How the necrotrophic fungus Alternaria brassicicola kills plant cells remains an enigma.

Alternaria species are mainly saprophytic fungi, but some are plant pathogens. Seven pathotypes of Alternaria alternata use secondary metabolites of host-specific toxins as pathogenicity factors. These toxins kill host cells prior to colonization. Genes associated with toxin synthesis reside on conditionally dispensable chromosomes, supporting the notion that pathogenicity might have been acquired several times by A. alternata. Alternaria brassicicola, however, seems to employ a different mechanism. Evidence on the use of host-specific toxins as pathogenicity factors remains tenuous, even after a diligent search aided by full-genome sequencing and efficient reverse-genetics approaches. Similarly, no individual genes encoding lipases or cell wall-degrading enzymes have been identified as strong virulence factors, although these enzymes have been considered important for fungal pathogenesis. This review describes our current understanding of toxins, lipases, and cell wall-degrading enzymes and their roles in the pathogenesis of A. brassicicola compared to those of other pathogenic fungi. It also describes a set of genes that affect pathogenesis in A. brassicicola. They are involved in various cellular functions that are likely important in most organisms and probably indirectly associated with pathogenesis. Deletion or disruption of these genes results in weakly virulent strains that appear to be sensitive to the defense mechanisms of host plants. Finally, this review discusses the implications of a recent discovery of three important transcription factors associated with pathogenesis and the putative downstream genes that they regulate.

The phytotoxin fusicoccin promotes platelet aggregation via 14-3-3-glycoprotein Ib-IX-V interaction.

The fungal toxin fusicoccin induces plant wilting by affecting ion transport across the plasma membrane of plant cell. The activity of this toxin is so far unknown in humans. In the present study we show that fusicoccin is able to affect the platelet aggregation process. The toxin associates with platelet intracellular binding sites and induces aggregation in platelet-rich plasma in a dose-dependent manner. We identified the adhesion receptor glycoprotein Ib-IX-V as fusicoccin target. The toxin promotes the binding of the regulatory 14-3-3 proteins to glycoprotein Ibα and hampers that to glycoprotein Ibß subunit. As a result, platelet adhesion to von Willebrand factor is stimulated, leading to platelet spreading and integrin αIIbß3 activation. We anticipate the present study to be a starting point for future therapeutic use of fusicoccin in genetic bleeding diseases characterized by qualitative or quantitative abnormalities of the platelet membrane-adhesion receptors. Furthermore, the present study also sets the stage for future work to determine the potential pharmacological application of fusicoccin as a drug directed to other 14-3-3-target complexes.

SnTox3 acts in effector triggered susceptibility to induce disease on wheat carrying the Snn3 gene.

The necrotrophic fungus Stagonospora nodorum produces multiple proteinaceous host-selective toxins (HSTs) which act in effector triggered susceptibility. Here, we report the molecular cloning and functional characterization of the SnTox3-encoding gene, designated SnTox3, as well as the initial characterization of the SnTox3 protein. SnTox3 is a 693 bp intron-free gene with little obvious homology to other known genes. The predicted immature SnTox3 protein is 25.8 kDa in size. A 20 amino acid signal sequence as well as a possible pro sequence are predicted. Six cysteine residues are predicted to form disulfide bonds and are shown to be important for SnTox3 activity. Using heterologous expression in Pichia pastoris and transformation into an avirulent S. nodorum isolate, we show that SnTox3 encodes the SnTox3 protein and that SnTox3 interacts with the wheat susceptibility gene Snn3. In addition, the avirulent S. nodorum isolate transformed with SnTox3 was virulent on host lines expressing the Snn3 gene. SnTox3-disrupted mutants were deficient in the production of SnTox3 and avirulent on the Snn3 differential wheat line BG220. An analysis of genetic diversity revealed that SnTox3 is present in 60.1% of a worldwide collection of 923 isolates and occurs as eleven nucleotide haplotypes resulting in four amino acid haplotypes. The cloning of SnTox3 provides a fundamental tool for the investigation of the S. nodorum-wheat interaction, as well as vital information for the general characterization of necrotroph-plant interactions.

Viruses activate a genetically conserved cell death pathway in a unicellular organism.

