Biotechnologically relevant enzymes and proteins

The mold Aspergillus giganteus produces a basic, low molecular weight protein showing antifungal properties against economically important plant pathogens, the AFP (Antifungal Protein). In this study, we investigated the mechanisms by which AFP exerts its antifungal activity against Magnaporthe grisea. M. grisea is the causal agent of rice blast, one of the most devastating diseases of cultivated rice worldwide. AFP was purified from the extracellular medium of A. giganteus cultures. The AFP protein was found to induce membrane permeabilization in M. grisea cells. Electron microscopy studies revealed severe cellular degradation and damage of plasma membranes in AFP-treated fungal cells. AFP however failed to induce membrane permeabilization on rice or human HeLa cells. Furthermore, AFP enters the fungal cell and targets to the nucleus, as revealed by co-localization experiments of Alexa-labeled AFP with the SYTOX Green dye. Finally, AFP binds to nucleic acids, including M. grisea DNA. Our results suggest that the combination of fungal cell permeabilization, cell-penetrating ability and nucleic acid-binding activity of AFP determines its potent antifungal activity against M. grisea. These results are discussed in relation to the potential of the AFP protein to enhance crop protection against fungal diseases.     

Exploring antifungal activities of acetone extract of selected Indian medicinal plants against human dermal fungal pathogens

A broad spectrum of medicinal plants was used as traditional remedies for various infectious diseases. Fungal infectious diseases have a significant impact on public health. Fungi cause more prevalent infections in immunocompromised individuals mainly patients undergoing transplantation related therapies, and malignant cancer treatments. The present study aimed to investigate the in vitro antifungal effects of the traditional medicinal plants used in India against the fungal pathogens associated with dermal infections. Indian medicinal plants (Acalypha indica, Lawsonia inermis Allium sativum and Citrus limon) extract (acetone/crude) were tested for their antifungal effects against five fungal species isolated from skin scrapings of fungal infected patients were identified as including Alternaria spp., Curvularia spp., Fusarium spp., Trichophyton spp. and Geotrichum spp. using well diffusion test and the broth micro dilution method. All plant extracts have shown to have antifungal efficacy against dermal pathogens. Particularly, Allium sativum extract revealed a strong antifungal effect against all fungal isolates with the minimum fungicidal concentration (MFC) of 50–100 μg/mL. Strong antifungal activity against Curvularia spp., Trichophyton spp., and Geotrichum spp. was also observed for the extracts of Acalypha indica, and Lawsonia inermis with MFCs of 50–800 μg/mL respectively. The extracts of Citrus limon showed an effective antifungal activity against most of the fungal strains tested with the MFCs of 50–800 μg/mL. Our research demonstrated the strong evidence of conventional plants extracts against clinical fungal pathogens with the most promising option of employing natural-drugs for the treatment of skin infections. Furthermore, in-depth analysis of identifying the compounds responsible for the antifungal activity that could offer alternatives way to develop new natural antifungal therapeutics for combating resistant recurrent infections.     

Synthesis and antifungal activity of functionalized 2,3-spirostane isomers

Invasive fungal infections are a major complication for individuals with compromised immune systems. One of the most significant challenges in the treatment of invasive fungal infections is the increased resistance of many organisms to widely used antifungals, making the development of novel antifungal agents essential. Many naturally occurring products have been found to be effective antimicrobial agents. In particular, saponins with spirostane glycosidic moieties—isolated from plant or marine species—have been shown to possess a range of antimicrobial properties. In this report, we outline a novel approach to the synthesis of a number of functionalized spirostane molecules that can be further used as building blocks for novel spirostane-linked glycosides and present results from the in vitro screenings of the antifungal potential of each derivative against four fungal species, including Candida albicans, Cryptococcus neoformans, Candida glabrata, and the filamentous fungus Aspergillus fumigatus.     

