Yeast Killer Toxin Research Articles

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.

Inhibition of DNA Synthesis in Saccharomyces cerevisiae by Yeast Killer Toxin KT28

Treatment of sensitive cells of Saccharomyces cerevisiae with killer toxin KT28 affected cell viability after 2 h: the effect was dependent upon the availability of a utilizable energy source. Treatment led to an interruption of cell growth. The mother cells contained nuclear DNA, whereas their daughter buds did not. Using a killer-toxin-sensitive thymidine auxotroph of S. cerevisiae carrying a temperature-sensitive thymidylate uptake mutation, it was shown that the incorporation of dTMP at the permissive temperature was inhibited within 30 min of the addition of KT28. When cells labelled at the permissive temperature were incubated at the restrictive temperature, the level of radioactivity declined in the absence but not in the presence of KT28. No other effects of KT28 were observed within 2 h of its addition, and it is concluded that the inhibition of DNA synthesis is an early effect of the action of KT28.

Purification of yeast killer toxin KT28 by receptor-mediated affinity chromatography

Purification of yeast killer toxin KT28 by receptor-mediated affinity chromatography

Role of protein processing, intracellular trafficking and endocytosis in production of and immunity to yeast killer toxin.

Yeast strains harboring M1-dsRNA and its packaging virus ScV-L secrete a disulfide-linked, heterodimeric toxin which kills sensitive yeast cells by disrupting plasma membrane function. The mature toxin is derived from a precursor (preprotoxin) which undergoes post-translational processing steps during export via the established yeast secretory pathway. Cleavage by both the KEX1 and KEX2 endopeptidases is required for expression of killing activity. The same 1.0 kb open reading frame on M1-dsRNA directs the expression of immunity to toxin. Differentially processed derivatives of protoxin, as well as protoxin itself, have been proposed to serve as mediators of immunity. To understand the mechanisms by which the killing and immunity phenotypes can be derived from a common precursor, we have: 1) studied cellular processes implicated in expression of the phenotypes; and 2) developed a system to produce mutants defective in immunity, killing, or both. In the first approach, the role played by both endocytosis and vesicular traffiking in expression of killing and immunity was examined. Strains defective in endocytosis (end1, end2) or vacuolar protein localization (vpl3, vpl6) were transformed with a plasmid encoding killer toxin under control of the pho5 promoter. When induced by phosphate starvation, both end mutants and all vpl mutants expressed killing activity. Immunity to exogenous toxin, however, was significantly decreased in strains carrying both vpl mutant alleles and in one of the endocytosis mutants (end1]. This suicidal phenotype (rex for resistance expression) has been described previously in M1-containing strains as a leaky phenocopy.(ABSTRACT TRUNCATED AT 250 WORDS)

Yeast killer toxin-like anti-idiotypic antibodies

Anti-idiotypic antibodies (anti-Ids) were raised in a rabbit against a murine monoclonal antibody (MAb) neutralizing the yeast killer toxin produced by a strain of Pichia (Hansenula) anomala. In an immunodiffusion test, the anti-Ids produced in the rabbit recognized the antigen-binding site of the MAb used as the immunogen (KT4) but not that of another heterologous MAb. The absence of any significant cross-reactivity among the anti-Ids raised in a rabbit for a heterologous MAb suggested that the anti-Ids were highly specific for unique variable-region determinants. Furthermore, the P. anomala killer toxin proved to be competing with anti-Ids for the binding site of MAb KT4. Anti-Ids against the MAb to yeast killer toxin inhibited the growth of Candida albicans, thereby mimicking the effect of the yeast killer toxin. These results suggest that, in some cases, anti-Ids might be useful tools for elucidating structure-function relationships for sensitive cell receptors.

Biotyping of micelial fungus cultures by the killer system.

