A Potent Kalihinol Analogue Disrupts Apicoplast Function and Vesicular Trafficking in P. falciparum Malaria.

Here we report the discovery of MED6-189, a new analogue of the kalihinol family of isocyanoterpene (ICT) natural products. MED6-189 is effective against drug-sensitive and -resistant P. falciparum strains blocking both intraerythrocytic asexual replication and sexual differentiation. This compound was also effective against P. knowlesi and P. cynomolgi. In vivo efficacy studies using a humanized mouse model of malaria confirms strong efficacy of the compound in animals with no apparent hemolytic activity or apparent toxicity. Complementary chemical biology, molecular biology, genomics and cell biological analyses revealed that MED6-189 primarily targets the parasite apicoplast and acts by inhibiting lipid biogenesis and cellular trafficking. Genetic analyses in P. falciparum revealed that a mutation in PfSec13, which encodes a component of the parasite secretory machinery, reduced susceptibility to the drug. The high potency of MED6-189 in vitro and in vivo, its broad range of efficacy, excellent therapeutic profile, and unique mode of action make it an excellent addition to the antimalarial drug pipeline.

Cercosporin-deficient mutants by plasmid tagging in the asexual fungus Cercospora nicotianae.

We have successfully adapted plasmid insertion and restriction enzyme-mediated integration (REMI) to produce cercosporin toxin-deficient mutants in the asexual phytopathogenic fungus Cercospora nicotianae. The use of pre-linearized plasmid or restriction enzymes in the transformation procedure significantly decreased the transformation frequency, but promoted a complicated and undefined mode of plasmid integration that leads to mutations in the C. nicotianae genome. Vector DNA generally integrated in multiple copies, and no increase in single-copy insertion was observed when enzymes were added to the transformation mixture. Out of 1873 transformants tested, 39 putative cercosporin toxin biosynthesis ( ctb) mutants were recovered that showed altered levels of cercosporin production. Seven ctb mutants were recovered using pre-linearized plasmids without the addition of enzymes, and these were considered to be non-REMI mutants. The correlation between a specific insertion and a mutant phenotype was confirmed using rescued plasmids as gene disruption vectors in the wild-type strain. Six out of fifteen rescued plasmids tested yielded cercosporin-deficient transformants when re-introduced into the wild-type strain, suggesting a link between the insertion site and the cercosporin-deficient phenotype. Sequence analysis of a fragment flanking the insert site recovered from one insertion mutant showed it to be disrupted in sequences with high homology to the acyl transferase domain of polyketide synthases from other fungi. Disruption of this polyketide synthase gene ( CTB1) using a rescued plasmid resulted in mutants that were defective in cercosporin production. Thus, we provide the first molecular evidence that cercosporin is synthesized via a polyketide pathway as previously hypothesized.

Isolation of PDX2, a second novel gene in the pyridoxine biosynthesis pathway of eukaryotes, archaebacteria, and a subset of eubacteria.

In this paper we describe the isolation of a second gene in the newly identified pyridoxine biosynthesis pathway of archaebacteria, some eubacteria, fungi, and plants. Although pyridoxine biosynthesis has been thoroughly examined in Escherichia coli, recent characterization of the Cercospora nicotianae biosynthesis gene PDX1 led to the discovery that most organisms contain a pyridoxine synthesis gene not found in E. coli. PDX2 was isolated by a degenerate primer strategy based on conserved sequences of a gene specific to PDX1-containing organisms. The role of PDX2 in pyridoxine biosynthesis was confirmed by complementation of two C. nicotianae pyridoxine auxotrophs not mutant in PDX1. Also, targeted gene replacement of PDX2 in C. nicotianae results in pyridoxine auxotrophy. Comparable to PDX1, PDX2 homologues are not found in any of the organisms with homologues to the E. coli pyridoxine genes, but are found in the same archaebacteria, eubacteria, fungi, and plants that contain PDX1 homologues. PDX2 proteins are less well conserved than their PDX1 counterparts but contain several protein motifs that are conserved throughout all PDX2 proteins.

Vitamin B6 (pyridoxine) and its derivatives are efficient singlet oxygen quenchers and potential fungal antioxidants.

