Claude P. Selitrennikoff

Zeamatin, an antifungal protein from maize with membrane-permeabilizing activity.

Summary: A 22 kDa antifungal protein (zeamatin) was purified from Zea mays seeds. It was identified and assayed by its unusual property of acting synergistically with nikkomycin to inhibit growth of Candida albicans. Alone, it inhibited growth in suspension culture of C. albicans, Neurospora crassa and Trichoderma reesei. Zeamatin contained no detectable chitinase, 1,3-β-glucanase or ribosome-inactivating protein activity, enzymes present in a variety of plants that have been shown to have antifungal properties. At low concentrations zeamatin caused the rapid release of cytoplasmic material from C. albicans and N. crassa. This was confirmed microscopically by observing zeamatin-induced hyphal rupture of these fungi. These results suggest that zeamatin permeabilizes the fungal plasma membrane. We believe zeamatin to be a representative of a previously unrecognized class of plant antifungal proteins.

Isolation and partial characterization of two antifungal proteins from barley

We have developed a simple assay for detecting antifungal compounds utilizing impregnated paper discs on agar to inhibit mycelial spread of an indicator organism, Trichoderma reesei. Using this assay we have isolated and purified to apparent homogeneity two antifungal proteins from dehusked barley grain. Both proteins are present at high concentrations: over 10 mg of each protein can be isolated per 100 g of grain. The first protein has a molecular weight of 30 000 and is identical to the 30 kDa ribosome-inactivating protein previously isolated from barley. This protein very effectively inactivates fungal ribosomes and this may explain its antifungal activity and biological role. The second antifungal protein has a molecular weight of 28 000 and is 20-fold more potent than the 30 kDa protein in inhibiting growth of Trichoderma. In addition to Trichoderma, the 28 kDa protein also efficiently inhibits growth of Phycomyces blakesleeanus, Alternaria alternaria and a protoplast-forming mutant of Neurospora crassa. The 28 kDa protein does not inactivate fungal ribosomes and we are currently investigating other possible enzymatic activities of this protein.

Thaumatin-like pathogenesis-related proteins are antifungal

Tobacco pathogenesis-related (PR) proteins of group 5, namely PR-S and osmotin, were shown to be serologically related to zeamatin, an antifungal protein of maize seeds. These PR proteins, dubbed thaumatin-like because they show sequence homology with thaumatin, a sweet-tasting protein from fruits of Thaumatococcus daniellii, were demonstrated to have a direct antifungal activity, with specificity for different fungal species. Osmotin was particularly effective in inhibiting the growth of Candida albicans, Neurospora crassa and Trichoderma reesei. PR-S had no detectable activity against these fungi but was found the most potent antifungal protein against the plant pathogen Cercospora beticola. Osmotin caused rapid bursting of the hyphal tips of N. crassa, suggesting that tobacco thaumatin-like PR proteins are antifungal by a membrane permeabilization mechanism similar to that demonstrated previously for zeamatin. These results confirm and extend observations made by Woloshuk et al. [1] who showed that osmotin and a related protein from tomato had antifungal activity against the phytopathogen, Phytophthora infestans.

Cell wall integrity is dependent on the PKC1 signal transduction pathway in Cryptococcus neoformans

Cell wall biogenesis and integrity are crucial for fungal growth, pathogenesis and survival, and are attractive targets for antifungal therapy. In this study, we identify, delete and analyse mutant strains for 10 genes involved in the PKC1 signal transduction pathway and its regulation in Cryptococcus neoformans. The kinases Bck1 and Mkk2 are critical for maintaining integrity, and deletion of each of these causes severe phenotypes different from each other. In stark contrast to results seen in Saccharomyces cerevisiae, a deletion in LRG1 has severe repercussions for the cell, and one in ROM2 has little effect. Also surprisingly, the phosphatase Ppg1 is crucial for cell integrity. These data indicate that the mechanisms of maintaining cell integrity differ between the two fungi. Deletions in SSD1 and PUF4, potential alternative regulators of cell integrity, also exhibit phenotypes. This is the first comprehensive analysis examining genes involved the maintenance of cell integrity in C. neoformans and sets the foundation for future biochemical and virulence studies.