Investigation of aryl isonitrile compounds with potent,broad-spectrum antifungal activity

Invasive fungal infections present a formidable global public health challenge due to the limited number of approved antifungal agents and the emergence of resistance to the frontline treatment options, such as fluconazole. Three fungal pathogens of significant concern are Candida, Cryptococcus, and Aspergillus given their propensity to cause opportunistic infections in immunocompromised individuals. New antifungal agents composed of unique chemical scaffolds are needed to address this public health challenge. The present study examines the structure-activity relationship of a set of aryl isonitrile compounds that possess broad-spectrum antifungal activity primarily against species of Candida and Cryptococcus. The most potent derivatives are capable of inhibiting growth of these key pathogens at concentrations as low as 0.5 µM. Remarkably, the most active compounds exhibit an excellent safety profile and are non-toxic to mammalian cells even at concentrations up to 256 µM. The present study lays the foundation for further investigation of aryl isonitrile compounds as a new class of antifungal agents.     

Design,synthesis, and structure-activity relationship studies of novel triazole agents with strong antifungal activity against Aspergillus fumigatus

The incidence of invasive fungal infections has dramatically increased for several decades. In order to discover novel antifungal agents with broad spectrum and anti-Aspergillus efficacy, a series of novel triazole derivatives containing 1,2,3-benzotriazin-4-one was designed and synthesized. Most of the compounds exhibited stronger in vitro antifungal activities against tested fungi than fluconazole. Moreover, 6m showed comparable antifungal activity against seven pathogenic strains as voriconazole and albaconazole, especially against Aspergillus fumigatus (MIC = 0.25 μg/ml), and displayed moderate antifungal activity against fluconazole-resistant strains of Candida albicans. A clear SAR study indicated that compounds with groups at the 7-position resulted in novel antifungal triazoles with more effectiveness and a broader-spectrum.     

Broad-spectrum antifungal activity of spirooxindolo-pyrrolidine tethered indole/imidazole hybrid heterocycles against fungal pathogens

Invasive fungal infections are one of the leading causes of nosocomial bloodstream infections with a limited treatment option. A series of derivatized spirooxindolo-pyrrolidine tethered indole and imidazole heterocyclic hybrids have been synthesized, and their antifungal activity against fungal strains were determined. Here we characterize the antifungal activity of a specific spirooxindolo-pyrrolidine hybrid, dubbed compound 9c, a spirooxindolo-pyrrolidine tethered imidazole synthesized with a 2-chloro and trifluoromethoxy substituent. The compound 9c exhibited no cytotoxicity against mammalian cell line at concentrations that inhibited fungal strains. Compound 9c also significantly inhibited the fungal hyphae and biofilm formation. Our results indicate that spirooxindolo-pyrrolidine heterocyclic hybrids potentially represent a broad class of chemical agents with promising antifungal potential.     

Synthesis and antifungal activity of substituted 2,4,6-pyrimidinetrione carbaldehyde hydrazones

Opportunistic fungal infections caused by the Candida spp. are the most common human fungal infections, often resulting in severe systemic infections—a significant cause of morbidity and mortality in at-risk populations. Azole antifungals remain the mainstay of antifungal treatment for candidiasis, however development of clinical resistance to azoles by Candida spp. limits the drugs’ efficacy and highlights the need for discovery of novel therapeutics. Recently, it has been reported that simple hydrazone derivatives have the capability to potentiate antifungal activities in vitro. Similarly, pyrimidinetrione analogs have long been explored by medicinal chemists as potential therapeutics, with more recent focus being on the potential for pyrimidinetrione antimicrobial activity. In this work, we present the synthesis of a class of novel hydrazone-pyrimidinetrione analogs using novel synthetic procedures. In addition, structure–activity relationship studies focusing on fungal growth inhibition were also performed against two clinically significant fungal pathogens. A number of derivatives, including phenylhydrazones of 5-acylpyrimidinetrione exhibited potent growth inhibition at or below 10 μM with minimal mammalian cell toxicity. In addition, in vitro studies aimed at defining the mechanism of action of the most active analogs provide preliminary evidence that these compound decrease energy production and fungal cell respiration, making this class of analogs promising novel therapies, as they target pathways not targeted by currently available antifungals.     

Benzimidazole tethered pyrrolo[3,4-b]quinoline with broad-spectrum activity against fungal pathogens

Fungal infections caused by Candida and Cryptococcus are particularly dangerous for immunocompromised individuals. In this study, we identified that benzimidazole fused pyrrolo[3,4-b]quinoline compounds have potent antifungal activity against several clinical isolates of pathogenic fungal strains. Specifically, the compound 6a did not show cytotoxicity against mammalian cells at a concentration that inhibits the growth of fungal strains. In addition, the compound 6a also significantly reduced the metabolic activity of fungal cells in the Candida albicans biofilms. Collectively, our results indicate that benzimidazole fused quinoline compounds have a potential to develop as an antifungal agents.     