Vitamin B6 (pyridoxine, 1) and its derivatives: pyridoxal (2), pyridoxal 5-phosphate (3) and pyridoxamine (4) are important natural compounds involved in numerous biological functions. Pyridoxine appears to play a role in the resistance of the filamentous fungus Cercospora nicotianae to its own abundantly produced strong photosensitizer of singlet molecular oxygen (1O2), cercosporin. We measured the rate constants (kq) for the quenching of 1O2 phosphorescence by 1-4 in D2O. The respective total (physical and chemical quenching) kq values are: 5.5 x 10(7) M-1 s-1 for 1; 7.5 x 10(7) M-1 s-1 for 2, 6.2 x 10(7) M-1 s-1 for 3 and 7.5 x 10(7) M-1 s-1 for 4, all measured at pD 6.2. The quenching efficacy increased up to five times in alkaline solutions and decreased approximately 10 times in ethanol. Significant contribution to total quenching by chemical reaction(s) is suggested by the degradation of all the vitamin derivatives by 1O2, which was observed as declining absorption of the pyridoxine moiety upon aerobic irradiation of RB used to photosensitize 1O2. This photodegradation was completely stopped by azide, a known physical quencher of 1O2. The pyridoxine moiety can also function as a redox quencher for excited cercosporin by forming the cercosporin radical anion, as observed by electron paramagnetic resonance. All B6 vitamers fluoresce upon UV excitation. Compounds 1 and 4 emit fluorescence at 400 nm, compound 2 at 450 nm and compound 3 at 550 nm. The fluorescence intensity of 3 increased approximately 10 times in organic solvents such as ethanol and 1,2-propanediol compared to aqueous solutions, suggesting that fluorescence may be used to image the distribution of 1-4 in Cercospora to understand better the interactions of pyridoxine and 1O2 in the living fungus.

Dihydrocercosporin singlet oxygen production and subcellular localization: a possible defense against cercosporin phototoxicity in Cercospora.

Fungi in the genus Cercospora produce cercosporin, a potent singlet oxygen (1O2)-generating photosensitizer that plays a critical role in the ability of these fungi to parasitize plants. Although plants, mice, bacteria and many fungi are sensitive to cercosporin, Cercospora species are resistant to its toxicity. The cellular resistance of these fungi to cercosporin has been correlated with fungal cell surface reducing ability and the ability to maintain cercosporin in a chemically reduced state. As a model for reduced cercosporin we employed a reduced, acetylated derivative (hexaacetyl-dihydrocercosporin, HAC) that we tested for 1O2 production in a range of solvents. We found that as a 1O2 photosensitizer, HAC was only moderately effective in organic solvents (phi SO = 0.14-0.18) and very poor in water (phi SO = 0.02-0.04). By contrast, the 1O2 quantum yield of cercosporin itself was unaffected by solvent (phi SO = 0.84-0.97). To investigate the localization of reduced cercosporin in fungal cells, we developed a fluorescence assay using laser scanning confocal microscopy. This assay showed a uniform green fluorescence, indicative of reduced cercosporin, in the cytoplasm of hyphal cells treated with cercosporin. We hypothesize that the main protection mechanism against cercosporin phototoxicity in the fungus consists of transformation of cercosporin to a reduced state and localization of this reduced form in the aqueous compartment of the cell, thus decreasing intracellular 1O2 production to levels that can be tolerated by the fungus. In addition, we have, for the first time, directly detected 1O2 phosphorescence from fungal culture, either stained with the photosensitizer rose bengal or actively synthesizing cercosporin, demonstrating 1O2 production in vivo and from cercosporin in culture.

Nucleocapsid Gene-Mediated Transgenic Resistance Provides Protection Against Tomato spotted wilt virus Epidemics in the Field.

ABSTRACT Transformation of plants with the nucleocapsid (N) gene of Tomato spotted wilt tospovirus (TSWV) provides resistance to disease development; however, information is lacking on the response of plants to natural inoculum in the field. Three tobacco cultivars were transformed with the N gene of a dahlia isolate of TSWV (TSWV-D), and plants were evaluated over several generations in the greenhouse. The resistant phenotype was more frequently observed in 'Burley 21' than in 'KY-14' or 'K-326', but highly resistant 'Burley 21' transgenic lines were resistant to only 44% of the heterologous TSWV isolates tested. Advanced generation (R(3) and R(4)) transgenic resistant lines of 'Burley 21' and a 'K-326' F(1) hybrid containing the N genes of two TSWV isolates were evaluated in the field near Tifton, GA, where TSWV is endemic. Disease development was monitored by symptom expression and enzyme-linked immunosorbent assay (ELISA) analysis. Whereas incidence of TSWV infection in 'Burley 21' susceptible controls was 20% in 1996 and 62% in 1997, the mean incidence in transgenic lines was reduced to 4 and 31%, respectively. Three transgenic 'Burley 21' lines were identified that had significantly lower incidence of disease than susceptible controls over the two years of the study. In addition, the rate of disease increase at the onset of the 1997 epidemic was reduced for all the 'Burley 21' transgenic lines compared with the susceptible controls. The 'K-326' F(1) hybrid was as susceptible as the 'K-326' nontransformed control. ELISA analysis demonstrated that symptomless plants from the most resistant 'Burley 21' transgenic lines accumulated detectable nucleocapsid protein, whereas symptomless plants from more susceptible lines did not. We conclude that transgenic resistance to TSWV is effective in reducing incidence of the disease in the field, and that accumulation of transgene protein may be important in broad-spectrum resistance.

