RAMAN MICROSPECTROSCOPY OF THE YEAST VACUOLES

 

L. Bednárová2, Š. Gregorová1, V. Bauerová2, O. Hrušková-Heidingsfeldová2,
J. Palacký1 and P. Mojzeš1

 

1Institute of Physics, Charles University in Prague, Prague, Czech Republic

2Institute of Organic Chemistry and Biochemistry, AS CR, Prague, Czech Republic

 


Extensive effort was undertaken recently to understand pathogenicity and virulence of some Candida species [1]. Their ability to escape immune defense of the host and survive even under the severe nutrition limitations and in the presence of various stress factors or antimycotics seems to be related, among others, to preservation of their vacuolar functions. Non-invasive methods providing information about chemical composition of the vacuoles within living cells exposed to various external factors are thus of great importance for development of novel antifungal strategies.

Especially, concentration and structural properties of polyphosphates accumulated in the yeast vacuoles are of particular interest, since they are sensitive to nutritional limitations and environmental stresses, and can thus reflect physiological state of the cell [2]. Recently, Raman microspectroscopy was suggested as practical alternative to laborious and time-consuming chemical analysis of intracellular polyphosphate inclusions in polyphosphate-accumulating bacteria [3]. In the present work, the method was used to monitor polyphosphates and other chemical compounds dissolved in the vacuoles of living Candida yeasts of different physiological state and/or under various stress conditions. It was shown that using proper immobilization protocols, the spatially-resolved Raman spectra from individual yeasts throughout statistically sound sets of living cells can be collected routinely. Abundance distributions of actual concentration and polymerization degree of polyphosphates can be assessed employing advanced multivariate methods for spectral analysis of structurally sensitive polyphosphate bands (~688 and ~1154 cm-1) properly normalized with respect to the water OH stretches at 3400 cm-1 [4].


Ministry of Education of the Czech Republic (MSM0021620835) and the Grant Agency of the Czech Republic (P208/10/0376) are acknowledged for financial support.

[1] B. Hube, Curr. Opin. Microbiol. 7 (2004) 336.

[2] J. W. McGrath, J. P. Quinn, Appl. Environ. Microbiol. 66 (2000) 4068.

[3] N. Majed, C. Matthäus, M. Diem M, A. Z. Gu, Environ. Sci. Technol. 43 (2009) 5436.

[4] J. Palacký, P. Mojzeš, J. Bok, J. Raman Spectrosc. 42 (2011) 1528.