Based on the occurrence of the yeast killer phenomenon in hyphomycetes, the toxic effect of 37 selected killer yeasts was studied on eleven strains of Pseudallescheria boydii, six strains of Aspergillus niger, 18 strains of Penicillium camemberti and nine strains of Sporothrix schenckii. The demonstration of different biotypes within the species of P. boydii and P. camemberti proves that the killer system also is a practical and effective method for epidemiological studies among hyphomycetes. Based on the system used, it was not possible to observe markedly different biotypes among the A. niger and S. schenckii strains studied. The first evidence that an isolated, concentrated (50X) and partially purified yeast killer toxin may display a lethal activity against mycelial fungus cultures is also given in this paper.

Production and characterization of yeast killer toxin monoclonal antibodies

Monoclonal antibodies were obtained after fusion of mouse myeloma cells with spleen cells isolated from mice primed with a crude extract of yeast killer toxin produced by a strain of Hansenula anomala. Hybridomas were selected by specific immunoassay reaction of their fluid with crude yeast killer toxin extract. Among the monoclonal antibodies, which were characterized by the Western blot technique, one (designated KT4) proved to have precipitating properties, thus permitting the neutralization of the killer activity of the toxin. Experiments in double immunodiffusion showed that monoclonal antibody KT4 produced homologous precipitin bands by reacting with either the crude toxin used as immunogen or a toxic extract of Hansenula mrakii. It is suggested that these monoclonal antibodies will be useful for the purification, characterization, and understanding of the bioactions of yeast killer toxins.

Potential therapeutic effect of yeast killer toxin.

Experimental infections were produced in guinea pigs, rabbits and dogs with lesions similar to those seen in human seborrheic dermatitis and otitis externa by cutaneous application of cultures of Malassezia furfur and M. pachydermatis. Infected animals were treated by topical application of a concentrated yeast killer toxin (Hansenula anomala UCSC 25F). Clinical recovery as well as negative mycological test cultures of infected animals proved to the clearly associated with the treatment by the killer toxin.

Immobilized cell wall as a biospecific sorbent

Isolated cell walls of a killer-toxin-sensitive yeast have been covalently linked to modified hydroxyalkyl methacrylate gel. The resulting preparation is a particularly convenient biosorbent for yeast killer toxin.

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 α and β subunits of the secreted toxin, and a glycosylated γ 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 α subunit into the N-terminal part of the γ 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.

On a New Genus of the Laboulbeniales: Majewskia

4. Middlebeek, E. J., J. M. H. Hermans, C. Stumm, and H. L. Muytjens. 1980. Incidence of sen? sitivity to yeast killer toxins among isolates of human origin Candida and Torulopsis. Antimicrob. Agents Chemother. 17: 350-354. 5. Morace, G., C. Archibusacci, M. Sestito, and L. Polonelli. 1984. Strain differentiation of pathogenic yeasts by the killer system. Mycopathologia 84: 81-85. Stumm, C, J. M. H. Hermans, E. J. Middlebeek, A. F. Croes, and G. J. M. K. de Vries. 1977. Killer-sensitive relationships in yeasts from nat? ural habitats. Antonie van Leeuwenhoek J. MU crobiol Serol 43: 125-128.

The expression of cDNA clones of yeast M1 double-stranded RNA in yeast confers both killer and immunity phenotypes.

Two cDNA clones of the segment of Saccharomyces cerevisiae M1 double-stranded RNA, which codes for the yeast killer toxin, have been expressed in yeast using the expression vector pYT760. Toxin expression and secretion depended upon the presence of a yeast promoter. Transformants not only contain an authentic preprotoxin precursor, as determined by precipitation of intracellular proteins with antitoxin antisera, but also display an immunity phenotype. The evidence is that the immunity protein is part of the preprotoxin and may act by masking toxin binding sites. Neither cDNA clone had a complete 5' terminus and the preprotoxin translational start was missing. The promoter and the initiator ATG were supplied by the expression vector. One clone with a full-length preprotoxin but altered N-terminal amino acids gave a normal glycosylated intracellular precursor. A clone with an N-terminal nine amino acid deletion gave a precursor which was not glycosylated but toxin was still secreted.

