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Glycine and its N-methylated analogues cause pH-dependent membrane damage to enterotoxigenic Escherichia coli

Donna Vanhauteghem UGent, Geert Janssens UGent, Angelo Lauwaerts, Stanislas Sys UGent, Filip Boyen UGent, Isabelle Kalmar and Evelyne Meyer UGent (2011) Cytométrie/Cytometry 2011 : livre des résumés.
abstract
The current study investigates the emulsifying potential of glycine and its N-methylated derivatives N-methylglycine (sarcosine), N,N-dimethylglycine (DMG) and N,N,N-trimethylglycine (betaine) under varying pH conditions. Subsequently, the effect of these test compounds on the membrane integrity of enterotoxigenic Escherichia coli (ETEC) was evaluated. Oil in water emulsions show that DMG is a more potent enhancer of emulsification than glycine, sarcosine and betaine. Flow cytometry was used to investigate whether this emulsifying potential is associated with an effect on ETEC membrane integrity. Bacteria were exposed to each of the test compounds under varying pH conditions and membrane integrity was assessed using the LIVE/DEAD BacLight kit. This kit consists of two nucleic acid stains: green fluorescent SYTO 9 is cell-permeable and can freely enter all ETEC, either live or dead, and red fluorescent propidium iodide (PI) can only enter membrane-comprised cells. Membrane integrity analysis with SYTO 9/PI revealed a unique fluorescence pattern directly related to the degree of membrane damage. Three bacterial subpopulations could be identified: live, intermediate and dead. With increasing membrane permeability there was a shift in green and red fluorescence. These data clearly show pH- and time-dependent ETEC membrane deterioration due to exposure to glycine, sarcosine and DMG, but not betaine, with an onset at pH 9.0. Conventional plate counts confirmed concomitant changes in culturability of the membrane comprised bacteria. The decrease in number of culturable bacteria when incubated with glycine, sarcosine or DMG at high alkalinity follows the same trend as the decrease in the percentage of live bacteria shown by flow cytometry. However, in the samples incubated for 20h at pH 10.0 with each of these three test compounds the portion of live bacteria is <1%, while there are still culturable ETEC counted after plating. This observation suggests that the subpopulation of intermediate bacteria is heterogeneous consisting of culturable and non-culturable ETEC, with varying degrees of membrane damage. This heterogeneity confirms the presence of various viability states within this intermediate population. In conclusion, the current results show a decrease in ETEC viability due to membrane damage caused by glycine, sarcosine and DMG under alkaline stress conditions, while only DMG could by identified as a weak enhancer of emulsification. In contrast, no emulsifying nor membrane altering properties could be established for betaine. Even though the pH-dependent membrane altering effect of glycine, sarcosine and DMG on ETEC is clearly demonstrated, the underlying mechanism remains to be elucidated
Please use this url to cite or link to this publication:
author
organization
year
type
conference
publication status
published
subject
keyword
Escherichia coli, glycine, membrane permeability, flow cytometry, bacterial viability, sarcosine, DMG, betaine
in
Cytométrie/Cytometry 2011 : livre des résumés
publisher
Association française de Cytométrie (AFC)
place of publication
Pont-Evêque, France
conference name
15e Congrès annuel de Cytométrie (Cytométrie/Cytometry 2011)
conference location
Paris, France
conference start
2011-10-26
conference end
2011-10-28
language
English
UGent publication?
yes
classification
C3
additional info
oral presentation
id
3263450
handle
http://hdl.handle.net/1854/LU-3263450
date created
2013-06-19 14:11:48
date last changed
2017-01-02 09:53:01
@inproceedings{3263450,
  abstract     = {The current study investigates the emulsifying potential of glycine and its N-methylated derivatives N-methylglycine (sarcosine), N,N-dimethylglycine (DMG) and N,N,N-trimethylglycine (betaine) under varying pH conditions. Subsequently, the effect of these test compounds on the membrane integrity of enterotoxigenic Escherichia coli (ETEC) was evaluated. Oil in water emulsions show that DMG is a more potent enhancer of emulsification than glycine, sarcosine and betaine. Flow cytometry was used to investigate whether this emulsifying potential is associated with an effect on ETEC membrane integrity. Bacteria were exposed to each of the test compounds under varying pH conditions and membrane integrity was assessed using the LIVE/DEAD BacLight kit. This kit consists of two nucleic acid stains:  green fluorescent SYTO 9 is cell-permeable and can freely enter all ETEC, either live or dead, and red fluorescent propidium iodide (PI) can only enter membrane-comprised cells. Membrane integrity analysis with SYTO 9/PI revealed a unique fluorescence pattern directly related to the degree of membrane damage. Three bacterial subpopulations could be identified: live, intermediate and dead. With increasing membrane permeability there was a shift in green and red fluorescence.
