, with the 12 cysteines characteristic from this peptide family at the GASA domain. The mature PdSN1 coding sequence was successfully expressed in Escherichia coli. The purified recombinant peptide inhibits the growth of important plant and human pathogens, like the economically relevant potato pathogen Streptomyces scabies and the opportunistic fungi Candida albicans and Aspergillus niger. Finally, homology and ab initio modeling techniques coupled to extensive molecular dynamics simulations were used to gain insight on the 3D structure of PdSN1, which exhibited a helix-turn-helix motif conserved in both native and recombinant peptides. We found this motif to be strongly coded in the sequence of PdSN1, as it is stable under different patterns of disulfide bonds connectivity, and even when the 12 cysteines are considered in their reduced form, explaining the previous experimental evidences.

Functional characterization of two novel purine transporters from the Basidiomycota Phanerochaete chrysosporium.

Purine transporters as substrate entry points in organisms, are involved in a number of cellular processes such as nitrogen source uptake, energy metabolism and synthesis of nucleic acids. In this study, two nucleobase transporter genes (phZ, phU) from Phanerochaete chrysosporium were cloned, identified, and functionally characterized. Our results show that PhZ is a transporter of adenine and hypoxanthine, and a protein belonging to the AzgA-like family, whilst PhU belongs to the NAT/NCS2 family, transporting xanthine and uric acid. No other sequences belonging to these families were detected in P. chrysosporium's genome. Phylogenetic analyses show that AzgA-like sequences form monophyletic groups for each major lineage (Ascomycota, Basidiomycota and Zygomycota). In contrast, Ascomycota and Basidiomycota NAT/NCS2 sequences do not form monophyletic groups and several copies of this protein are distributed across the tree. Expression of phU was significantly downregulated in the presence of a primary source like ammonium, and enhanced if purines were present or if the mycelium was nitrogen starved. phZ was clearly induced by its substrates (hypoxanthine, adenine), very lightly induced by xanthine, suppressed by urea and amino acids and expressed at a basal level when uric acid or ammonium was the nitrogen source or when the mycelium was starved for nitrogen. In order to perform substrate analyses, both P. chrysosporium proteins (PhZ, PhU) were expressed in Aspergillus nidulans. Epifluorescent microscopy showed that under inducing conditions, PhZ-GFP and PhU-GFP were present at the plasma membrane of A. nidulans transformed strains, and were internalized in repressed conditions. Our results suggest that in the white-rot fungus P. chrysosporium, phU has a catabolic role and phZ, (less dependent of the nitrogen source), plays a key role in purine acquisition to provide biosynthetic components. These are the first purine transporters characterized in Basidiomycota.

Screening for Antimicrobial Activity of Wood Rotting Higher Basidiomycetes Mushrooms from Uruguay against Phytopathogens.

In this work, the antimicrobial activity of extracts of wood rotting higher Basidiomycetes mushrooms isolated from Eucalyptus plantations in Uruguay was studied using bacterial and fungal phytopathogens as targets. Fifty-one extracts from mycelia and growth broth were prepared from higher Basidiomycetes mushrooms, from which eight extracts (from Ganoderma resinaceum, Laetiporus sulphureus, Dictyopanus pusillus, and Bjerkandera adusta) showed antimicrobial activity against Xanthomonas vesicatoria, Aspergillus oryzae, Penicillium expansum, Botrytis cinerea, and Rhizopus stolonifer as assayed in the qualitative test. The minimum inhibitory concentration (MIC) for those fungal extracts was determined and the results showed that L. sulphureus deserved further study, with low MIC values against X. vesicatoria. The antimicrobial activity of L. sulphureus culture broth extracts grown under different culture conditions was evaluated against X. vesicatoria. From the results of these assays, larger-scale cultures for the production of the compound(s) with antimicrobial activity should be performed using malt extract broth, at pH 5, at 20°C and static culture conditions.

Mutations in the basic loop of the Zn binuclear cluster of the UaY transcriptional activator suppress mutations in the dimerisation domain.

UaY is the specific ZnII(2)Cys(6) transcriptional activator of the purine utilisation pathway in Aspergillus nidulans. Previous work has determined the consensus binding sequence by EMSA and foot-printing. We determine here that it binds as a dimer to its cognate CGG-N(6)-CCG sites. We identify the uaY109 mutation, which has been shown to affect differentially the expression of a number of UaY-regulated genes, as a F112I substitution in the DNA-binding motif dimerisation domain. We isolated back mutants, revertants carrying different residues at the same position (I112N and I112M) and intragenic suppressors mapping in the first loop of the Zn cluster (N75T and N75K). We have analysed the original mutant and its revertants by growth tests and by their effects on the mRNA steady states of five UaY-regulated genes. We have determined the effect of the different mutations on UaY dimerisation, on the apparent Kdiss of the UaY DNA-binding domain to appropriate DNA sequences and on the methylation interference pattern. We have attempted to rationalise these phenotypes by modelling the UaY DNA binding domain on the structure of the highly similar Ppr1p. However, modelling of the wild-type and mutant proteins provides only a partial explanation for the observed phenotypes. This suggests that the mutated residues may have other roles besides the obvious ones inferred from their position in the sequence and by the similarity of UaY and Ppr1p.