Mode of action of yeast killer toxins: channel formation in lipid bilayer membranes.

The toxic action of yeast killer proteins seems to involve selective functional damage to the plasma membrane of the sensitive cell. Physiological effects include leakage of K+ (refs 1, 2), inhibition of active transport of amino acids and acidification of the cell interior. These effects are strikingly similar to the effects of certain bacterial colicins which have been demonstrated previously to form channels in membranes. Proposed mechanisms of action have usually postulated a limited permeability change induced by the toxin in the plasma membrane. We report here that a killer toxin from the yeast Pichia kluyveri forms ion-permeable channels in phospholipid bilayer membranes, and we propose that the in vitro electrophysiological properties of these channels account for the morbid effects observed in intoxicated cells. A preliminary account of this work has appeared elsewhere.

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.

Primary effects of yeast killer toxin

Killer toxin from Saccharomyces cerevisiae binds to sensitive cells immediately after addition to the cells. However, 50% mortality was obtained only after 40 minutes. Although it is thought that a lag phase is required for the killer to exert its action, we report experiments showing that the killer starts affecting the cell immediately after binding. Thus, shortly after addition the toxin was able to inhibit the transport of L-|; 3H|; leucine as well as that of protons which are cotransported with this amino-acid or with histidine. Moreover, killer toxin inhibited the pumping of protons to the medium by cells which were actively metabolizing glucose. These effects were a function of the concentration of toxin used. The results suggest that killer toxin acts by affecting the electrochemical proton gradient across the plasma membrane of yeast.

High incidence of sensitivity to yeast killer toxins among Candida and Torulopsis isolates of human origin.

Among yeast strains of human origin belonging to the genera Candida, Cryptococcus, Torulopsis, and Rhodotorula which were examined for killer and sensitive characteristics with killer and sensitive strains of Cryptococcus, Hansenula, Kluyveromyces, Pichia, Saccharomyces, and Torulopsis as screening organisms, a high incidence of sensitivity to killer toxins was observed within the genera Candida and Torulopsis. Of 142 strains tested, 116 strains distributed over all Candida and Torulopsis species examined were sensitive to one or more killers. Several new intergeneric killer-sensitive relationships are described. Furthermore, killing activity was exhibited by six strains of Candida (C. krusei, C. guilliermondii) and three strains of Torulopsis (T. glabrata).

Binding of yeast killer toxin to a cell wall receptor on sensitive Saccharomyces cerevisiae

35S-labeled killer toxin protein bound to cells of sensitive Saccharomyces cerevisiae S14a. Strains that were resistant to toxin through mutation in the nuclear genes kre1 kre2 bound toxin only weakly. Non-radioactive toxin competed effectively with 35S-labeled toxin for binding to S14a, but did not compete significantly in the binding to mutant kre1-1. This implied that binding to kre1-1 was nonspecific. A Scatchard analysis of the specific binding to S14a gave a linear plot, with an association constant of 2.9 x 10(6) M-1 and a receptor number of 1.1 x 10(7) per cell. Killer toxin receptors were solubilized from the cell wall by zymolyase digestion. Soluble, non-dialyzable cell wall digest from S14a competed with sensitive yeast cells for 35S-labeled toxin binding and reduced toxin-dependent killing of a sensitive strain. Wall digest from kre1-1 competed only weakly for toxin binding with sensitive cells and caused little reduction of toxin-dependent killing. Although the abundant (1.1 x 10(7) per cell) receptor appeared necessary for toxin action, as few as 2.8 x 10(4) toxin molecules were necessary to kill a sensitive cell of S14a. The kinetics killing of S14a suggested that some component was saturated with toxin at a concentration 50-fold lower than that needed to saturate the wall receptor.