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. 2017 Dec 19;16(1):228.
doi: 10.1186/s12934-017-0844-0.

Production of fluorescent and cytotoxic K28 killer toxin variants through high cell density fermentation of recombinant Pichia pastoris

Esther Giesselmann  1 , Björn Becker  1 , Manfred J Schmitt  2
Affiliations

Production of fluorescent and cytotoxic K28 killer toxin variants through high cell density fermentation of recombinant Pichia pastoris

Esther Giesselmann et al. Microb Cell Fact. .

Abstract

Background: Virus infected killer strains of the baker's yeast Saccharomyces cerevisiae secrete protein toxins such as K28, K1, K2 and Klus which are lethal to sensitive yeast strains of the same or related species. K28 is somewhat unique as it represents an α/β heterodimeric protein of the A/B toxin family which, after having bound to the surface of sensitive target cells, is taken up by receptor-mediated endocytosis and transported through the secretory pathway in a retrograde manner. While the current knowledge on yeast killer toxins is largely based on genetic screens for yeast mutants with altered toxin sensitivity, in vivo imaging of cell surface binding and intracellular toxin transport is still largely hampered by a lack of fluorescently labelled and biologically active killer toxin variants.

Results: In this study, we succeeded for the first time in the heterologous K28 preprotoxin expression and production of fluorescent K28 variants in Pichia pastoris. Recombinant P. pastoris GS115 cells were shown to successfully process and secrete K28 variants fused to mCherry or mTFP by high cell density fermentation. The fluorescent K28 derivatives were obtained in high yield and possessed in vivo toxicity and specificity against sensitive yeast cells. In cell binding studies the resulting K28 variants caused strong fluorescence signals at the cell periphery due to toxin binding to primary K28 receptors within the yeast cell wall. Thereby, the β-subunit of K28 was confirmed to be the sole component required and sufficient for K28 cell wall binding.

Conclusion: Successful production of fluorescent killer toxin variants of S. cerevisiae by high cell density fermentation of recombinant, K28 expressing strains of P. pastoris now opens the possibility to study and monitor killer toxin cell surface binding, in particular in toxin resistant yeast mutants in which toxin resistance is caused by defects in toxin binding due to alterations in cell wall structure and composition. This novel approach might be easily transferable to other killer toxins from different yeast species and genera. Furthermore, the fluorescent toxin variants described here might likewise represent a powerful tool in future studies to visualize intracellular A/B toxin trafficking with the help of high resolution single molecule imaging techniques.

Keywords: A/B toxins; Fluorescence labelling; Heterologous protein expression; High cell density fermentation; Killer toxin; Pichia pastoris; Saccharomyces cerevisiae.

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Figures

Fig. 1
Fig. 1
a Schematic structure of K28-mCherryHDEL. The K28 chimera contains an N-terminal signal peptide derived from the α-mating factor which is removed by signal peptidase (SP) cleavage after ER import. In the cis-Golgi, endopeptidase Kex2p cleavage removes the pro- and γ-sequence which leads to the separation of α and β. Both subunits remain connected by a single disulphide which was previously formed in the ER. Carboxypeptidase Kex1p removes the C-terminal arginine residue in a trans-Golgi compartment and, thereby, unmasks the HDEL motif. b Western blot analysis of secreted K28-mCherryHDEL from P. pastoris. Cell-free culture supernatants of the recombinant strain GS115 [pPIC9 K28-mCherryHDEL] and the negative control strain GS115 [pPIC9] grown for 120 h in BMM and ×125 concentrated by centrifugal concentrators were separated under non-reducing (left) and reducing (right) conditions by SDS-PAGE and probed with an anti-DsRed antibody. Position and size of the correctly processed toxin is indicated. c Detection of fluorescent K28-mCherryHDEL. GS115 [pPIC9 K28-mCherryHDEL] was grown for 120 h in BMM medium (pH 7) in the presence of protease inhibitors. SDS-PAGE of the ×125 concentrated cell-free culture supernatant under non-reducing conditions shows the pink fluorescent protein and its fluorescence under UV illumination
Fig. 2
Fig. 2
a Growth curve of the recombinant P. pastoris strain GS115 [pPIC9 K28-mCherryHDEL] during fermentation. The starting point of methanol feeding (K28 expression induction) is marked by an arrow. b Fluorescence development of mCherry-tagged K28. Cell-free culture supernatant samples from different fermentation time points were separated by SDS-PAGE under non-reducing conditions. Fusion proteins were detected by UV illumination. Position and size of the fusion proteins are indicated. c Killer activity of the fermentation product K28-mCherryHDEL. Cell-free culture supernatants were tested against the sensitive S. cerevisiae strain 192.2d in an agar diffusion assay (t0 = starting point of methanol feeding; tend = 120 h post methanol induction). Cell-free zones of growth inhibition were determined and killer activity was compared to a ×200 concentrated culture supernatant of the K28 killer strain S. cerevisiae MS300b
Fig. 3
Fig. 3
a Fluorescence distribution of S. cerevisiae cells after incubation with K28-mCherryHDEL. S. cerevisiae BY4742 was washed twice after 2 h incubation with K28-mCherryHDEL and analyzed by confocal laser scanning microscopy. b Same cells analysed by structured illumination microscopy (SIM). The hypersensitive yeast strain 192.2d was treated with K28-mCherryHDEL as described in a and subjected to SIM. (c) Fluorescence quenching was performed on cells of S. cerevisiae BY4742 after incubation with K28-mTFPHDEL as described in a, however extracellular mTFP fluorescence was subsequently quenched by the addition of 2 mM bromophenol blue (BPB)
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References

    1. Bevan EA, Makower M. The physiological basis of the killer character in yeast. Proc Xlth Int Congr Genet. 1963;1:202–203.
    1. Starmer WT, Ganter PF, Aberdeen V, Lachance MA, Phaff HJ. The ecological role of killer yeasts in natural communities of yeasts. Can J Microbiol. 1987;33:783–796. doi: 10.1139/m87-134. - DOI - PubMed
    1. Schmitt MJ, Breinig F. Yeast viral killer toxins: lethality and self-protection. Nat Rev Microbiol. 2006;4(3):212–221. doi: 10.1038/nrmicro1347. - DOI - PubMed
    1. Rodriguez-Cousino N, Maqueda M, Ambrona J, Zamora E, Esteban R, Ramirez M. A new wine Saccharomyces cerevisiae killer toxin (Klus), encoded by a double-stranded rna virus, with broad antifungal activity is evolutionarily related to a chromosomal host gene. Appl Environ Microbiol. 2011;77(5):1822–1832. doi: 10.1128/AEM.02501-10. - DOI - PMC - PubMed
    1. Riffer F, Eisfeld K, Breinig F, Schmitt MJ. Mutational analysis of K28 preprotoxin processing in the yeast Saccharomyces cerevisiae. Microbiology. 2002;148:1317–1328. doi: 10.1099/00221287-148-5-1317. - DOI - PubMed

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