Yeast killer toxins: from ecological significance to application

Killer toxins are proteins that are often glycosylated and bind to specific receptors on the surface of their target microorganism, which is then killed through a target-specific mode of action. The killer phenotype is widespread among yeast and about 100 yeast killer species have been described to date. The spectrum of action of the killer toxins they produce targets spoilage and pathogenic microorganisms. Thus, they have potential as natural antimicrobials in food and for biological control of plant pathogens, as well as therapeutic agents against animal and human infections. In spite of this wide range of possible applications, their exploitation on the industrial level is still in its infancy. Here, we initially briefly report on the biodiversity of killer toxins and the ecological significance of their production. Their actual and possible applications in the agro-food industry are discussed, together with recent advances in their heterologous production and the manipulation for development of peptide-based therapeutic agents.     

Pichia anomala and Kluyveromyces wickerhamii killer toxins as new tools against Dekkera/Brettanomyces spoilage yeasts

Two yeast killer toxins active on spoilage yeasts belonging to the genus Dekkera/Brettanomyces are here described for the first time. The two toxins produced by Pichia anomala (DBVPG 3003) and Kluyveromyces wickerhamii (DBVPG 6077), and named Pikt and Kwkt, respectively, differ for molecular weight and biochemical properties. Interestingly, the fungicidal effect exerted by Pikt and Kwkt against Dekkera bruxellensis is stable for at least 10 days in wine. Thus, a potential application for the two toxins as antimicrobial agents active on Dekkera/Brettanomyces during wine ageing and storage can be hypothesised.     

Bacterial toxins and their application

The review deals with the structure of protein bacterial toxins, steps of the toxin molecule interaction with the target cell, molecular mechanisms of the toxic effect, as well as with the fields of application of toxins as research tools and as medicinal preparations.     

Serological study of yeast killer toxins by monoclonal antibodies

Yeast killer toxins coded by determined and undetermined killer plasmids or presumptive nuclear gene(s) in various genera (Saccharomyces, Kluyveromyces, Pichia and Candida) have been serologically investigated by a monoclonal antibody (KT4), produced against the yeast killer toxin of Pichia (Hansenula) anomala UCSC 25F. Double immunodiffusion with the killer toxins as antigens and indirect immunofluorescence on whole cells of the corresponding killer yeast have been used. In both the serological procedures, monoclonal antibody KT4 proved to be reacting only with the killer toxins and the whole cells of yeasts belonging to the genus Pichia.     

Fingerprinting of yeasts at the strain level by differential sensitivity responses to a panel of selected killer toxins

We used differential sensitivities to a panel of twenty-five cell-free crude killer toxins to fingerprint forty-four Saccharomyces cerevisiae strains of different origin and all taxonomically certified by nDNA-nDNA reassociation. Cluster analysis of numerical data obtained by different growth inhibition areas observed in Petri dishes allowed the complete and reproducible discrimination of all S. cerevisiae strains.     

Role of membrane gangliosides in the binding and action of bacterial toxins

Summary Gangliosides are complex glycosphingolipids that contain from one to several residues of sialic acid. They are present in the plasma membrane of vertebrate cells with their oligosaccharide chains exposed to the external environment. They have been implicated as cell surface receptors and several bacterial toxins have been shown to interact with them. Cholera toxin, which mediates its effects on cells by activating adenylate cyclase, bind with high affinity and specificity to ganglioside G_M1. Toxin-resistant cells which lack G_M1 can be sensitized to cholera toxin by treating them with G_M1. Cholera toxin specifically protects G_M1 from cell surface labeling procedures and only G_M1 is recovered when toxin-receptor complexes are isolated by immunoadsorption. These results clearly demonstrate that G_M1 is the specific and only receptor for cholera toxin. Although cholera toxin binds to G_M1 on the external side of the plasma membrane, it activates adenylate cyclase on the cytoplasmic side of the membrane by ADP-ribosylation of the regulatory component of the cyclase. G_M1 in addition to functioning as a binding site for the toxin appears to facilitate its transmembrane movement. The heat-labile enterotoxin ofE. coli is very similar to cholera toxin in both form and function and can also use G_M1 as a cell surface receptor. The potent neurotoxin, tetanus toxin, has a high affinity for gangliosides G_D1b and G_T1b and binds to neurons which contain these gangliosides. It is not yet clear whether these gangliosides are the physiological receptors for tetanus toxin. By applying the techniques that established G_M1 as the receptor for cholera toxin, the role of gangliosides as receptors for tetanus toxin as well as physiological effectors may be elucidated.     