A novel gene required for cercosporin toxin resistance in the fungus Cercospora nicotianae.

Cercosporin, a photosensitizing perylenequinone toxin produced by the plant pathogenic Cercospora fungi, generates the highly toxic singlet oxygen (1O2) upon exposure to light. Cercosporin shows broad toxicity against a wide range of organisms, including bacteria, fungi, plants, and animals; however, Cercospora fungi are resistant to its effects. A novel gene, crg1 (cercosporin-resistance gene) was isolated from a wild-type strain of C. nicotianae by genetic complementation of a C. nicotianae mutant (CS10) which is cercosporin sensitive and down-regulated in cercosporin production. Sequence analysis indicated that crg1 encodes a putative protein of 550 amino acids with four putative transmembrane helical regions, however CRG1 shows no strong similarity to any other protein in sequence databases. Northern analysis identified two transcripts (4.5 and 2.6 kb) that are unaffected by the presence of light or cercosporin. Southern analysis demonstrated that crg1 is present in a single copy in the C. nicotianae genome and can be detected only in Cercospora species. Targeted disruption of crg1 resulted in mutants that, like CS10, are sensitive to cercosporin. However, unlike CS10, crg1 disruption mutants are not down-regulated in toxin production. Both CS10 and the crg1 disruption mutants are unaffected in their response to other 1O2-generating photosensitizers, suggesting that CRG1 functions specifically against cercosporin, rather than against 1O2.

A highly conserved sequence is a novel gene involved in de novo vitamin B6 biosynthesis.

The Cercospora nicotianae SOR1 (singlet oxygen resistance) gene was identified previously as a gene involved in resistance of this fungus to singlet-oxygen-generating phototoxins. Although homologues to SOR1 occur in organisms in four kingdoms and encode one of the most highly conserved proteins yet identified, the precise function of this protein has, until now, remained unknown. We show that SOR1 is essential in pyridoxine (vitamin B6) synthesis in C. nicotianae and Aspergillus flavus, although it shows no homology to previously identified pyridoxine synthesis genes identified in Escherichia coli. Sequence database analysis demonstrated that organisms encode either SOR1 or E. coli pyridoxine biosynthesis genes, but not both, suggesting that there are two divergent pathways for de novo pyridoxine biosynthesis in nature. Pathway divergence appears to have occurred during the evolution of the eubacteria. We also present data showing that pyridoxine quenches singlet oxygen at a rate comparable to that of vitamins C and E, two of the most highly efficient biological antioxidants, suggesting a previously unknown role for pyridoxine in active oxygen resistance.

Functional characterization of SOR1, a gene required for resistance to photosensitizing toxins in the fungus Cercospora nicotianae.

The Cercospora nicotianae SOR1 gene is required for resistance to singlet oxygen-generating photosensitizers. SOR1 was characterized in the wild-type and in five photosensitizer-sensitive mutant strains which are complemented to photosensitizer resistance by transformation with SOR1. Sequence analysis determined that three of the mutants contain SOR1 copies with mutations encoding substitutions in the protein-coding sequence; however, two other mutants had wild-type SOR1 protein and promoter sequences. All five mutants accumulate SOR1 mRNA at levels comparable to that of the wild-type strain. In the wild-type strain, SOR1 accumulation is enhanced two-fold by light, but is unaffected by the presence of cercosporin, the photosensitizer synthesized by C. nicotianae. Southern analysis indicates that SOR1 is present in other fungi that synthesize structurally related perylenequinone photosensitizers.

SOR1, a gene required for photosensitizer and singlet oxygen resistance in Cercospora fungi, is highly conserved in divergent organisms.

