Patrick H.K. Ngai

Proteins with antifungal properties and other medicinal applications from plants and mushrooms

Living organisms produce a myriad of molecules to protect themselves from fungal pathogens. This review focuses on antifungal proteins from plants and mushrooms, many of which are components of the human diet or have medicinal value. Plant antifungal proteins can be classified into different groups comprising chitinases and chitinase-like proteins, chitin-binding proteins, cyclophilin-like proteins, defensins and defensin-like proteins, deoxyribonucleases, embryo-abundant protein-like proteins, glucanases, lectins, lipid transfer proteins, peroxidases, protease inhibitors, ribonucleases, ribosome-inactivating proteins, storage 2S albumins, and thaumatin-like proteins. Some of the aforementioned antifungal proteins also exhibit mitogenic activity towards spleen cells, nitric oxide inducing activity toward macrophages, antiproliferative activity toward tumor cells, antibacterial activity, and inhibitory activity toward HIV-1 reverse transcriptase. In contrast to the large diversity of plant antifungal proteins, only a small number of mushroom antifungal proteins have been reported. Mushroom antifungal proteins are distinct from their plant counterparts in N-terminal sequence. Nevertheless, some of the mushroom antifungal proteins have been shown to inhibit HIV-1 reverse transcriptase activity and tumor cell proliferation.

Agrocybin, an antifungal peptide from the edible mushroom Agrocybe cylindracea.

An antifungal peptide with a molecular mass of 9 kDa was isolated from fresh fruiting bodies of the mushroom Agrocybe cylindracea. The isolation procedure comprised ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography by fast protein liquid chromatography (FPLC) on Mono S, and FPLC-gel filtration on a Superdex 75 column. The antifungal peptide, designated as agrocybin, was unadsorbed on DEAE-cellulose, and adsorbed on Affi-gel blue gel and Mono S. Agrocybin exerted antifungal activity against several fungal species but lacked inhibitory activity against bacteria when tested up to 300 microM. The activity of HIV-1 reverse transcriptase was attenuated in the presence of agrocybin. It exhibited weaker mitogenic activity than Con A on isolated murine splenocytes, but was devoid of antiproliferative activity on Hep G2 (hepatoma) cells when tested at 110 microM.

A ribonuclease with antimicrobial, antimitogenic and antiproliferative activities from the edible mushroom Pleurotus sajor-caju

A 12 kDa ribonuclease preferential for poly U and with much lower activity toward poly A, poly G and poly C was isolated from fresh fruiting bodies of the mushroom Pleurotus sajor-caju. A purification procedure involving ion exchange chromatography on CM-cellulose, affinity chromatography on Red-Sepharose and Heparin-Sepharose, and fast protein liquid chromatography-gel filtration on Superdex 75 was used. The ribonuclease was adsorbed on all of the first three types of chromatographic media. It exhibited some activity toward herring sperm DNA and calf thymus DNA. The ribonuclease activity was unaffected in the presence of KCl (10 and 100 mM) and NaCl (100 mM and 1 M), but was strongly inhibited by CuSO4 (0.01 and 0.1 mM) and less potently inhibited by other divalent salts including MgCl2, CaCl2, ZnCl2, ZnSO4 and FeSO4. The optimal pH was 5.5 and the ribonuclease was stable up to 60 degrees C for 1 h. The ribonuclease inhibited mycelial growth in the fungi Fusarium oxysporum and Mycosphaerella arachidicola with an IC50 value of 95 and 72 microM, respectively. Out of the 12 species of bacteria tested, only Pseudomonas aeruginosa and Staphylococcus aureus were inhibited in growth by the ribonuclease. Viability of the tumor cells HepG2 (hepatoma) and L1210 (leukemia) was reduced with an IC50 of 0.22 and 0.1 microM, respectively in the presence of the ribonuclease. The ribonuclease inhibited translation in a cell-free rabbit reticulocyte lysate system with an IC50 of 158 nM and 3H-methyl-thymidine uptake by murine splenocytes with an IC50 of 65 nM.

Purification and characterization of a ubiquitin-like peptide with macrophage stimulating, antiproliferative and ribonuclease activities from the mushroom Agrocybe cylindracea

A peptide, with a molecular mass of 9.5kDa and demonstrating an N-terminal sequence similar to ubiquitin, was isolated from fruiting bodies of the mushroom Agrocybe cylindracea. The peptide was isolated with a purification protocol involving ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, FPLC-ion exchange chromatography on Mono S and FPLC-gel filtration on Superdex 75. The peptide was unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel and Mono S. It showed antiproliferative activity on leukemia cell line (M1) and hepatoma cell line (HepG2), and enhanced nitric oxide production in murine peritoneal macrophages with a potency comparable to that of lipopolysaccharide. A pH of 6.0 was required for optimal RNase activity. Its RNase activity was stable over the temperature range of 0-60 degrees C. It exerted ribonucleolytic activity preferentially on polyC, much lower activity on polyU, and negligible activity on polyA and polyG.

