Mikrobiol. Z. 2020; 82(2):3-13.
doi: https://doi.org/10.15407/microbiolj82.02.003

The Role of PPN1 and PPX1 Polyphosphatases in the Stress-Induced Changes of the Polysaccharide
Composition of Cell Wall and Extracellular Matrix of Saccharomyces cerevisiae Cells

S.I. Voychuk, O.M. Gromozova

Zabolotny Institute of Microbiology and Virology, NAS of Ukraine
154 Akad. Zabolotny Str., Kyiv, 03143, Ukraine

Polysaccharides (PS) are important structural elements of all living organisms. They perform many important functions and protect cells from the action of various stresses. The in vivo synthesis of PS is an energy-consuming process that requires phosphates, as well as the structure of PS requires the phosphates and polyphosphates (poly(P)) as binding elements. However, the role of enzymes that metabolize poly(P) (polyphosphatases, poly(P)ases) in the processes of cell wall components and extracellular matrix synthesis is poorly understood. Aim. The aim was to study the role of PPN1 and PPX1 poly(P)ases in the processes of cell wall and extracellular matrix formation. Methods. Saccharomyces cerevisiae yeast cells with deletions of PPN1 (Δppn1) and PPX1 (Δppx1) were used in the study. Cells were exposed to hydrogen peroxide (25–100 mM), acetic acid (25–100 mM) and sorbitol (0.25–1.0 M) to induce stress reactions. RF-EMF (40.68 MHz, 15W power, 30 min) was applied separately and 30 min before treatment with other stress factors to induce an adaptive response. The influence of stress factors was evaluated by changes in the content of sugars. The sugars and sugar residues in the cell walls and extracellular matrix of the yeast cells were detected using GC/MS and lectin-gold binding test. The content of D-mannose/Dglucose (Man/Glu), D-galactose (Gal), N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc) and N-acetylneuraminic acid (NANA) was assessed. Results. The defects in PPN1 and PPX1 affect the content of sugars in the cell walls and extracellular matrix. The amounts of glucosamine (according to GC/MS analysis) decreased in 3–5 times in the cell walls of PPN1 and PPX1 defective cells. The lectingold test showed that the Man/Glu content was the most stable (27–38%) among all the yeast cells, while the amounts of other sugars varied significantly. The deletion of poly(P)ases had different effects on the content of sugars in the cell walls and extracellular matrix: the extracellular matrix showed a significant decrease in GlcNAc, GalNAc and an increase in NANA, while the content of GalNAc in the cell walls remained almost constant, and the content of NANA decreased in case of PPN1 deletion and increased in case of PPX1 deletion. Correlation analysis showed a potentially high (up to 97%) correlation between Man/Glu, GlcNAc, and GalNAc amount in cell walls, and cell viability (stress-resistance). However, only one of these sugars, GlcNAc, showed correlation with defectiveness in PPN1 and PPX1. The differences between the effects observed in cells with single and double deletions of poly(P)ases indicate that both enzymes positively regulate GlcNAc biosynthesis of cell walls and extracellular matrix and the biosynthesis of extracellular GalNAc. Conclusions. Both poly(P)ases (PPN1 and PPX1) are involved in the assembly of the cell wall and extracellular matrix and influence mainly the content of their minor constituents: Gal, GalNAc, GlcNAc, and NANA. The change in stresses resistance of PPN1 and PPX1 defective cells correlate with the content of Man/Glu, GalNAc and GlcNAc, which is regulated by both poly(P)ases. The marked differences and changes in the content of the PS may indicate a decrease of the cell wall rigidity and a decrease of the GPI-bound proteins portion in it, as well as the conformational changes of the PS in the extracellular matrix that resulted from the deletion of the poly(P)ases.

Keywords: polysaccharides, cell wall, extracellular matrix, polyphosphatases, stresses, Saccharomyces cerevisiae.

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