Mikrobiol. Z. 2020; 82(2):3-13.
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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- Lesage G, Bussey H. Cell wall assembly in Saccharomyces cerevisiae. Microbiol Mol Biol Rev. 2006; 70(2):317–43. https://doi.org/10.1128/MMBR.00038-05
- Kulaev IS, Vagabov VM, Kulakovskaya TV. The biochemistry of inorganic polyphosphates, 2nd ed. Chichester, UK: John Wiley & Sons, Ltd. 2004. https://doi.org/10.1002/0470858192
- Zvonarev AN, Crowley DE, Ryazanova LP, Lichko LP, Rusakova TG, Kulakovskaya TV, Dmitriev VV. Cell wall canals formed upon growth of Candida maltosa in the presence of hexadecane are associated with polyphosphates. FEMS Yeast Research. 2017; 17(3):fox026. https://doi.org/10.1093/femsyr/fox026
- García R, Bermejo C, Grau C, Pérez R, Rodríguez-Peña JM, Francois J, Nombela C, Arroyo J. The global transcriptional response to transient cell wall damage in Saccharomyces cerevisiae and its regulation by the cell integrity signaling pathway. J Biol Chem. 2004; 279(15):15183–95. https://doi.org/10.1074/jbc.M312954200
- Bru S, Martinez JM, Hernandez-Ortega S, Quandt E, Torres-Torronteras J, Marti RR, Canadell D, Arino J, Sharma S, Jimenez J, Clotet J. Polyphosphate is involved in cell cycle progression and genomic stability in Saccharomyces cerevisiae. Mol Microbiol. 2016; 101(3):367–80. https://doi.org/10.1111/mmi.13396
- Kalebina TS, Egorov SN, Arbatskii NP, Bezsonov EE, Gorkovskii AA, Kulaev IS. [The role of high-molecular-weight polyphosphates in activation of glucan transferase Bgl2p from Saccharomyces cerevisiae cell wall]. Doklady Biochemistry and Biophysics. 2008; 420(1):142–45. Russian. https://doi.org/10.1134/S1607672908030125
- Kalebina TS, Kulaev IS. [The role of proteins in the formation of the molecular structure of the cell wall of yeast]. Adv Biol Chem. 2001; 4:105–30. Russian.
- Voychuk SI, Gromozova OM. The functional role of PPN1 and PPX1 polyphosphatases under stresses action and for adaptive response development. Mikrobiol Z. 2020; 82(1):3–12. https://doi.org/10.15407/microbiolj82.01.003
- Pitarch A, Nombela C, Gil C. Cell wall fractionation for yeast and fungal proteomics. 2D PAGE: Sample preparation and fractionation. 2008; 217–39. https://doi.org/10.1007/978-1-60327-210-0_19
- Dallies N, François J, Paquet V. A new method for quantitative determination of polysaccharides in the yeast cell wall. Application to the cell wall defective mutants of Saccharomyces cerevisiae. Yeast. 1998; 14:1297–1306. https://doi.org/10.1002/(SICI)1097-0061(1998100)14:14<1297::AID-YEA310>3.0.CO;2-L
- François JM. A simple method for quantitative determination of polysaccharides in fungal cell walls. Nature Protocols. 2007; 1:2995–3000. https://doi.org/10.1038/nprot.2006.457
- Pinto M, Coelho E, Nunes A, Brandão T, Coimbra MA. Valuation of brewers spent yeast polysaccharides: A structural characterization approach. Carbohydrate Polymers. 2015; 116:215–22. https://doi.org/10.1016/j.carbpol.2014.03.010
- Kulakovskaya TV, Trilisenko LV, Lichko LP, Vagabov VM, Kulaev IS. The effect of inactivation of the exo- and endopolyphosphatase genes PPX1 andPPN1 on the level of different polyphosphates in the yeast Saccharomyces cerevisiae. Microbiology. 2006; 75(1):25–8. https://doi.org/10.1134/S002626170601005X
- Klis FM, Mol P, Hellingwerf K, Brul S. Dynamics of cell wall structure in Saccharomyces cerevisiae. FEMS Microbiology Reviews. 2002; 26(3):239– 56. https://doi.org/10.1111/j.1574-6976.2002.tb00613.x
- Soares RM, de A Soares RM, Alviano DS, Angluster J, Alviano CS, Travassos LR. Identification of sialic acids on the cell surface of Candida albicans. Biochim Biophys Acta. 2000; 1474(2):262–8. https://doi.org/10.1016/S0304-4165(00)00003-9