Differential physiological and developmental expression of the UapA and AzgA purine transporters in Aspergillus nidulans.

In this article we study the cellular expression of UapA and AzgA, the two major purine transporters of Aspergillus nidulans, by constructing strains expressing, from their native promoters, fully functional fluorescent (UapA-sGFP, AzgA-sGFP) or immunological (UapA-His) chimeric transporters. Epifluorescence microscopy and immunodetection showed that under different physiological conditions and during Aspergillus development: (i) UapA and AzgA expression in the plasma membrane becomes evident early during germination and remains at a significant basal level in mycelium, (ii) Neither of the two transporters is expressed in the stalk, the vesicle, the phialides and the conidiospores, but surprisingly, UapA is specifically and strongly expressed in the periphery of metulae, (iii) Both transporters are expressed in ascogenous hyphae and in hülle cells but not in cleistothecia or ascospores, (iv) Purine induction leads to approximately 4-fold increase in UapA and AzgA protein content in mycelium, compatible with an analogous increase at the transcriptional level, (v) Ammonium leads to removal of UapA, but not of AzgA, from the plasma membrane by sorting of the protein to the vacuole.

Transcription of purine transporter genes is activated during the isotropic growth phase of Aspergillus nidulans conidia.

Aspergillus nidulans possesses three well-characterized purine transporters encoded by the genes uapA, uapC and azgA. Expression of these genes in mycelium is induced by purines and repressed by ammonium or glutamine through the action of the pathway-specific UaY regulator and the general GATA factor AreA respectively. Here, we describe the regulation of expression of purine transporters during conidiospore germination and the onset of mycelium development. In resting conidiospores, mRNA steady-state levels of purine transporter genes and purine uptake activities are undetectable or very low. Both mRNA steady-state levels and purine transport activities increase substantially during the isotropic growth phase of conidial germination. Both processes occur in the absence of purine induction and independently of the nitrogen source present in the medium. The transcriptional activator UaY is dispensable for the germination-induced expression of the three transporter genes. AreA, on the other hand, is essential for the expression of uapA, but not for that of azgA or uapC, during germination. Transcriptional activation of uapA, uapC and azgA during germination is also independent of the presence of a carbon source in the medium. This work establishes the presence of a novel system triggering purine transporter transcription during germination. Similar results have been found in studies on the expression of other transporters in A. nidulans, suggesting that global expression of transporters might operate as a general system for sensing solute availability.

The AzgA purine transporter of Aspergillus nidulans. Characterization of a protein belonging to a new phylogenetic cluster.

The azgA gene of Aspergillus nidulans encodes a hypoxanthine-adenine-guanine transporter. It has been cloned by a novel transposon methodology. The null phenotype of azgA was defined by a number of mutations, including a large deletion. In mycelia, the azgA gene is, like other genes of purine catabolism, induced by uric acid and repressed by ammonium. Its transcription depends on the pathway-specific UaY zinc binuclear cluster protein and the broad domain AreA GATA factor. AzgA is not closely related to any other characterized membrane protein, but many close homologues of unknown function are present in fungi, plants, and prokaryotes but not metazoa. Two of three data bases and the phylogeny presented in this article places proteins of this family in a cluster clearly separated (but perhaps phylogenetically related) from the NAT family that includes other eukaryotic and prokaryotic nucleobase transporters. Thus AzgA is the first characterized member of this family or subfamily of membrane proteins.

PrnA, a Zn2Cys6 activator with a unique DNA recognition mode, requires inducer for in vivo binding.

The PrnA transcriptional activator of Aspergillus nidulans binds as a dimer to CCGG-N-CCGG inverted repeats and to CCGG-6/7N-CCGG direct repeats. The binding specificity of the PrnA Zn cluster differs from that of the Gal4p/Ppr1p/UaY/Put3p group of proteins. Chimeras with UaY, a protein that strictly recognizes a CGG-6N-CCG motif, show that the recognition of the direct repeats necessitates the PrnA dimerization and linker elements, but the recognition of the CCGG-N-CCGG inverted repeats depends crucially on the PrnA Zn binuclear cluster and/or on residues amino-terminal to it. Three high-affinity sites in two different promoters have been visualized by in vivo methylation protection. Proline induction is essential for in vivo binding to these three sites but, as shown previously, not for nuclear entry. Simultaneous repression by ammonium and glucose does not affect in vivo binding to these high-affinity sites. PrnA differs from the isofunctional Saccharomyces cerevisiae protein Put3p, both in its unique binding specificity and in the requirement of induction for in vivo DNA binding.