Given the importance of apoptosis in the pathogenesis of virus infections in mammals, we investigated the possibility that unicellular organisms also respond to viral pathogens by activating programmed cell death. The M1 and M2 killer viruses of Saccharomyces cerevisiae encode pore-forming toxins that were assumed to kill uninfected yeast cells by a nonprogrammed assault. However, we found that yeast persistently infected with these killer viruses induce a programmed suicide pathway in uninfected (nonself) yeast. The M1 virus-encoded K1 toxin is primarily but not solely responsible for triggering the death pathway. Cell death is mediated by the mitochondrial fission factor Dnm1/Drp1, the K+ channel Tok1, and the yeast metacaspase Yca1/Mca1 encoded by the target cell and conserved in mammals. In contrast, cell death is inhibited by yeast Fis1, a pore-forming outer mitochondrial membrane protein. This virus-host relationship in yeast resembles that of pathogenic human viruses that persist in their infected host cells but trigger programmed death of uninfected cells.

Viral killer toxins induce caspase-mediated apoptosis in yeast.

In yeast, apoptotic cell death can be triggered by various factors such as H2O2, cell aging, or acetic acid. Yeast caspase (Yca1p) and cellular reactive oxygen species (ROS) are key regulators of this process. Here, we show that moderate doses of three virally encoded killer toxins (K1, K28, and zygocin) induce an apoptotic yeast cell response, although all three toxins differ significantly in their primary killing mechanisms. In contrast, high toxin concentrations prevent the occurrence of an apoptotic cell response and rather cause necrotic, toxin-specific cell killing. Studies with Deltayca1 and Deltagsh1 deletion mutants indicate that ROS accumulation as well as the presence of yeast caspase 1 is needed for apoptosis in toxin-treated yeast cells. We conclude that in the natural environment of toxin-secreting killer yeasts, where toxin concentration is usually low, induction of apoptosis might play an important role in efficient toxin-mediated cell killing.

Transport of mycotoxins across human gastric NCI-N87 and intestinal Caco-2 cell models.

Aflatoxin B1 (AFB1), deoxynivalenol (DON), fumonisin B1 (FB1), and ochratoxin A (OTA) are prevalent mycotoxins co-occurring in food, and their oral intake is conceivable to occur in the gastrointestinal epithelium. The intestinal absorption of some mycotoxins has been studied but only considering their isolated intake, while their gastric absorption in humans has not been explored. This study evaluated the bidirectional in vitro transport of four mycotoxins, isolated and in mixture, across gastric NCI-N87 and intestinal Caco-2 monolayers. AFB1 and DON were bidirectionally transported, more rapidly for AFB1; whereas OTA and FB1 were only transported in the absorptive direction, the first in both monolayers, and the second only in the gastric epithelium. The mixture of four mycotoxins exhibited some differences in cell uptake/excretion ratios. AFB1 presented the highest fraction absorbed (>96%) isolated and in mixture, followed by DON (72.8 and 82.9%); and OTA (11 and 66%) when transported isolated and in mixture, respectively. Different absorptive patterns on both epithelia were found when mycotoxins are transported isolated or in mixture. Further investigation on combined ingestion of toxins and their mixed transport should be considered for the proper evaluation of human absorption and toxicity of those mycotoxins considering their frequent co-occurrence and consequent co-exposure.

Application of Low-Fermenting Yeast Lachancea thermotolerans for the Control of Toxigenic Fungi Aspergillus parasiticus, Penicillium verrucosum and Fusarium graminearum and Their Mycotoxins.

Mycotoxins are important contaminants of food and feed. In this study, low fermenting yeast (Lachancea thermotolerans) and its derivatives were applied against toxigenic fungi and their mycotoxins. A. parasiticus, P. verrucosum and F. graminearum and their mycotoxins were exposed to yeast volatile organic compounds (VOCs) and cells, respectively. VOCs reduced significantly the fungal growth (up to 48%) and the sporulation and mycotoxin synthesis (up to 96%). Very interestingly, it was shown that even 7 yeast colonies reduced Fusarium’s growth and the synthesis of its mycotoxin, deoxynivalenol (DON). Moreover, decreasing yeast nutrient concentrations did not affect the inhibition of fungal growth, but reduced DON synthesis. In addition, inactivated yeast cells were able to remove up to 82% of the ochratoxin A (OTA). As an application of these findings, the potentialities of the VOCs to protect tomatoes inoculated with F. oxysporum was explored and showed that while in the presence of VOCs, no growth was observed of F. oxysporum on the inoculated surface areas of tomatoes, in the absence of VOCs, F. oxysporum infection reached up to 76% of the tomatoes’ surface areas. These results demonstrate that the application of yeasts and their derivatives in the agriculture and food industry might be considered as a very promising and safe biocontrol approach for food contamination.