These data clearly show pH- and time-dependent ETEC membrane deterioration due to exposure to glycine, sarcosine and DMG, but not betaine, with an onset at pH 9.0. Conventional plate counts confirmed concomitant changes in culturability of the membrane comprised bacteria. The decrease in number of culturable bacteria when incubated with glycine, sarcosine or DMG at high alkalinity follows the same trend as the decrease in the percentage of live bacteria shown by flow cytometry. However, in the samples incubated for 20h at pH 10.0 with each of these three test compounds the portion of live bacteria is {\textlangle}1\%, while there are still culturable ETEC counted after plating. This observation suggests that the subpopulation of intermediate bacteria is heterogeneous consisting of culturable and non-culturable ETEC, with varying degrees of membrane damage. This heterogeneity confirms the presence of various viability states within this intermediate population. In conclusion, the current results show a decrease in ETEC viability due to membrane damage caused by glycine, sarcosine and DMG under alkaline stress conditions, while only DMG could by identified as a weak enhancer of emulsification. In contrast, no emulsifying nor membrane altering properties could be established for betaine. Even though the pH-dependent membrane altering effect of glycine, sarcosine and DMG on ETEC is clearly demonstrated, the underlying mechanism remains to be elucidated},
  author       = {Vanhauteghem, Donna and Janssens, Geert and Lauwaerts, Angelo and Sys, Stanislas and Boyen, Filip and Kalmar, Isabelle and Meyer, Evelyne},
  booktitle    = {Cytom{\'e}trie/Cytometry 2011 : livre des r{\'e}sum{\'e}s},
  keyword      = {Escherichia coli,glycine,membrane permeability,flow cytometry,bacterial viability,sarcosine,DMG,betaine},
  language     = {eng},
  location     = {Paris, France},
  publisher    = {Association fran\c{c}aise de Cytom{\'e}trie (AFC)},
  title        = {Glycine and its N-methylated analogues cause pH-dependent membrane damage to enterotoxigenic Escherichia coli},
  year         = {2011},
}

Chicago
Vanhauteghem, Donna, Geert Janssens, Angelo Lauwaerts, Stanislas Sys, Filip Boyen, Isabelle Kalmar, and Evelyne Meyer. 2011. “Glycine and Its N-methylated Analogues Cause pH-dependent Membrane Damage to Enterotoxigenic Escherichia Coli.” In Cytométrie/Cytometry 2011 : Livre Des Résumés. Pont-Evêque, France: Association française de Cytométrie (AFC).
APA
Vanhauteghem, D., Janssens, G., Lauwaerts, A., Sys, S., Boyen, F., Kalmar, I., & Meyer, E. (2011). Glycine and its N-methylated analogues cause pH-dependent membrane damage to enterotoxigenic Escherichia coli. Cytométrie/Cytometry 2011 : livre des résumés. Presented at the 15e Congrès annuel de Cytométrie (Cytométrie/Cytometry 2011), Pont-Evêque, France: Association française de Cytométrie (AFC).
Vancouver
1.
Vanhauteghem D, Janssens G, Lauwaerts A, Sys S, Boyen F, Kalmar I, et al. Glycine and its N-methylated analogues cause pH-dependent membrane damage to enterotoxigenic Escherichia coli. Cytométrie/Cytometry 2011 : livre des résumés. Pont-Evêque, France: Association française de Cytométrie (AFC); 2011.
MLA
Vanhauteghem, Donna, Geert Janssens, Angelo Lauwaerts, et al. “Glycine and Its N-methylated Analogues Cause pH-dependent Membrane Damage to Enterotoxigenic Escherichia Coli.” Cytométrie/Cytometry 2011 : Livre Des Résumés. Pont-Evêque, France: Association française de Cytométrie (AFC), 2011. Print.