Characterization and Inhibitory Effects of an Antifungal Protein from the Bacillus cereus Strain YN917

In this research, a type of antifungal protein derived from the Bacillus cereus strain YN917, was precipitated by ammonium sulfate after fermentation. We investigated the antifungal effects and antifungal mechanism of the protein as well as its stability under different stress conditions such as the variation of temperature, pH value, exposure to ultraviolet radiation and proteases. Furthermore, the in vitro needle inoculation method was carried out to explore the antagonistic effects of 40% isoprothiolane Wettable powder (WP), 20% tricyclazole WP and the YN917 antifungal protein against the rice blast pathogen, Magnaporthe oryzae. Results showed that 20% tricyclazole WP and 40% isoprothiolane EC treatments had significant antagonistic effects on rice blast, and that the YN917 protein also had a dual inhibitory effect on mycelium growth and conidiospore germination in M. oryzae, which can cause this mycelium to swell and twist. The YN917 protein had a broad inhibitory spectrum with different effects on 15 plant- associated pathogenic fungi and oomycetes, such as Botrytis cinerea, Bipolaris maydis and M. oryzae. In addition, the physical and chemical properties of this antifungal protein were relatively stable and have potential for application in the biocontrol of fungal pathogens in plants. Overall, the results revealed that this protein has a wide tolerance to different temperatures, pH values, exposures to ultraviolet radiation and proteases, which is required for it to function as a bio-control agent against rice blast under a wide range of environmental conditions. © 2021 Friends Science Publishers

An Antifungal Chitosanase from Bacillus subtilis SH21

Bacillus subtilis SH21 was observed to produce an antifungal protein that inhibited the growth of F. solani. To purify this protein, ammonium sulfate precipitation, gel filtration chromatography, and ion-exchange chromatography were used. The purity of the purified product was 91.33% according to high-performance liquid chromatography results. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis revealed that the molecular weight of the protein is 30.72 kDa. The results of the LC–MS/MS analysis and a subsequent sequence-database search indicated that this protein was a chitosanase, and thus, we named it chitosanase SH21. Scanning and transmission electron microscopy revealed that chitosanase SH21 appeared to inhibit the growth of F. solani by causing hyphal ablation, distortion, or abnormalities, and cell-wall depression. The minimum inhibitory concentration of chitosanase SH21 against F. solani was 68 µg/mL. Subsequently, the corresponding gene was cloned and sequenced, and sequence analysis indicated an open reading frame of 831 bp. The predicted secondary structure indicated that chitosanase SH21 has a typical a-helix from the glycoside hydrolase (GH) 46 family. The tertiary structure shared 40% similarity with that of Streptomyces sp. N174. This study provides a theoretical basis for a topical cream against fungal infections in agriculture and a selection marker on fungi.

Solution Structure, Dynamics, and New Antifungal Aspects of the Cysteine-Rich Miniprotein PAFC

The genome of Penicillium chrysogenum Q176 contains a gene coding for the 88-amino-acid (aa)-long glycine- and cysteine-rich P. chrysogenum antifungal protein C (PAFC). After maturation, the secreted antifungal miniprotein (MP) comprises 64 aa and shares 80% aa identity with the bubble protein (BP) from Penicillium brevicompactum, which has a published X-ray structure. Our team expressed isotope (15N, 13C)-labeled, recombinant PAFC in high yields, which allowed us to determine the solution structure and molecular dynamics by nuclear magnetic resonance (NMR) experiments. The primary structure of PAFC is dominated by 14 glycines, and therefore, whether the four disulfide bonds can stabilize the fold is challenging. Indeed, unlike the few published solution structures of other antifungal MPs from filamentous ascomycetes, the NMR data indicate that PAFC has shorter secondary structure elements and lacks the typical β-barrel structure, though it has a positively charged cavity and a hydrophobic core around the disulfide bonds. Some parts within the two putative γ-core motifs exhibited enhanced dynamics according to a new disorder index presentation of 15N-NMR relaxation data. Furthermore, we also provided a more detailed insight into the antifungal spectrum of PAFC, with specific emphasis on fungal plant pathogens. Our results suggest that PAFC could be an effective candidate for the development of new antifungal strategies in agriculture.

The Neosartorya fischeri Antifungal Protein 2 (NFAP2): A New Potential Weapon against Multidrug-Resistant Candida auris Biofilms

Candida auris is a potential multidrug-resistant pathogen able to persist on indwelling devices as a biofilm, which serve as a source of catheter-associated infections. Neosartorya fischeri antifungal protein 2 (NFAP2) is a cysteine-rich, cationic protein with potent anti-Candida activity. We studied the in vitro activity of NFAP2 alone and in combination with fluconazole, amphotericin B, anidulafungin, caspofungin, and micafungin against C. auris biofilms. The nature of interactions was assessed utilizing the fractional inhibitory concentration index (FICI), a Bliss independence model, and LIVE/DEAD viability assay. NFAP2 exerted synergy with all tested antifungals with FICIs ranging between 0.312–0.5, 0.155–0.5, 0.037–0.375, 0.064–0.375, and 0.064–0.375 for fluconazole, amphotericin B, anidulafungin, caspofungin, and micafungin, respectively. These results were confirmed using a Bliss model, where NFAP2 produced 17.54 μM2%, 2.16 μM2%, 33.31 μM2%, 10.72 μM2%, and 111.19 μM2% cumulative synergy log volume in combination with fluconazole, amphotericin B, anidulafungin, caspofungin, and micafungin, respectively. In addition, biofilms exposed to echinocandins (32 mg/L) showed significant cell death in the presence of NFAP2 (128 mg/L). Our study shows that NFAP2 displays strong potential as a novel antifungal compound in alternative therapies to combat C. auris biofilms.