[Putative virulence factors of Aspergillus species]. / Aspergillus cinsi mantarlarin olasi virülans faktörleri.

Members of the genus Aspergillus are filamentous, ubiquitous fungi found in nature. Aspergillus species can cause clinical settings in man, such as opportunistic infections, allergic states and toxicoses. Immunosuppression is the major factor predisposing to development of opportunistic mycoses. To invade the host, Aspergillus species must be able to adhere to and penetrate the host tissues. Much work has focused on the search for virulence determinants that allow the establishment of the fungus within the host. Adhesins, pigment production, toxic metabolites and extracellular enzymes are thought to be the putative virulence factors of Aspergillus species. Adhesion systems provide binding of conidia to various circulating or basement membrane-associated host proteins; toxic molecules inhibit macrophage phagocytosis and mucociliary action; pigment production aids in longer survival in tissues and also overwhelms the host defence systems; extracellular enzymes degrade the structural barriers in the host. In this review, putative virulence factors of Aspergillus species and their potential role in aspergillosis have been discussed.

A conditionally dispensable chromosome controls host-specific pathogenicity in the fungal plant pathogen Alternaria alternata.

The filamentous fungus Alternaria alternata contains seven pathogenic variants (pathotypes), which produce host-specific toxins and cause diseases on different plants. Previously, the gene cluster involved in host-specific AK-toxin biosynthesis of the Japanese pear pathotype was isolated, and four genes, named AKT genes, were identified. The AKT homologs were also found in the strawberry and tangerine pathotypes, which produce AF-toxin and ACT-toxin, respectively. This result is consistent with the fact that the toxins of these pathotypes share a common 9,10-epoxy-8-hydroxy-9-methyl-decatrienoic acid structural moiety. In this study, three of the AKT homologs (AFT1-1, AFTR-1, and AFT3-1) were isolated on a single cosmid clone from strain NAF8 of the strawberry pathotype. In NAF8, all of the AKT homologs were present in multiple copies on a 1.05-Mb chromosome. Transformation-mediated targeting of AFT1-1 and AFT3-1 in NAF8 produced AF-toxin-minus, nonpathogenic mutants. All of the mutants lacked the 1.05-Mb chromosome encoding the AFT genes. This chromosome was not essential for saprophytic growth of this pathogen. Thus, we propose that a conditionally dispensable chromosome controls host-specific pathogenicity of this pathogen.

Genes and molecules involved in Aspergillus fumigatus virulence.

Aspergillus fumigatus causes a wide range of diseases that include mycotoxicosis, allergic reactions and systemic diseases (invasive aspergillosis) with high mortality rates. Pathogenicity depends on immune status of patients and fungal strain. There is no unique essential virulence factor for development of this fungus in the patient and its virulence appears to be under polygenetic control. The group of molecules and genes associated with the virulence of this fungus includes many cell wall components, such as beta-(1-3)-glucan, galactomannan, galactomannanproteins (Afmp1 and Afmp2), and the chitin synthetases (Chs; chsE and chsG), as well as others. Some genes and molecules have been implicated in evasion from the immune response, such as the rodlets layer (rodA/hyp1 gene) and the conidial melanin-DHN (pksP/alb1 gene). The detoxifying systems for Reactive Oxygen Species (ROS) by catalases (Cat1p and Cat2p) and superoxide dismutases (MnSOD and Cu, ZnSOD), had also been pointed out as essential for virulence. In addition, this fungus produces toxins (14 kDa diffusible substance from conidia, fumigaclavin C, aurasperon C, gliotoxin, helvolic acid, fumagilin, Asp-hemolysin, and ribotoxin Asp fI/mitogilin F/restrictocin), allergens (Asp f1 to Asp f23), and enzymatic proteins as alkaline serin proteases (Alp and Alp2), metalloproteases (Mep), aspartic proteases (Pep and Pep2), dipeptidyl-peptidases (DppIV and DppV), phospholipase C and phospholipase B (Plb1 and Plb2). These toxic substances and enzymes seems to be additive and/or synergistic, decreasing the survival rates of the infected animals due to their direct action on cells or supporting microbial invasion during infection. Adaptation ability to different trophic situations is an essential attribute of most pathogens. To maintain its virulence attributes A. fumigatus requires iron obtaining by hydroxamate type siderophores (ornitin monooxigenase/SidA), phosphorous obtaining (fos1, fos2, and fos3), signal transductional falls that regulate morphogenesis and/or usage of nutrients as nitrogen (rasA, rasB, rhbA), mitogen activated kinases (sakA codified MAP-kinase), AMPc-Pka signal transductional route, as well as others. In addition, they seem to be essential in this field the amino acid biosynthesis (cpcA and homoaconitase/lysF), the activation and expression of some genes at 37 degrees C (Hsp1/Asp f12, cgrA), some molecules and genes that maintain cellular viability (smcA, Prp8, anexins), etc. Conversely, knowledge about relationship between pathogen and immune response of the host has been improved, opening new research possibilities. The involvement of non-professional cells (endothelial, and tracheal and alveolar epithelial cells) and professional cells (natural killer or NK, and dendritic cells) in infection has been also observed. Pathogen Associated Molecular Patterns (PAMP) and Patterns Recognizing Receptors (PRR; as Toll like receptors TLR-2 and TLR-4) could influence inflammatory response and dominant cytokine profile, and consequently Th response to infec tion. Superficial components of fungus and host cell surface receptors driving these phenomena are still unknown, although some molecules already associated with its virulence could also be involved. Sequencing of A. fumigatus genome and study of gene expression during their infective process by using DNA microarray and biochips, promises to improve the knowledge of virulence of this fungus.

