Ultrastructural immunodetection of a Pichia anomala killer toxin: a preliminary study

Abstract

Summary— A monoclonal antibody (mAb KT4), produced against a Pichia anomala killer toxin, was used to study the secretion process of toxin producing cells. The indirect immunofluorescence assay, performed with large concentrations of mAb KT4, showed a homogeneous distribution of the epitope at the cell surface of the P anomala cells. When increasing dilutions of mAb KT4 were employed, a ‘punctuated’ labeling appeared on the yeast's cell wall which suggested a heterogeneous secretion of the killer toxin. Similar labeling was also observed by immunodetection on live yeast cells held in buffered suspension. These results confirmed that 'punctuated' labeling was not an artefact due to a distortion of the cell's shape by having been dried on glass slides. Indirect immunodetection was performed in electron microscopy on ultra‐thin sections of cells embedded in Araldite resin. The labeling thus obtained showed both the presence of the epitope in the cytoplasm and its sensitivity to strong glutaraldehyde fixation. Indirect immunodetection, performed on ultra‐thin frozen sections, showed a cytoplasmic and cell wall labelling. However, the amount of gold particles observed in the cell wall was too low to confirm the heterogeneous killer toxin secretion observed in immunofluorescence. In this case, killer cells were fixed with a low concentration of glutaraldehyde which preserved the structure of the epitope complementary with mAb KT4.

A Wickerhamomyces anomalus Killer Strain in the Malaria Vector Anopheles stephensi

“…By in vitro IFA assay on free cells, we verified that cultivated Wa F17.12 yeasts also produce a molecule recognized by mAbKT4, concentrated at the surface of the yeast. This is in accordance with published results indicating toxin secretion through the cell wall in W. anomalus Wa KT-producing strains [36], [37]. Overall, the above results strongly suggest that Wa F17.12 yeast strain from mosquitoes produces a killer toxin.…”

Isolation of Yeasts from Various Food Products and Detection of Killer Toxin Activity <i>In vitro</i>

“…Though the killer phenotype was discovered in the 1963 [2,3,[7][8][9], there is yet a lot to be explored on the killer toxin production of non-pathogenic strains. The probability that a killer toxin produced by yeast may kill certain susceptible yeasts would also depend on the ecological characters such as the region, the host plant and the habitat from which the killer yeasts were collected [10].…”

Pichia anomalakiller toxin secretion in the presence of tunicamycin

“…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.…”

“…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.…”