Therapeutic potential of yeast killer toxin-like antibodies and mimotopes

This review focuses on the potential of yeast killer toxin (KT)-like antibodies (KTAbs), that mimic a wide-spectrum KT through interaction with specific cell wall receptors (KTR) and their molecular derivatives (killer mimotopes), as putative new tools for transdisease anti-infective therapy. KTAbs are produced during the course of experimental and natural infections caused by KTR-bearing micro-organisms. They have been produced by idiotypic vaccination with a KT-neutralizing mAb, also in their monoclonal and recombinant formats. KTAbs and KTAbs-derived mimotopes may exert a strong therapeutic activity against mucosal and systemic infections caused by eukaryotic and prokaryotic pathogenic agents, thus representing new potential wide-spectrum antibiotics.     

Bacteria used in the biological control of plant-parasitic nematodes: populations, mechanisms of action, and future prospects

As a group of important natural enemies of nematode pests, nematophagous bacteria exhibit diverse modes of action: these include parasitizing; producing toxins, antibiotics, or enzymes; competing for nutrients; inducing systemic resistance of plants; and promoting plant health. They act synergistically on nematodes through the direct suppression of nematodes, promoting plant growth, and facilitating the rhizosphere colonization and activity of microbial antagonists. This review details the nematophagous bacteria known to date, including parasitic bacteria, opportunistic parasitic bacteria, rhizobacteria, Cry protein-forming bacteria, endophytic bacteria and symbiotic bacteria. We focus on recent research developments concerning their pathogenic mechanisms at the biochemical and molecular levels. Increased understanding of the molecular basis of the various pathogenic mechanisms of the nematophagous bacteria could potentially enhance their value as effective biological control agents. We also review a number of molecular biological approaches currently used in the study of bacterial pathogenesis in nematodes. We discuss their merits, limitations and potential uses.     

Characterization of the in vitro antimycotic activity of a novel killer protein from Williopsis saturnus DBVPG 4561 against emerging pathogenic yeasts

A novel killer toxin, labelled as KT4561, secreted by Williopsis saturnus DBVPG 4561, was found to possess a wide antimycotic activity against strains of Candida glabrata, Issatchenkia orientalis and Pichia guillermondii. KT4561 was precipitated by ethanol and purified by ion-exchange chromatography. The active protein migrated as a single band in SDS-PAGE and was characterized by a molecular weight of approximately 62 kDa. Purified KT4561 was active across wide ranges of temperature (5-45 degrees C) and pH (4.5-8.0) and displayed a rapid decrease in viability of yeast cells after 4-8 h. The in vitro activity of KT4561 against 102 yeast isolates (79% of clinical origin) was determined: MIC(50) and MIC(90) of strains were 0.08 and 0.15 microg/ml for C. glabrata, 0.03 and 0.23 microg/ml for I. orientalis and 1.50 and 2.25 microg/ml for P. guilliermondii. Comparative susceptibility tests showed that a high number of strains used in the present study were insensitive to selected azole and polyene antibiotics. The present study demonstrated the potential of KT4561 to be applied as novel control agent against pathogenic yeasts.     

玫瑰红鹅膏主要肽类毒素的HPLC 测定及其对白色念珠菌的抑制活性

【目的】检测玫瑰红鹅膏中所含肽类毒素及其含量,并对其肽类毒素的抑制白色念珠菌活性进行研究。【方法】采用HPLC和ESI-MS法从玫瑰红鹅膏中分离并鉴定出所含肽类毒素,并采用HPLC法测定其子实体、菌盖及菌柄和菌托混合部分中肽类毒素的含量。同时,采用纸片法研究了玫瑰红鹅膏粗毒液和分离到的单品肽类毒素对白色念珠菌JLC31680和JLC31681的抑菌作用。【结果】分离并鉴定出α-鹅膏毒肽(α-AMA)、β-鹅膏毒肽(β-AMA)和二羟鬼笔毒肽(PHD)等3种肽类毒素。玫瑰红鹅膏子实体中α-AMA、β-AMA、PHD的含量分别为30.3168、6.9932和9.9459 mg/g;菌盖中含量分别为44.9573、11.0798和11.3025 mg/g;菌柄和菌托混合部分中:α-AMA 11.6904 mg/g和PHD 7.9775 mg/g,β-AMA未检出。粗毒液、α-AMA、β-AMA和PHD对白色念珠菌JLC31680均具有很好的抑制作用,抑制率分别达到11.96%、32.52%、23.29%(p<0.01)和15.46%(p<0.05);粗毒液和β-AMA对白色念珠菌JLC31681的最高抑制率分别为10.16%和11.10%(p<0.01),α-AMA对白色念珠菌JLC31681最高抑菌率为6.89%(p<0.05)。【结论】玫瑰红鹅膏中的三种肽类毒素的含量较高,是制备肽类毒素的新资源;其具有抑制白色念珠菌的活性,可开发利用。     