[Killer toxins of clinically important yeasts]. / Killernye toksiny klinicheski znachimykh drozhzhei.

The review deals with some theoretical and applied aspects of the capacity of yeasts for synthesizing toxins. Similarly to antibiotic formation in micellar fungi and actinomycetes and the synthesis of bactericins in prokaryotes, yeast cells also have their mechanism of protection from other microorganisms. The substances, essentially of the same nature, synthesized by yeast are known for more than 30 years as mycocins or killer toxins. They are proteins or glycoproteins, active mainly against yeast microorganisms. Mycocins are not active against bacteria and protozoa exhibiting only fungicidal or fungistatic action. The formation of mycocins may be determined by nucleus or plasmid DNA. In this review information on killer toxins produced by clinically important yeasts of the genera Candida, Cryptococcus and Rhodotorula is systematized.

Sociobiology of the budding yeast.

Social theory has provided a useful framework for research with microorganisms. Here I describe the advantages and possible risks of using a well-known model organism, the unicellular yeast Saccharomyces cerevisiae, for sociobiological research. I discuss the problems connected with clear classification of yeast behaviour based on the fitnessbased Hamilton paradigm. Relevant traits include different types of communities, production of flocculins, invertase and toxins, and the presence of apoptosis.

Victorin triggers programmed cell death and the defense response via interaction with a cell surface mediator.

The host-selective toxin victorin is produced by Cochliobolus victoriae, the causal agent of victoria blight of oats. Victorin has been shown to bind to the P protein of the glycine decarboxylase complex (GDC) in mitochondria, and induce defense-related responses such as phytoalexin synthesis, extracellular alkalization and programmed cell death. However, evidence demonstrating that the GDC plays a critical role in the onset of cell death is still lacking, and the role of defense-like responses in the pathogenicity has yet to be elucidated. Here, cytofluorimetric analyses, using the fluorescein (VicFluor) or bovine serum albumin-fluorescein derivative of victorin (VicBSA), demonstrated that victorin-induced cell death occurs before these conjugates traverse the plasma membrane. As with native victorin, VicBSA clearly elicits apoptosis-like cell death, production of phytoalexin, extracellular alkalization, and generation of nitric oxide and reactive oxygen intermediates. These results suggest that the initial recognition of victorin takes place on the cell surface, not in mitochondria, and leads to the activation of a battery of victorin-induced responses. Pharmacological studies showed that extracellular alkalization is the essential regulator for both victorin- and VicBSA-induced cellular responses. We propose a model where victorin may kill the host cell by activating an HR-like response, independent of the binding to the GDC, through ion fluxes across the plasma membrane.

Yeast killer toxin: site-directed mutations implicate the precursor protein as the immunity component.