The Osmotic-1 Locus of Neurospora crassa Encodes a Putative Histidine Kinase Similar to Osmosensors of Bacteria and Yeast

Abstract. Osmotically sensitive mutants of Neurospora crassa are unable to grow on medium supplemented with 4% NaCl, have altered morphologies and cell-wall compositions, and are resistant to dicarboximide fungicides. Osmotic-1 (os-1) mutants have a unique characteristic of forming protoplasts that grow and divide in specialized liquid medium, suggesting that the os-1+ gene product is important for cell-wall assembly. A cosmid containing the os-1+ locus of N. crassa, isolated from a genomic cosmid library by chromosomal walk from a closely linked gene, was used to subclone the os-1+ gene by functional complementation of an os-1 mutant. Analysis of the sequence of complementing DNA predicts that os-1+ encodes a predicted protein similar to sensor-histidine kinases of bacteria and a yeast osmosensor-histidine kinase. Importantly, the predicted os-1+ protein is identical to the N. crassa nik-1 predicted protein that was identified by using polymerase chain reaction primers directed against histidine kinase consensus DNA sequences. Our results indicate that nik-1 and os-1 encode the same osmosensing histidine kinase that plays an important role in the regulation of cell-wall assembly and, probably, other cell responses to changes in external osmolarity.

Cryptococcus neoformans Resistance to Echinocandins: (1,3)β-Glucan Synthase Activity Is Sensitive to Echinocandins

ABSTRACT (1,3)β-d-Glucan synthase (EC 2.4.1.34. UDP-glucose: 1,3-β-d-glucan 3-β-glucosyltransferase) uses UDP-glucose as substrate and catalyzes the polymerization of glucose ([1,3]-β-linkages) to form the major carbohydrate component of the fungal cell wall. We have optimized in vitro assay conditions for (1,3)β-glucan synthase activity from Cryptococcus neoformans. Cells lysed in 50 mM Tris, pH 7.75, containing 20% glycerol, 2 mM NaF, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 5 mM MgCl2, 0.1% protease and phosphatase inhibitor cocktails, and 60 μM GTPγS produced maximum specific activity in vitro. We tested in vitro C. neoformans (1,3)β-glucan synthase activity against the (1,3)β-glucan synthase inhibitors, caspofungin and cilofungin, and have determined that (1,3)β-glucan synthase activity is very sensitive (apparent Ki of 0.17 ± 0.02 μM and 22 ± 5.7 μM, respectively) to these echinocandins. Taken together with high MICs for C. neoformans (caspofungin MIC, 16 μg/ml; cilofungin MIC, 64 μg/ml), our results indicate that C. neoformans is resistant to caspofungin and cilofungin by a mechanism(s) unrelated to (1,3)β-glucan synthase resistance.

Cilofungin (LY121019) inhibits Candida albicans (1-3)-beta-D-glucan synthase activity.

Cilofungin (LY121019) inhibited Candida albicans growth and activity of (1-3)-beta-glucan synthase, for which it was a noncompetitive inhibitor with a Ki-app of 2.5 microM. Cilofungin had no effect on chitin synthase activity. Based on these and other data, it seems likely that cilofungin inhibits fungal growth by inhibiting (1-3)-beta-glucan synthase activity. Images

Zeamatin Inhibits Trypsin and α-Amylase Activities

ABSTRACT Zeamatin is a 22-kDa protein isolated from Zea maysthat has antifungal activity against human and plant pathogens. Unlike other pathogenesis-related group 5 proteins, zeamatin inhibits insect α-amylase and mammalian trypsin activities. It is of clinical significance that zeamatin did not inhibit human α-amylase activity and inhibited mammalian trypsin activity only at high molar concentrations.

Neurospora crassa FKS Protein Binds to the (1,3)β-Glucan Synthase Substrate, UDP-Glucose

The essential fungal cell-wall polymer (1,3)β-glucan is synthesized by the enzyme (1,3)β-glucan synthase. This enzyme, which is the target of the echinocandin and pneumocandin families of fungicidal antibiotics, is a complex composed of at least two proteins, Rho1p and Fks1p. Homologs of the yeast FKS1 gene have been discovered in numerous fungi, and existing evidence points to, but has not yet proved, Fks1p being the catalytic subunit of (1,3)β-glucan synthase. We have purified (1,3)β-glucan synthase from Neurospora crassa ∼400-fold enrichment and labeled the substrate-binding protein by using a UDP-glucose analog, 5-azido-[β-32P]-UDP-glucose. UDP-glucose-binding proteins were photo-crosslinked to the substrate analog and identified from SDS-PAGE gels by Quadrupole time-of-flight mass spectrometry by sequencing the tryptic peptides. Two plasma membrane proteins were labeled FKS and H+-ATPase. These results suggest that FKS appears to be the substrate-binding subunit of (1,3)β-glucan synthase.

Novel Compounds Active against Leishmania major

ABSTRACT Leishmania major is an important trypanosomatid pathogen that causes leishmaniasis, which is a serious disease in much of the Old World. Current treatments include a small number of antimony compounds that, while somewhat effective, are limited by serious side effects. We have screened a small portion of a unique chemical library and have found at least three novel compounds that are effective against L. tarentolae and L. major in vitro and in a murine macrophage model of L. major infection. These compounds were effective in both assays at doses significantly lower than those of sodium stibogluconate (Pentostam) and represent possible candidates for drug development.