Filamentous Cercospora fungi are resistant to photosensitizing compounds that generate singlet oxygen. C. nicotianae photosensitizer-sensitive mutants were restored to full resistance by transformation with SOR1 (Singlet Oxygen Resistance 1), a gene recovered from a wild-type genomic library. SOR1 null mutants generated via targeted gene replacement confirmed the requirement for SOR1 in photosensitizer resistance. SOR1 RNA is present throughout the growth cycle. Although resistance to singlet oxygen is rare in biological systems, SOR1, a gene with demonstrated activity against singlet-oxygen-generating photosensitizers, is highly conserved in organisms from widely diverse taxa. The characterization of SOR1 provides an additional phenotype to this large group of evolutionarily conserved genes.

Isolation, sequence, and characterization of the Cercospora nicotianae phytoene dehydrogenase gene.

We have cloned and sequenced the Cercospora nicotianae gene for the carotenoid biosynthetic enzyme phytoene dehydrogenase. Analysis of the derived amino acid sequence revealed it has greater than 50% identity with its counterpart in Neurospora crassa and approximately 30% identity with prokaryotic phytoene dehydrogenases and is related, but more distantly, to phytoene dehydrogenases from plants and cyanobacteria. Our analysis confirms that phytoene dehydrogenase proteins fall into two groups: those from plants and cyanobacteria and those from eukaryotic and noncyanobacter prokaryotic microbes. Southern analysis indicated that the C. nicotianae phytoene dehydrogenase gene is present in a single copy. Extraction of beta-carotene, the sole carotenoid accumulated by C. nicotianae, showed that both light- and dark-grown cultures synthesize carotenoids, but higher levels accumulate in the light. Northern (RNA) analysis of poly(A)+ RNA, however, showed no differential accumulation of phytoene dehydrogenase mRNA between light- and dark-grown fungal cultures.

Reductive detoxification as a mechanism of fungal resistance to singlet oxygen-generating photosensitizers.

Fungi that are resistant or sensitive to the singlet oxygen-generating toxin cercosporin were assayed for their ability to detoxify it by reduction. Cercosporin reduction was assayed microscopically by using bandpass filters to differentiate between fluorescence emission from cercosporin and reduced cercosporin. Hyphae of the resistant Cercospora and Alternaria species emitted a green fluorescence, indicative of reduced cercosporin. Hyphae of nonviable cultures and of cercosporin-sensitive fungi did not reduce cercosporin. Sensitive fungi occasionally reduced cercosporin when incubated with reducing agents that protect against cercosporin toxicity. Cercosporin could not be efficiently photoreduced in the absence of the fungus. Cercospora species were also resistant to eosin Y but were sensitive to rose bengal. Microscopic observation demonstrated that Cercospora species were not capable of reducing rose bengal but were capable of reducing eosin Y. These observations were supported by in vitro electrochemical measurements that revealed the following order with respect to ease of reduction: cercosporin >> eosin Y > rose bengal. The formal redox potential (E 0') of cercosporin at pH 7.5 was found to be -0.14 V vs. the normal hydrogen electrode. We conclude that Cercospora species protect themselves against cercosporin by the reduction and detoxification of the toxin molecule.

Cell surface redox potential as a mechanism of defense against photosensitizers in fungi.

The phytotoxin cercosporin, a singlet oxygen-generating photosensitizer, is toxic to plants, mice, and many fungi, yet the fungi that produce it, Cercospora spp., are resistant. We hypothesize that resistance to cercosporin may result from a reducing environment at the cell surface. Twenty tetrazolium dyes differing in redox potential were used as indicators of cell surface redox potential of seven fungal species differing in resistance to cercosporin. Resistant fungi were able to reduce significantly more dyes than were sensitive fungi. A correlation between dye reduction and cercosporin resistance was also observed when resistance levels of Cercospora species were manipulated by growth on different media. The addition of the reducing agents ascorbate, cysteine, and reduced glutathione (GSH) to growth media decreased cercosporin toxicity for sensitive fungi. None of these agents directly reduced cercosporin at the concentrations at which they protected fungi. Spectral and thin-layer chromatographic analyses of cercosporin solutions containing the different reducing agents indicated that GSH, but not cysteine or ascorbate, reacted with cercosporin. Resistant and sensitive fungi did not differ in endogenous levels of cysteine, GSH, or total thiols. On the basis of data from this and other studies, this report presents a model which proposes that cercosporin resistance results from the production of reducing power at the surfaces of resistant cells, leading to transient reduction and detoxification of the cercosporin molecule.