Yeast killer toxin and a component giving immunity to it are both encoded by a gene specifying a single 35 kd precursor polypeptide. This precursor is composed of a leader peptide, the alpha and beta subunits of the secreted toxin, and a glycosylated gamma peptide separating the latter. The toxin subunits are proteolytically processed from the precursor during toxin secretion. Using site-directed mutagenesis, we have identified a region of the precursor gene necessary for expression of the immunity phenotype. This immunity-coding region extends through the C-terminal half of the alpha subunit into the N-terminal part of the gamma glycopeptide. Mutations in other parts of the gene allow full immunity but produce precursors that fail to be processed. The precursor can therefore confer immunity, and we propose that it does so in the wild type by competing with mature toxin for binding to a membrane receptor.

Influence of killer strains of Saccharomyces cerevisiae on wine fermentation.

The effect of killer strains of Saccharomyces cerevisiae on the growth of sensitive strains during must fermentation was studied by using a new method to monitor yeast populations. The capability of killer yeast strains to eliminate sensitive strains depends on the initial proportion of killer yeasts, the susceptibility of sensitive strains, and the treatment of the must. In sterile filtered must, an initial proportion of 2-6% of killer yeasts was responsible for protracted fermentation and suppression of isogenic sensitive strains. A more variable initial proportion was needed to get the same effect with non-isogenic strains. The suspended solids that remain in the must after cold-settling decreased killer toxin effect. The addition of bentonite to the must avoided protracted fermentation and the suppression of sensitive strains; however, the addition of yeast dietary nutrients with yeast cell walls did not, although it decreased fermentation lag.

Effect of yeast killer toxin on sensitive cells of Saccharomyces cerevisiae.

Killer toxin from Saccharomyces cerevisiae inhibited the pumping of protons into the medium by metabolically active sensitive cells. Such inhibition coincided with that of the uptake of potassium ions which are thought to be accumulated by yeast cells in order to neutralize the membrane potential created because of the extrusion of protons. The consumption of glucose, however, was identical in killer-treated and untreated cells. These alterations can be explained by the ability of the toxin to reduce the chemical proton gradient across the plasma membrane as measured by the accumulation of the weak permeable [14C]propionic acid. With this method, an internal pH of 6.42 was calculated from normal cells (the external pH was 4.6) while that of toxin-treated cells was decreased as a function of time. The proton concentration gradient was reduced from 66- to 17-fold. It is shown that the toxin-induced alteration of the proton gradient is due to an enhanced proton permeability of the yeast plasma membrane upon binding of the toxin. It is suggested that killer toxin acts as a macromolecular proton conductor similar in some respects to the known proton conductors 2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone, since all the described effects are also observed with these substances.

Role of the arginyl-glycyl-aspartic motif in the action of Ptr ToxA produced by Pyrenophora tritici-repentis.

A fundamental problem of plant science is to understand the biochemical basis of plant/pathogen interactions. The foliar disease tan spot of wheat (Triticum aestivum), caused by Pyrenophora tritici-repentis, involves Ptr ToxA, a proteinaceous host-selective toxin that causes host cell death. The fungal gene ToxA encodes a 17.2-kD pre-pro-protein that is processed to produce the mature 13.2-kD toxin. Amino acids 140 to 142 of the pre-pro-protein form an arginyl-glycyl-aspartic (RGD) sequence, a motif involved in the binding of some animal proteins and pathogens to transmembrane receptor proteins called integrins. Integrin-like proteins have been identified in plants recently, but their role in plant biology is unclear. Our model for Ptr ToxA action predicts that toxin interacts with a putative host receptor through the RGD motif. Mutant clones of a ToxA cDNA, created by polymerase chain reaction such that the RGD in the pro-toxin was changed to arginyl-alanyl-aspartic or to arginyl-glycyl-glutamic, were expressed in Escherichia coli. Extracts containing mutated forms of toxin failed to cause host cell death, but extracts from E. coli expressing both a wild-type pro-protein cDNA and a control mutation away from RGD were active in cell death development. In competition experiments, 2 mM RGD tripeptide reduced the level of electrolyte leakage from wheat leaves by 63% when co-infiltrated with purified Ptr ToxA (15 microg mL(-1)) obtained from the fungus, but the control peptide arginyl-glycyl-glutamyl-serine provided no protection. These experiments indicate that the RGD motif of Ptr ToxA is involved with toxin action, possibly by interacting with a putative integrin-like receptor in the host.