Abstract
In 2016, the first peptide toxin in any human fungal pathogen was identified. It was discovered in Candida albicans and was named candidalysin. Candidalysin is an amphipathic cationic peptide that damages cell membranes. Like most lytic peptides, candidalysin shows alpha-helical secondary structure. As the helicity and the membrane lytic activity of candidalysin are key factors for pathogenicity, here we describe in vitro approaches to monitor both its membrane-lytic function and the secondary structure. First, membrane permeabilization activity of candidalysin is measured in real time by direct electrical recording. Second, the secondary structure and helicity of candidalysin are determined by circular dichroism spectroscopy. These biophysical methods provide a means to characterize the activity and physical properties of candidalysin in vitro and will be useful in determining the structural and functional features of candidalysin and other similar cationic membrane-active peptides.
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References
Jacobsen ID, Wilson D, Wächtler B, Brunke S, Naglik JR, Hube B (2012) Candida albicans dimorphism as a therapeutic target. Expert Rev Anti-Infect Ther 10:85–93
Naglik JR, Challacombe SJ, Hube B (2003) Candida albicans secreted aspartyl proteinases in virulence and pathogenesis. Microbiol Mol Biol Rev 67:400–428
Wächtler B, Wilson D, Haedicke K, Dalle F, Hube B (2011) From attachment to damage: defined genes of Candida albicans mediate adhesion, invasion and damage during interaction with oral epithelial cells. PLoS One 6:e17046
Mogavero S, Sauer FM, Brunke S, Allert S, Schulz D, Wisgott S, Jablonowski N, Elshafee O, Krüger T, Kniemeyer O, Brakhage AA, Naglik JR, Dolk E, Hube B (2021) Candidalysin delivery to the invasion pocket is critical for host epithelial damage induced by Candida albicans. Cell Microbiol 23:e13378
Moyes DL, Wilson D, Richardson JP, Mogavero S, Tang SX, Wernecke J, Höfs S, Gratacap RL, Robbins J, Runglall M, Murciano C, Blagojevic M, Thavaraj S, Förster TM, Hebecker B, Kasper L, Vizcay G, Iancu SI, Kichik N, Häder A, Kurzai O, Luo T, Krüger T, Kniemeyer O, Cota E, Bader O, Wheeler RT, Gutsmann T, Hube B, Naglik JR (2016) Candidalysin is a fungal peptide toxin critical for mucosal infection. Nature 532:64–68
Richardson JP, Mogavero S, Moyes DL, Mariana B, Krüger T, Verma AH, Coleman BM, De La Cruz Diaz J, Schulz D, Ponde NO, Carrano G, Kniemeyer O, Naglik JR (2018) Processing of Candida albicans Ece1p is critical for candidalysin maturation and fungal virulence. mBio 9:e02178-17
Richardson JP, Willems HME, Moyes DL, Shoaie S, Barker KS, Tan L, Palmer GE, Hube B, Naglik JR, Peters BM (2018) Candidalysin drives epithelial signaling, neutrophil recruitment, and immunopathology at the vaginal mucosa. Infect Immun 86:e00645-17
Allert S, Förster TM, Svensson C, Richardson JP, Pawlik T, Hebecker B, Rudolphi S, Juraschitz M, Schaller M, Blagojevic M, Morschhäuser J, Figge T, Jacobsen ID, Naglik JR, Kasper L, Mogavero S (2018) Candida albicans-induced epithelial damage mediates translocation through intestinal barriers. mBio 9:e00915-18
Martins RM, Sforça ML, Amino R, Juliano MA, Oyzama S, Juliano L, Pertinhez TA, Spisni A, Schenkman S (2006) Lytic activity and structural differences of amphipathic peptides derived from trialysin. Biochemistry 45:1765–1774
Sani MA, Separovic F (2016) How membrane-active peptides get into lipid membranes. Acc Chem Res 49:1130–1138
Lee TH, Hofferek V, Separovic F, Reid GE, Aguilar MI (2019) The role of bacterial lipid diversity and membrane properties in modulating antimicrobial peptide activity and drug resistance. Curr Opin Chem Biol 52:85–92
Oren Z, Shai Y (1997) Selective lysis of bacteria but not mammalian cells by diastereomers of melittin: structure-function study. Biochemistry 36:1826–1835
Allende D, Simon SA, McIntosh TJ (2005) Melittin-induced bilayer leakage depends on lipid material properties: evidence for toroidal pores. Biophys J 88:1828–1837
van den Bogaart G, Guzmán JV, Mika JT, Poolman B (2008) On the mechanism of pore formation by melittin. J Biol Chem 283:33854–33857
Lee MT, Sun TL, Hung WC, Huang HW (2013) Process of inducing pores in membranes by melittin. Proc Natl Acad Sci U S A 110:14243–14248
Sani MA, Le Brun AP, Separovic F (1862) The antimicrobial peptide maculatin self assembles in parallel to form a pore in phospholipid bilayers. Biochim Biophys Acta Biomembr 2020:183204
Naglik JR, Gaffen SL, Hube B (2019) Candidalysin: discovery and function in Candida albicans infections. Curr Opin Microbiol 52:100–109
Frey CM, Barth H, Kranz C, Mizaikoff B (2018) Horizontal black lipid bilayer membranes for studying pore-forming toxins. Anal Methods 10:3153–3161
Baaken G, Sondermann M, Schlemmer C, Rühe J, Behrends JC (2008) Planar microelectrode-cavity array for high-resolution and parallel electrical recording of membrane ionic currents. Lab Chip 8:938–944
Austermeier S, Pekmezović M, Porschitz P, Lee S, Kichik N, Moyes DL, Ho J, Kotowicz NK, Naglik JR, Hube B, Gresnigt MS (2021) Albumin neutralizes hydrophobic toxins and modulates Candida albicans pathogenicity. mBio 12:1–17
Greenfield NJ (2007) Using circular dichroism spectra to estimate protein secondary structure. Nat Protoc 1:2876–2890
Micsonai A, Wien F, Kernya L, Lee YH, Goto Y, Réfrégiers M, Kardos J (2015) Accurate secondary structure prediction and fold recognition for circular dichroism spectroscopy. Proc Natl Acad Sci U S A 112:E3095–E3103
Garavito RM, Ferguson-Miller S (2001) Detergents as tools in membrane biochemistry. J Biol Chem 276:32403–32406
Domínguez A, Fernández A, Gonzalez N, Iglesias E, Montenegro L (1997) Determination of critical micelle concentration of some surfactants by three techniques. J Chem Educ 74:1227–1231
Curtis HJ, Cole KS (1942) Membrane resting and action potentials from the squid giant axon. J Cell Physiol 19:135–144
Sikorska E, Wyrzykowski D, Szutkowski K, Greber K, Lubecka EA, Zhukov I (2016) Thermodynamics, size, and dynamics of zwitterionic dodecylphosphocholine and anionic sodium dodecyl sulfate mixed micelles. J Therm Anal Calorim 123:511–523
Acknowledgments
This work was supported by Wellcome Trust Investigator Award 214229_Z_18_Z.
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Lee, S., Kichik, N., Hepworth, O.W., Richardson, J.P., Naglik, J.R. (2022). In Vitro Biophysical Characterization of Candidalysin: A Fungal Peptide Toxin. In: Calderone, R. (eds) Candida Species. Methods in Molecular Biology, vol 2542. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2549-1_12
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DOI: https://doi.org/10.1007/978-1-0716-2549-1_12
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