Mutants of Cercospora kikuchii Altered in Cercosporin Synthesis and Pathogenicity.

We have obtained spontaneous and UV-induced stable mutants, altered in the synthesis of cercosporin, of the fungal soybean pathogen Cercospora kikuchii. The mutants were isolated on the basis of colony color on minimal medium. The UV-induced mutants accumulated, at most, 2% of wild-type cercosporin levels on all media tested. In contrast, cercosporin accumulation by the spontaneous mutants was strongly medium regulated, occurring only on potato dextrose medium but at concentrations comparable to those produced by the wild-type strain. UV-induced mutants unable to synthesize cercosporin on any medium were unable to incite lesions when inoculated onto the soybean host. Cercosporin was reproducibly isolated from all inoculated leaves showing lesions. Although cercosporin involvement in disease has been indirectly suggested by many previous studies, this is the first report in which mutants blocked in cercosporin synthesis have been used to demonstrate that cercosporin is a crucial pathogenicity factor for this fungal genus.

Effects of Singlet Oxygen Quenchers and pH on the Bacterially Induced Hypersensitive Reaction in Tobacco Suspension Cell Cultures.

Suspension cell cultures of Nicotiana tabacum L. inoculated with the incompatible pathogen Pseudomonas syringae pv pisi undergo a hypersensitive reaction. Addition of the singlet oxygen quencher bixin to cell suspensions had no effect on hypersensitive cell death. Addition of the singlet oxygen quencher 1,4-diazabicyclo octane (DABCO) increased the medium pH and delayed the onset of cell death. This delay was eliminated when cell suspensions were buffered, and could also be induced by increasing medium pH with KOH. Bixin and DABCO also did not suppress the hypersensitive reaction in tobacco leaves. These data do not support a role for singlet oxygen in the hypersensitive reaction. Medium pH, however, appears to be a critical factor in cell suspension cultures.

Light-induced production of singlet oxygen and superoxide by the fungal toxin, cercosporin.

Cercosporin, a toxin produced by members of the fungal genus Cercospora, is a photosensitizing compound which rapidly kills plant cells in the light. We have found that cercosporin, when activated by light in the presence of oxygen, is able to generate both singlet oxygen and superoxide ions. Cercosporin, when illuminated in the presence of O(2), reacted with cholesterol to form the 5alpha-hydroperoxide of cholesterol which is only produced by reaction with singlet oxygen. Cercosporin, in the presence of light, O(2), and a reducing substrate, was also able to reduce p-nitro blue tetrazolium chloride, a compound readily reduced by superoxide. Superoxide dismutase, a scavenger of superoxide, inhibited this reaction. Production of both singlet oxygen and superoxide by cercosporin must be considered when studying the possible mechanisms of resistance to cercosporin.

Changes in tobacco cell membrane composition and structure caused by cercosporin.

Cercosporin, a toxin produced by Cercospora species, rapidly kills plant cells in the light. Previous work has shown that cercosporin treatment causes products of lipid peroxidation to be released. We have found that the unsaturated acyl chains of lipids in tobacco (Nicotiana tabacum) cell membranes are destroyed when cells are treated with cercosporin. Concomitant with this change in composition is a change in structure of the membranes as detected by two different fatty acid spin labels, 2-(3-carboxypropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinyloxyl (denoted I[12,3]) and 2-(14-carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolidinyloxyl (denoted I[1,14]). Cercosporin causes the membranes to become more rigid at all temperatures tested and increases the membrane phase transformation temperature from 12.7 degrees C to 20.8 degrees C.

Peroxidation of tobacco membrane lipids by the photosensitizing toxin, cercosporin.

Cercosporin, a nonspecific toxin from Cercospora species, is a photosensitizing compound which rapidly kills plant cells in the light. Cell death appears to be due to a cercosporin-mediated peroxidation of membrane lipids. Tobacco leaf discs treated with cercosporin showed a large increase in electrolyte leakage 1 to 2 minutes after irradiation with light. All tobacco protoplasts exposed to cercosporin in the light were damaged within 45 minutes. Chloroform:methanol extracts of toxin-treated suspension cultures gave positive reactions for lipid hydroperoxides in the thiobarbituric acid test. Cercosporin-treated leaf discs emitted high concentrations of ethane 12 to 24 hours after incubation in the light. Cercosporin also oxidized solutions of methyl linolenate as determined by the thiobarbituric acid assay and the emission of ethane. alpha-Tocopherol had an inhibitory effect on the cercosporin-mediated lipid peroxidation.