Antifungal and antiviral products of marine organisms

Marine organisms including bacteria, fungi, algae, sponges, echinoderms, mollusks, and cephalochordates produce a variety of products with antifungal activity including bacterial chitinases, lipopeptides, and lactones; fungal (-)-sclerotiorin and peptaibols, purpurides B and C, berkedrimane B and purpuride; algal gambieric acids A and B, phlorotannins; 3,5-dibromo-2-(3,5-dibromo-2-methoxyphenoxy)phenol, spongistatin 1, eurysterols A and B, nortetillapyrone, bromotyrosine alkaloids, bis-indole alkaloid, ageloxime B and (-)-ageloxime D, haliscosamine, hamigeran G, hippolachnin A from sponges; echinoderm triterpene glycosides and alkene sulfates; molluscan kahalalide F and a 1485-Da peptide with a sequence SRSELIVHQR; and cepalochordate chitotriosidase and a 5026.9-Da antifungal peptide. The antiviral compounds from marine organisms include bacterial polysaccharide and furan-2-yl acetate; fungal macrolide, purpurester A, purpurquinone B, isoindolone derivatives, alterporriol Q, tetrahydroaltersolanol C and asperterrestide A, algal diterpenes, xylogalactofucan, alginic acid, glycolipid sulfoquinovosyldiacylglycerol, sulfated polysaccharide p-KG03, meroditerpenoids, methyl ester derivative of vatomaric acid, lectins, polysaccharides, tannins, cnidarian zoanthoxanthin alkaloids, norditerpenoid and capilloquinol; crustacean antilipopolysaccharide factors, molluscan hemocyanin; echinoderm triterpenoid glycosides; tunicate didemnin B, tamandarins A and B and; tilapia hepcidin 1-5 (TH 1-5), seabream SauMx1, SauMx2, and SauMx3, and orange-spotted grouper β-defensin. Although the mechanisms of antifungal and antiviral activities of only some of the aforementioned compounds have been elucidated, the possibility to use those known to have distinctly different mechanisms, good bioavailability, and minimal toxicity in combination therapy remains to be investigated. It is also worthwhile to test the marine antimicrobials for possible synergism with existing drugs. The prospects of employing them in clinical practice are promising in view of the wealth of these compounds from marine organisms. The compounds may also be used in agriculture and the food industry.

An agglutinin with mitogenic and antiproliferative activities from the mushroom Flammulina velutipes

A hemagglutinin with a molecular mass of 12 kDa was isolated from the fruiting bodies of the mushroom Flammulina velutipes. Its molecular mass is similar to that of the fungal immunomodulatory protein isolated from F. velutipes (FIP-fve) with ice-cold 5% acetic acid and 50 mM 2-mercaptoethanol as extraction medium and to that of the larger 12 kDa subunit of F. velutipes lectin isolated with phosphate buffer as extraction medium. Its hemagglutinating activity cannot be inhibited by a variety of carbohydrates tested. The activity is stable between pH 4 and pH 11. Loss in activity occurred when the temperature is raised to 60 C and 70 C. Activity is indiscernible at and above 80 C. Its N-terminal sequence shows differences from that of FIP-fve. F. velutipes hemagglutinin stimulates [3H-methyl] thymidine uptake by mouse splenocytes. It inhibits proliferation of leukemia L1210 cells with an IC50 of 13 microM.

A napin-like polypeptide from dwarf Chinese white cabbage seeds with translation-inhibitory, trypsin-inhibitory, and antibacterial activities