寄生性膜翅目昆虫毒素的生态机制及应用前景

膜翅目昆虫利用高效的毒素进行自身防卫、攻击猎物和调节寄主生长发育.从寄生性膜翅目昆虫毒素的产生、类别、组份、性质、毒素的生态功能以及毒素的作用机制等方面综述了寄生性膜翅目昆虫毒素的研究概况.膜翅目的泌毒器官起源于外胚层,由生殖系统的附腺演化而来.毒液由成熟雌蜂的毒腺或酸腺所分泌,并贮于毒囊中.昆虫毒素是成分复杂的混合物,已知膜翅目昆虫毒素中含有烃类、醇类、醛类、酮类、羧酸类、酯类、内酯类、酶类等多种化合物.寄生性膜翅目昆虫的毒素在提高自身适应能力方面的作用是巨大的,如通过麻痹寄主提高产卵成功的概率、通过抑制寄主的生长发育和免疫功能提高后代的存活率、通过干扰寄主的生理活动改善后代的营养需求等.体外寄生蜂毒素可造成寄主幼虫停止发育、永久性的麻痹甚至死亡,这类毒素常为抑性的、广谱的,一般作用于中枢神经系统或神经-肌肉连接点.而体内寄生蜂多为容性寄生,其毒液中含有多分DNA病毒（PDV）,PDV通过抑制寄主免疫系统而巧妙地调节寄主的生理活动和发育,影响寄主的正常变态,大多数种类直到寄主结茧或做好蛹室时才将其杀死在安全的场所,从而使寄生蜂后代能够顺利完成发育.容性寄生蜂毒素对PDV的功能具有显著的增效或协同作用,而不会使寄主产生永久性麻痹.PDV对寄生蜂本身是非致病性的,与寄生蜂是一种分子水平上的共生或依生关系.寄生性膜翅目昆虫毒素显示了良好的应用前景,特别是在开发人类医药和特异性生物杀虫剂方面.但分离和纯化毒液中各个活性成分是应用的前提,也是生化和毒理研究的需要.     

Effects and mechanisms of Bacillus thuringiensis crystal toxins for mosquito larvae

Bacillus thuringiensis is a Gram‐positive aerobic bacterium that produces insecticidal crystalline inclusions during sporulation phases of the mother cell. The virulence factor, known as parasporal crystals, is composed of Cry and Cyt toxins. Most Cry toxins display a common 3‐domain topology. Cry toxins exert intoxication through toxin activation, receptor binding and pore formation in a suitable larval gut environment. The mosquitocidal toxins of Bt subsp. israelensis (Bti) were found to be highly active against mosquito larvae and are widely used for vector control. Bt subsp. jegathesan is another strain which possesses high potency against broad range of mosquito larvae. The present review summarizes characterized receptors for Cry toxins in mosquito larvae, and will also discuss the diversity and effects of 3‐D mosquitocidal Cry toxin and the ongoing research for Cry toxin mechanisms generated from investigations of lepidopteran and dipteran larvae.     

Different action of killer toxins K1 and K2 on the plasma membrane and the cell wall of Saccharomyces cerevisiae

Study of Saccharomyces cerevisiae killer toxin-sensitive strains with the deltakre2 phenotype (resistant to toxin K1, sensitive to toxin K2) showed that the phenotype is complemented by the KRE2 gene not only in intact cells but also in spheroplasts, and resistance to K1 thus resides very probably in the plasma membrane. deltakre1 deletant displays a faulty interaction with both K1 and K2 toxin. Hence, Kre1p probably serves as plasma membrane receptor for both toxins. Deletants in seven other genes (GDA1, SAC1, LUV1, KRE23, SAC2, KRE21, ERG4) exhibit different degrees of the deltakre2-like resistance pattern, but the phenotype in deltagda1 and deltasac1 is not connected with a defect in K1 toxin interaction with the plasma membrane, similarly as in deltakre6 and deltakre11 strains with a higher resistance to K2 toxin. Differences between the K1 and K2 killer toxin thus occur on the level of both the plasma membrane and the cell wall.     

Isolation and characterization of Saccharomyces cerevisiae mutants with a different degree of resistance to killer toxins K1 and K2

Killer toxin K1 of Saccharomyces cerevisiae kills sensitive cells of the same species by disturbing the ion gradient across the plasma membrane after binding to the receptor at cell wall beta-1,6-glucan. Killer protein K2 is assumed to act by a similar mechanism. To identify the putative plasma membrane receptors for both toxins we mutagenized three sensitive S. cerevisiae strains and searched for clones with killer-resistant spheroplasts. The well diffusion assay identified three phenotypically different groups of clones: clones resistant simultaneously to both toxins, clones with lowered sensitivity to only K1 toxin and those with strongly lowered sensitivity to K2 and partially lowered sensitivity to K1 toxin. These phenotypes are controlled by recessive mutations that belong to at least four different complementation groups. This indicates certain differences at the level of interaction of K1 and K2 toxin with sensitive cells.     

