Pichia anomalakiller toxin secretion in the presence of tunicamycin

Abstract

The role of N-glycosylation in the secretion and activity of a Pichia anomala killer toxin was studied using tunicamycin. Secretion of the yeast toxin was localized using a specific monoclonal antibody. Preservation of the toxin killer effect was evaluated with two sensitive yeast strains. Tunicamycin did not affect the secretion of active toxin, suggesting that N-glycosylation does not appear to be involved in either toxin secretion or in killer activity.The role of the polysaccharidic chains in the secretion of yeast glycoproteins has been studied using tunicamycin on species such as Saccharomyces cerevisiae [9,12] and Candida albicans [3,4]. Tunicamycin is an antibiotic which inhibits the N-glycosylation of proteins by preventing the formation of asparagine-linked precursors from which all N-linked oligosaccharides are derived [10]. Therefore, newly synthesized glycoproteins (except for those synthesized before complete incorporation of the drug) do not carry N-glycosylated oligosaccharides [23].In yeast, particular secreted glycoproteins have been shown to possess a killer activity against a specific panel of sensitive micro-organisms [13,16,18,21]. It has been reported that glycosylation may be a prerequisite for chanelling some of them through the secretion pathway [24].In this work, a killer strain of Pichia anomala was grown in the presence of tunicamycin. Indirect immunofluorescence assays (IFA) were carried out with a monoclonal antibody (MAb KT4) neutralizing the toxin activity [2,17]. IFA were performed on normal and drug-treated P. anomala K36 cells to observe the effect of tunicamycin on the yeast surface expression of the toxin. The killer properties of the toxin secreted by the drug-treated yeasts were compared to those of the toxin produced in the absence of tunicamycin. Viable cell count, recovered as colony forming units (CFU) and growth inhibition assays, were performed with both culture supernatants on sensitive strains of P. anomala (UM3 strain) and C. albicans (CDC B385 strain) [14].Production of the toxin was carried out as described previously [2,15]. Where indicated, yeast cultures were grown with 3 pg ml-1 of tunicamycin. Toxin activity was

Anti-beta-glucan-like immunoprotective candidacidal antiidiotypic antibodies

“…A neutralizing mAb produced against PaKT (mAb KT4) specifically can label the surface of both KT producing strains, suggesting that they secrete KTs bearing a common epitope (126). PaKT is a glycoprotein encoded by nuclear genes, which is heterogeneously secreted in the presence of tunicamycin, thus excluding the involvement of N-glycosylation in toxin secretion and killer activity (127)(128)(129). PaKT killer activity is mediated by direct interaction with specific cell wall KTRs, distributed mainly in budding cells and germination tubes, constituted by BGs.…”

Yeast killer systems

“…Killer cells are immune to their own toxin while possessing specific cell wall receptors, but the mechanisms of killing and immunity still remain unknown. A fully active toxin is secreted in the presence of tunicamycin, suggesting that N glycosylation is not involved in either toxin secretion or killer activity (56). Ultrastructural studies on the secretion of the toxin, carried out by immunoelectron microscopy with a specific monoclonal antibody, suggest that it undergoes heterogeneous secretion by the killer cells and concentration in the cell wall layers before being exported outside the cells (55,57).…”

Involvement of β-Glucans in the Wide-Spectrum Antimicrobial Activity of Williopsis saturnus var. mrakii MUCL 41968 Killer Toxin

“…Optimal WmKT activity is observed in acidic conditions (pH 4.6) and at temperatures around 26-28 ЊC. WmKT is specifically recognized by mAbKT4, a mouse monoclonal antibody (mAB) that neutralizes the killer activity of the toxin (8). Recently, anti-idiotypic antibodies displaying killer toxin activity (KTIdAbs) were produced by immunizing mice with mAbKT4.…”