Napins are 1:1 disulfide-linked complexes of a smaller (ca. 4kDa) subunit and a larger (ca. 10kDa) subunit. The intent of the present study was to ascertain the production of napin by the seeds of a Brassica species that has not been examined previously, and also to explore new biological activities of the napin. A heterodimeric 11-kDa napin-like polypeptide has been isolated from Chinese white cabbage (Brassica chinensis cv dwarf) seeds with a protocol comprising ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, fast protein liquid chromatography (FPLC)-ion exchange chromatography on Mono S and FPLC-gel filtration on Superdex 75. The N-terminal sequence of the 7-kDa subunit manifests striking similarity to napin large chain, albumin and trypsin inhibitor. The N-terminal sequence of the 4-kDa subunit is homologous to napin large chain and an antimicrobial peptide. The napin-like polypeptide inhibited translation in the rabbit reticulocyte system with an IC50 of 18.5nM. This translation-inhibitory activity was stable between pH 4 and 11, and between 10 and 40 degrees C. The polypeptide inhibited trypsin with a higher potency ( IC50 = 8.5 microM) than it inhibited chymotrypsin (IC50 = 220 microM), but was devoid of ribonuclease and antifungal activities. It manifested antibacterial activity against Pseudomonas aeruginosia, Bacillus subtilis, Bacillus cereus, and Bacillus megaterium. The results revealed that the napin-like polypeptide from Chinese white cabbage seeds exhibited some potentially exploitable activities.

Pharmacotherapy approaches to antifungal prophylaxis

Introduction: Invasive fungal infection (IFI) is a serious problem due to difficulties in early diagnosis and high mortality. Different approaches are adopted for the treatment and management of IFI, including prophylactic, empiric, preemptive and directed strategies. Areas covered: This paper reviews the type of pharmacotherapy used for antifungal prophylaxis in infants with extremely low birth weights, pediatric patients with cardiac disease, preterm neonates, pediatric oncology patients, adult cancer patients with neutropenia, adult patients with hematologic malignancy, hematopoietic stem-cell transplantation recipients, organ transplant recipients, HIV-infected patients, immunosuppressed patients treated with moderate or high doses of corticosteroids, and patients with invasive fusariosis, candidemia, invasive candidiasis, systemic mycoses and immunocompromised patients. Expert opinion: Azole drugs are the drugs most often used in cost-effective antifungal prophylaxis of patients with conditions such as immunodeficiency and cancer, which render them highly susceptible to IFI. Fluconazole is the most outstanding example. However, there are many azoles with different pharmacological characteristics that the physician can choose from. Echinocandins have favorable characteristics that make them useful for treating Candida infections. Antibodies, or their engineered derivatives directed against cell-wall polysaccharides and glycopeptides, and some protein epitopes of Candida albicans, appear to be a promising novel approach for prophylaxis against Candida infection and deserve further in-depth investigations.

Differential abilities of the mushroom ribosome-inactivating proteins hypsin and velutin to perturb normal development of cultured mouse embryos.

The teratogenicity of two fungal ribosome-inactivating proteins, hypsin from Hypsizigus mamoreus and velutin from Flammulina velutipes, was examined in this investigation using microinjection and postimplantation whole-embryo culture. The results demonstrated that hypsin induced abnormal embryonic development at 2.5 microM during the organogenesis period from E8.5 to E9.5. As its dosage increased, there was an increase in the total number of abnormal embryos, a drop in the final somite number, and a rise of abnormal structures. Structural abnormalities were detected: open cranial neural tube, abnormal branchial arches, absence of forelimb buds and twisted body axis. The otic and optic placodes were, however, less affected. Histological study of the abnormal embryos revealed a correlation of increased cell death with abnormal structures, suggesting that induction of cell death by hypsin may account for its teratogenicity. In contrast, velutin did not exert any adverse influence on mouse development.

Antifungal Protein from Passion Fruit ( Passiflora edulis ) Seeds

Publisher Summary This chapter outlines the usage of passion fruit seeds and its contribution to health and nutrition. Passion fruit has an abundance of vitamins A and C, folic acid, niacin, calcium, iron, potassium, and other nutrients. Two steroidal saponins with three and four glucose moieties, respectively, have been found to exert fungicidal effects on Aspergillus and Fusarium spp. An antifungal protein has been isolated from seeds of the passion fruit (Passiflora edulis). Passiflin specifically inhibits the fungus Rhizoctonia solani, with an IC50 of 16 mM, but no effect is seen on two other fungi, Fusarium oxysporum and Mycosphaerella arachidicola. It suppresses proliferation of breast cancer cells with an IC50 of 15 μM, but there is no effect on hepatoma HepG2 cells. It is devoid of ribonuclease, hemagglutinating, and HIV-1 reverse transcriptase inhibitory activities that may be present in some antifungal proteins. In addition to the aforementioned antifungal proteins and peptides, passion fruit has other therapeutic effects. Dietary fiber from passion-fruit rind may be promising for treatment of ailments such as diabetes, colon cancer, and other diverticular diseases. 2S albumin-like antifungal proteins and peptides distinct from passiflin in species specificity of antifungal activity have also been isolated from passion fruit.