Rust and Downy Mildew Resistance in Pearl Millet (Pennisetum glaucum) Mediated by Heterologous Expression of the afp Gene from Aspergillus giganteus

The cDNA encoding the antifungal protein AFP from the mould Aspergillus giganteus was introduced into two pearl millet (Pennisetum glaucum) genotypes by particle bombardment. Stable integration and expression of the afp gene was confirmed in two independent transgenic T₀ plants and their progeny using Southern blot and RT-PCR analysis. In vitro infection of detached leaves and in vivo inoculation of whole plants with the basidomycete Puccinia substriata, the causal agent of rust disease, and the oomycete Sclerospora graminicola, causal agent of downy mildew, resulted in a significant reduction of disease symptoms in comparison to wild type control plants. The disease resistance of pearl millet was increased by up to 90% when infected with two diverse, economically important pathogens. This is the first report of genetic enhancement of Pennisetum glaucum against fungal infections.     

A pathogenesis related protein, AhPR10 from peanut: an insight of its mode of antifungal activity

A pathogenesis related protein (AhPR10) is identified from a clone of 6-day old Arachis hypogaea L. (peanut) cDNA library. The clone expressed as a ∼20 kDa protein in E. coli. Nucleotide sequence derived amino acid sequence of the coding region shows its homology with PR10 proteins having Betv1 domain and P loop motif. Recombinant AhPR10 has ribonuclease activity, and antifungal activity against the peanut pathogens Fusarium oxysporum and Rhizoctonia solani. Mutant protein AhPR10-K54N where lys54 is mutated to asn54 loses its ribonuclease and antifungal activities. FITC labeled AhPR10 and AhPR10-K54N are internalized by hyphae of F. oxysporum and R. solani but the later protein does not inhibit the fungal growth. This suggests that the ribonuclease function of AhPR10 is essential for its antifungal activity. Energy and temperature dependent internalization of AhPR10 into sensitive fungal hyphae indicate that internalization of the protein occurs through active uptake.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .The nucleotide sequence of AhPR10 reported in this paper is submitted to NCBI Nucleotide Sequence Database under the Accession number AY726607.     

Antifungal activities of actinomycete strains associated with high-altitude sagebrush rhizosphere

The antifungal-producing potential of actinomycete populations from the rhizosphere of low-altitude sagebrush, Artemisia tridentata, has been examined. In a continued investigation of new sources of antifungal-producing microorganisms, this study examined the antifungal-producing potential of actinomycetes from the rhizosphere of high-altitude A. tridentata. With high-altitude sagebrush, rhizosphere soil actinomycete numbers were one to four orders of magnitude higher than those found in nonrhizosphere bulk soils and different from those found with the low-altitude plants. A total of 122 actinomycete isolates was screened against nine fungal species and six bacterial species for the production of antimicrobial compounds. Four rhizosphere isolates, Streptomyces amakusaensis, S. coeruleorubidus, S. hawaiiensis and S. scabies, showed broad-spectrum antifungal activity against three or more fungal species in plate assays. In liquid antagonism assays, mycelium production by Aspergillus niger was reduced by up to 50% by two of the actinomycete isolates. These results demonstrate the potential of rhizosphere microbiology in the search for new antimicrobials.     

Fungicidal activity of the human peptide hepcidin 20 alone or in combination with other antifungals against Candida glabrata isolates

Candida glabrata infections are often difficult to eradicate due to the intrinsically low susceptibility to azoles of this species. In addition, C. glabrata has also been shown to be insensitive to several cationic peptides, which have been shown to be promising novel therapeutic candidates for the treatment of fungal infection. In this study, the in vitro fungicidal activity of the human cationic peptide hepcidin 20 (Hep-20) was evaluated against clinical isolates of C. glabrata with different levels of fluconazole susceptibility. Interestingly, all isolates were susceptible to Hep-20 (100–200 μg/ml) at pH 7.4, whereas the fungicidal effect of the peptide was higher (50 μg/ml) at acidic pH values. In addition, an increased antifungal activity was observed for Hep-20 with amphotericin B and a synergistic effect was demonstrated for the Hep-20/fluconazole and Hep-20/caspofungin combinations.     

