RESUMO
Novosphingobium resinovorum SA1 was the first single isolate capable of degrading sulfanilic acid, a widely used representative of sulfonated aromatic compounds. The genome of the strain was recently sequenced, and here, we present whole-cell transcriptome analyses of cells exposed to sulfanilic acid as compared to cells grown on glucose. The comparison of the transcript profiles suggested that the primary impact of sulfanilic acid on the cell transcriptome was a starvation-like effect. The genes of the peripheral, central, and common pathways of sulfanilic acid biodegradation had distinct transcript profiles. The peripheral genes located on a plasmid had very high basal expressions which were hardly upregulated by sulfanilic acid. The genomic context and the codon usage preference of these genes suggested that they were acquired by horizontal gene transfer. The genes of the central pathways were remarkably inducible by sulfanilic acid indicating the presence of a substrate-specific regulatory system in the cells. Surprisingly, the genes of the common part of the metabolic pathway had low and sulfanilic acid-independent transcript levels. The approach applied resulted in the identification of the genes of proteins involved in auxiliary processes such as electron transfer, substrate and iron transports, sulfite oxidases, and sulfite transporters. The whole transcriptome analysis revealed that the cells exposed to xenobiotics had multiple responses including general starvation-like, substrate-specific, and substrate-related effects. From the results, we propose that the genes of the peripheral, central, and common parts of the pathway have been evolved independently.
Assuntos
Sphingomonadaceae/genética , Ácidos Sulfanílicos/metabolismo , Transcriptoma , Xenobióticos , Biodegradação Ambiental , Perfilação da Expressão Gênica , Genômica , Sphingomonadaceae/metabolismoRESUMO
Antifungal peptides offer promising alternative compounds for the treatment of fungal infections, for which new antifungal compounds are urgently needed. Constant and broad antifungal spectra of these peptides play essential roles in their reliable therapeutic application. It has been observed that rationally designed peptides using the evolutionarily conserved γ-core region (GXC-X3-9-C) of an antifungal protein from Neosartorya (Aspergillus) fischeri highly inhibit the growth of fungi. The cysteines in these peptides have free sulfhydryl groups, which allow cyclization and dimerization under oxidative conditions, thereby impairing antifungal efficacy. To overcome this problem, one or two cysteine residues were substituted by serines or S-tert-butyl was applied as a cysteine-protecting group. Furthermore, structural integrity and antifungal efficacy investigations before and after oxidative exposure revealed that substituting both cysteines with serines and S-tert-butylation helped maintain the structural integrity. However, it slightly decreased the antifungal efficacy against a yeast, Candida albicans. Interestingly, S-tert-butylation maintained the efficacy and could extend the antifungal activity to a mold, Aspergillus fumigatus. Usually, cyclization and dimerization did not influence the antifungal efficacy of most peptides. Additionally, hemolysis tests and Galleria mellonella toxicity model experiments indicated that none of the applied modifications made the peptides harmful to animals.
RESUMO
As a consequence of the fast resistance spreading, a limited number of drugs are available to treat fungal infections. Therefore, there is an urgent need to develop new antifungal treatment strategies. The features of a disulfide bond-stabilized antifungal protein, NFAP2 secreted by the mold Neosartorya (Aspergillus) fischeri render it to be a promising template for future protein-based antifungal drug design, which requires knowledge about the native disulfide linkage pattern as it is one of the prerequisites for biological activity. However, in the lack of tryptic and chymotryptic proteolytic sites in the ACNCPNNCK sequence, the determination of the disulfide linkage pattern of NFAP2 is not easy with traditional mass spectrometry-based methods. According to in silico predictions working with a preliminary nuclear magnetic resonance (NMR) solution structure, two disulfide isomers of NFAP2 (abbacc and abbcac) were possible. Both were chemically synthesized; and comparative reversed-phase high-performance liquid chromatography, electronic circular dichroism and NMR spectroscopy analyses, and antifungal susceptibility and efficacy tests indicated that the abbcac is the native pattern. This knowledge allowed rational modification of NAFP2 to improve the antifungal efficacy and spectrum through the modulation of the evolutionarily conserved γ-core region, which is responsible for the activity of several antimicrobial peptides. Disruption of the steric structure of NFAP2 upon γ-core modification led to the conclusions that this motif may affect the formation of the biologically active three-dimensional structure, and that the γ-core modulation is not an efficient tool to improve the antifungal efficacy or to change the antifungal spectrum of NFAP2.
Assuntos
Antifúngicos , Neosartorya , Antifúngicos/farmacologia , Antifúngicos/química , Neosartorya/química , Neosartorya/metabolismo , Nozes , Aspergillus , Dissulfetos/metabolismoRESUMO
Plant pathogenic fungi are responsible for enormous crop losses worldwide. Overcoming this problem is challenging as these fungi can be highly resistant to approved chemical fungicides. There is thus a need to develop and introduce fundamentally new plant and crop protection strategies for sustainable agricultural production. Highly stable extracellular antifungal proteins (AFPs) and their rationally designed peptide derivatives (PDs) constitute feasible options to meet this challenge. In the present study, their potential for topical application to protect plants and crops as combinatorial biofungicides is supported by the investigation of two Neosartorya (Aspergillus) fischeri AFPs (NFAP and NFAP2) and their γ-core PDs. Previously, the biofungicidal potential of NFAP, its rationally designed γ-core PD (γNFAP-opt), and NFAP2 was reported. Susceptibility tests in the present study extended the in vitro antifungal spectrum of NFAP2 and its γ-core PD (γNFAP2-opt) to Botrytis, Cladosporium, and Fusarium spp. Besides, in vitro additive or indifferent interactions, and synergism were observed when NFAP or NFAP2 was applied in combination with γNFAP-opt. Except for γNFAP2-opt, the investigated proteins and peptides did not show any toxicity to tomato plant leaves. The application of NFAP in combination with γNFAP-opt effectively inhibited conidial germination, biofilm formation, and hyphal extension of the necrotrophic mold Botrytis cinerea on tomato plant leaves. However, the same combination only partially impeded the B. cinerea-mediated decay of tomato fruits, but mitigated the symptoms. Our results highlight the feasibility of using the combination of AFP and PD as biofungicide for the fungal infection control in plants and crops. Supplementary Information: The online version contains supplementary material available at 10.1007/s10526-022-10132-y.