The Epichloë festucae Antifungal Protein Efe-AfpA Is Also a Possible Effector Protein Required for the Interaction of the Fungus with its Host Grass Festuca rubra subsp. rubra

Strong creeping red fescue (Festuca rubra subsp. rubra) is a commercially important low-maintenance turfgrass and is often naturally infected with the fungal endophyte Epichloë festucae. Epichloë spp. are endophytes of several cool-season grass species, often conferring insect resistance to the grass hosts due to the production of toxic alkaloids. In addition to insect resistance, a unique feature of the strong creeping red fescue/E. festucae symbiosis is the endophyte-mediated disease resistance to the fungal pathogen Clarireedia jacksonii, the causal agent of dollar spot disease. Such disease resistance is not a general feature of other grass/ Epichloë interactions. E. festucae isolates infecting red fescue have an antifungal protein gene Efe-afpA, whereas most other Epichloë spp. do not have a similar gene. The uniqueness of this gene suggests it may, therefore, be a component of the unique disease resistance seen in endophyte-infected red fescue. Here, we report the generation of CRISPR-Cas9 Efe-afpA gene knockouts with the goal of determining if absence of the protein in endophyte-infected Festuca rubra leads to disease susceptibility. However, it was not possible to infect plants with the knockout isolates, although infection was possible with the wild type E. festucae and with complemented isolates. This raises the interesting possibility that, in addition to having antifungal activity, the protein is required for the symbiotic interaction. The antifungal protein is a small secreted protein with high expression in planta relative to its expression in culture, all characteristics consistent with effector proteins. If Efe-AfpA is an effector protein it must be specific to certain interactions, since most Epichloë spp. do not have such a gene in their genomes.

Analysis of mutations of defensin protein using accelerated molecular dynamics simulations

Plant defensins possess diverse biological functions that include antifungal and antibacterial activities and α-amylase and trypsin inhibitory properties. Two mutations, G9R and V39R, were confirmed to increase the antifungal activity of Raphanus sativus antifungal protein 2 (RsAFP2). Accelerated Molecular Dynamics (aMD) were carried out to examine the conformational changes present in these RsAFP2 mutants, and its two closest homologs compared to the wild-type protein. Specifically, the root mean square fluctuation values for the eight cysteine amino acids involved in the four disulfide bonds were low in the V39R mutant compared to the wild-type. Additionally, analysis of the free energy change revealed that G9R and V39R mutations exert a neutral and stabilizing effect on RsAFP2 conformation, and this is supported by the observed lower total energy of mutants compared to the wild-type, suggesting that enhanced stability of the mutants. However, MD simulations to a longer time scale would aid in capturing more conformational state of the wild-type and mutants defensin protein. Furthermore, the aMD simulations on fungal mimic membranes with RsAFP2 and its mutants and homologs showed that the mutant proteins caused higher deformation and water diffusion than the native RsAFP2, especially the V39R mutant. The mutant variants seem to interact by specifically targeting the POPC and POPI lipids amongst others. This work highlights the stabilizing effect of mutations at the 9th and 39th positions of RsAFP2 and their increased membrane deformation activity.

Isolation, heterologous expression, and purification of a novel antifungal protein from Bacillus subtilis strain Z-14

Background Wheat sheath blight, a soil borne fungal disease caused by Rhizoctonia cerealis, is considered as one of the most serious threats to wheat worldwide. Bacillus subtilis Z-14 was isolated from soil sampled from a wheat rhizosphere and was confirmed to have strong antifungal activity against R. cerealis. Results An antifungal protein, termed F2, was isolated from the culture supernatant of Z-14 strain using precipitation with ammonium sulfate, anion exchange chromatography, and reverse phase chromatography. Purified F2 had a molecular mass of approximately 8 kDa, as assessed using sodium dodecyl sulfate polyacrylamide gel electrophoresis. Edman degradation was used to determine the amino acid sequence of the N-terminus, which was NH2ASGGTVGIYGANMRS. This sequence is identical to a hypothetical protein RBAM_004680 (YP_001420098.1) synthesized by B. amyloliquefaciens FZB42. The recombinant F2 protein (rF2) was heterologously expressed in the yeast host Pichia pastoris, purified using a Niaffinity column, and demonstrated significant antifungal activity against R. cerealis. The purified rF2 demonstrated broad spectrum antifungal activity against different varieties of fungi such as Fusarium oxysporum, Verticillium dahliae, Bipolaris papendorfii, and Fusarium proliferatum. rF2 was thermostable, retaining 91.5% of its activity when incubated for 30 min at 100 °C. Meanwhile, rF2 maintained its activity under treatment by proteinase K and trypsin and over a wide pH range from 5 to 10. Conclusions A novel antifungal protein, F2, was purified from biocontrol Bacillus subtilis Z-14 strain fermentation supernatant and heterologously expressed in Pichia pastoris to verify its antifungal activity against R. cerealis and the validity of the gene encoding F2. Considering its significant antifungal activity and stable characteristics, protein F2 presents an alternative compound to resist fungal infections caused by R. cerealis.