Prokaryotic expression of a constitutively expressed <Emphasis Type="Italic">Tephrosia villosa</Emphasis> defensin and its potent antifungal activity

Plant defensins are small, highly stable, cysteine-rich antimicrobial peptides produced by the plants for inhibiting a broad-spectrum of microbial pathogens. Some of the well-characterized plant defensins exhibit potent antifungal activity on certain pathogenic fungal species only. We characterized a defensin, TvD1 from a weedy leguminous herb, Tephrosia villosa. The open reading frame of the cDNA was 228 bp, which codes for a peptide with 75 amino acids. Expression analyses indicated that this defensin is expressed constitutively in T. villosa with leaf, stem, root, and seed showing almost similar levels of high expression. The recombinant peptide (rTvD1), expressed in the Escherichia coli expression system, exhibited potent in vitro antifungal activity against several filamentous soil-borne fungal pathogens. The purified peptide also showed significant inhibition of root elongation in Arabidopsis seedlings, subsequently affecting the extension of growing root hairs indicating that it has the potential to disturb the plant growth and development.     

Antifungal constituents of Northern prickly ash, Zanthoxylum americanum Mill.

Leaf, fruit, stem, bark and root of Zanthoxylum americanum were investigated for antifungal activity with 11 strains of fungi representing diverse opportunistic and systemic pathogens, including Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus. All extracts demonstrated a broad spectrum of antifungal activity and inhibited at least eight fungal species in a disk diffusion assay (600 microg/disk). Antifungal activity was light-dependent, with fruit and leaf extracts most active in general. The presence of light-mediated compounds, such as psoralen, 8-methoxypsoralen and imperatorin in extracts of different organs was confirmed by RP-HPLC. A high furanocoumarin content was detected in fruit and leaf and low furanocoumarin levels were found in bark and wood. A high positive correlation was observed between total furanocoumarin content and fungal inhibition zones (r2=0.902, p<0.001). The results provide a phytochemical basis for the very widespread use of Z. americanum in indigenous North American ethnomedical tradition for conditions that may be related to fungal infections.     

Design, synthesis, and evaluations of antifungal activity of novel phenyl(2H-tetrazol-5-yl)methanamine derivatives

Fungal infections pose a continuous and serious threat to human health and life. The intrinsic resistance has been observed in many genera of fungi. Many fungal infections are caused by opportunistic pathogens that may be endogenous (Candida infections) or acquired from the environment (Cryptococcus and Aspergillus infections). So, new therapeutic strategies are needed to combat various fungal infections. Fluconazole shows good antifungal activity with relatively low toxicity and is preferred as first line antifungal therapy, but it has suffered from severe drug resistance. So, there is a need to design novel analogues by modification of fluconazole-like structure. A novel series of phenyl(2H-tetrazol-5-yl)methanamine derivatives were synthesized by reaction of α-amino nitrile with sodium azide and ZnCl₂ in presence of isopropyl alcohol. They were evaluated for antifungal activity against Candida albicans and Aspergillus niger and subjected to docking study against 1EA1.     

Antifungal activity of 2α,3β-functionalized steroids stereoselectively increases with the addition of oligosaccharides

Invasive fungal infections pose a significant problem to the immune-compromised. Moreover, increased resistance to common antifungals requires development of novel compounds that can be used to treat invasive fungal infections. Naturally occurring steroidal glycosides have been shown to possess a range of functional antimicrobial properties, but synthetic methodology for their development hinders thorough exploration of this class of molecules and the structural components required for broad spectrum antifungal activity. In this report, we outline a novel approach to the synthesis of glycoside-linked functionalized 2α,3β-cholestane and spirostane molecules and present data from in vitro screenings of the antifungal activities against human fungal pathogens and as well as mammalian cell toxicity of these derivatives.     