Biotyping of pathogenic fungi by the killer system and with monoclonal antibodies

Biotyping of pathogenic yeasts and hyphomycetes based on their suceptibility to selected killer yeasts and their reactivity with monoclonal antibodies are described. Both methods were used to differentiate fungi isolated from patients providing valuable epidemiological information on mycotic infections. The functional biotyping obtained with the two systems and the conventional auxenographic biocoding approaches commercially available for opportunistic yeasts are comparatively evaluated. The potential for biotyping of industrial fungal isolates is also discussed.     

The costs and benefits of killer toxin production by the yeast Pichia kluyveri

Numerous yeast species in many genera are able to produce and excrete extracellular toxic proteins (mycocins) that can kill other specific sensitive yeasts. Natural distributions of killer yeasts suggest that they may be important in maintaining community composition and provide a benefit to the toxin producing cells. The fact that not all yeasts are killers and that polymorphisms exist within some killer species suggests there may be a cost associated with killer toxin production. This study focuses on the costs and benefits associated with toxin production by the yeast Pichia kluyveri. Strains differing in their ability to kill were obtained by tetrad dissection. One parent strain produced spores that exhibited a trade-off between killing ability and intrinsic growth rate. A killer clone from this strain was able to maintain a higher proportion of cells than a non-killer when grown with the same sensitive yeast under laboratory-simulated natural conditions. On the other hand, when grown with a yeast not sensitive to Pichia kluyveri toxin, the non-killer maintained a higher proportion of the total community than did the killer clone. The data support the hypothesis that there are both costs and benefits to producing killer toxin, and based on this, selection may favor different phenotypes in different conditions.     

Production and characteristics of Debaryomyces hansenii killer toxin

The optimal conditions for the production of the killer toxin of Debaryomyces hansenii CYC 1021 have been studied. The lethal activity of the killer toxin increased with the presence of NaCl in the medium used for testing the killing action. Production of the killer toxin was stimulated in the presence of proteins of complex culture media. Addition of nonionic detergents and other additives, such as dimethylsulfoxide enhanced killer toxin production significantly. Killer toxin secretion pattern followed the growth curve and reached its maximum activity at the early stationary phase. Optimal stability was observed at pH 4.5 and temperatures up to 20 °C. Above pH 4.5 a steep decrease of the stability was noted. The activity was hardly detectable at pH 5.1.     

Mating-type locus control of killer toxins from Kluyveromyces lactis and Pichia acaciae

Killer-toxin complexes produced by Kluyveromyces lactis and Pichia acaciae inhibit cell proliferation of Saccharomyces cerevisiae. Analysis of their actions in haploid MATalpha cells revealed that introduction of the opposite mating-type locus (MATa) significantly suppressed antizymosis. Together with resistance expressed by MATa/MATalpha diploids, the reciprocal action of MATa or MATalpha in haploids of opposite mating types suggests that these killer toxins may be subject to MAT locus control. Congruently, derepressing the silent mating-type loci, HMR and HML, by removing individual components of the histone deacetylase complex Sir1-4, either by transposon-tagging or by chemically inactivating the histone deacetylase catalytic subunit Sir2, yields toxin resistance. Consistent with MAT control of toxin action, killer-toxin-insensitive S. cerevisiae mutants (kti) become mating-compromised despite resisting the toxins' cell-cycle effects. Mating inhibition largely depends on the time point of toxin application to the mating mixtures and is less pronounced in Elongator mutants, whose resistance to the toxins' cell-cycle effects is the result of toxin-target process deficiencies. In striking contrast, non-Elongator mutants defective in early-response events such as toxin import/activation hardly recover from toxin-induced mating inhibition. This study reveals a novel effect of yeast killer toxins on mating and sexual reproduction that is independent of their impact on cellular proliferation and cell-cycle progression.     

Serpentine type receptors and heterotrimeric G-proteins in yeasts: Structural-functional organization and molecular mechanisms of action

The signal systems of the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, coupled to heterotrimeric G-proteins and sensitive to pheromones and alimentary molecules, are prototypes of hormonal signal systems of the higher vertebrate animals and are widely used in studies on molecular mechanisms of their functioning. This review summarizes and analyzes data on structural-functional organization of the first two components of these systems—receptors of the serpentine type and heterotrimeric G-proteins; mechanisms of functional coupling of receptors and G-proteins both between each other and to other signal proteins are discussed. It has been shown that at the early stages of evolution of signaling systems, at the yeast level, various models of transduction of signals into the cell were tested; many of them differ essentially from the classic model of the three-component, G-protein-coupled signal system of the higher vertebrates.