An overview of antifungal peptides derived from insect

Fungi are not classified as plants or animals. They resemble plants in many ways but do not produce chlorophyll or make their own food photosynthetically like plants. Fungi are useful for the production of beer, bread, medicine, etc. More complex than viruses or bacteria; fungi can be destructive human pathogens responsible for various diseases in humans. Most people have a strong natural immunity against fungal infection. However, fungi can cause diseases when this immunity breaks down. In the last few years, fungal infection has increased strikingly and has been accompanied by a rise in the number of deaths of cancer patients, transplant recipients, and acquired immunodeficiency syndrome (AIDS) patients owing to fungal infections. The growth rate of fungi is very slow and quite difficult to identify. A series of molecules with antifungal activity against different strains of fungi have been found in insects, which can be of great importance to tackle human diseases. Insects secrete such compounds, which can be peptides, as a part of their immune defense reactions. Active antifungal peptides developed by insects to rapidly eliminate infectious pathogens are considered a component of the defense munitions. This review focuses on naturally occurring antifungal peptides from insects and their challenges to be used as armaments against human diseases.     

Reassessing the Use of Undecanoic Acid as a Therapeutic Strategy for Treating Fungal Infections

Treating fungal infections is challenging and frequently requires long-term courses of antifungal drugs. Considering the limited number of existing antifungal drugs, it is crucial to evaluate the possibility of repositioning drugs with antifungal properties and to revisit older antifungals for applications in combined therapy, which could widen the range of therapeutic possibilities. Undecanoic acid is a saturated medium-chain fatty acid with known antifungal effects; however, its antifungal properties have not been extensively explored. Recent advances indicate that the toxic effect of undecanoic acid involves modulation of fungal metabolism through its effects on the expression of fungal genes that are critical for virulence. Additionally, undecanoic acid is suitable for chemical modification and might be useful in synergic therapies. This review highlights the use of undecanoic acid in antifungal treatments, reinforcing its known activity against dermatophytes. Specifically, in Trichophyton rubrum, against which the activity of undecanoic acid has been most widely studied, undecanoic acid elicits profound effects on pivotal processes in the cell wall, membrane assembly, lipid metabolism, pathogenesis, and even mRNA processing. Considering the known antifungal activities and associated mechanisms of undecanoic acid, its potential use in combination therapy, and the ability to modify the parent compound structure, undecanoic acid shows promise as a novel therapeutic against fungal infections.

     

The antifungal protein AFP from Aspergillus giganteus prevents secondary growth of different Fusarium species on barley

Secondary growth is a common post-harvest problem when pre-infected crops are attacked by filamentous fungi during storage or processing. Several antifungal approaches are thus pursued based on chemical, physical, or bio-control treatments; however, many of these methods are inefficient, affect product quality, or cause severe side effects on the environment. A protein that can potentially overcome these limitations is the antifungal protein AFP, an abundantly secreted peptide of the filamentous fungus Aspergillus giganteus. This protein specifically and at low concentrations disturbs the integrity of fungal cell walls and plasma membranes but does not interfere with the viability of other pro- and eukaryotic systems. We thus studied in this work the applicability of AFP to efficiently prevent secondary growth of filamentous fungi on food stuff and chose, as a case study, the malting process where naturally infested raw barley is often to be used as starting material. Malting was performed under lab scale conditions as well as in a pilot plant, and AFP was applied at different steps during the process. AFP appeared to be very efficient against the main fungal contaminants, mainly belonging to the genus Fusarium. Fungal growth was completely blocked after the addition of AFP, a result that was not observed for traditional disinfectants such as ozone, hydrogen peroxide, and chlorine dioxide. We furthermore detected reduced levels of the mycotoxin deoxynivalenol after AFP treatment, further supporting the fungicidal activity of the protein. As AFP treatments did not compromise any properties and qualities of the final products malt and wort, we consider the protein as an excellent biological alternative to combat secondary growth of filamentous fungi on food stuff.     

A protein from the mold Aspergillus giganteus is a potent inhibitor of fungal plant pathogens.

A purified preparation of antifungal protein (AFP) from Aspergillus giganteus exhibited potent antifungal activity against the phytopathogenic fungi Magnaporthe grisea and Fusarium moniliforme, as well as the oomycete pathogen Phytophthora infestans. Under conditions of total inhibition of fungal growth, no toxicity of AFP toward rice protoplasts was observed. Additionally, application of AFP on rice plants completely inhibited M. grisea growth. These results are discussed in relation to the potential of the afp gene to enhance crop protection against fungal pathogens in transgenic plants.