Advanced search
Add to list

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 (UGent) and Evelyne Meyer (UGent)
Author
Organization
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
Keywords
Escherichia coli, glycine, membrane permeability, flow cytometry, bacterial viability, sarcosine, DMG, betaine

Citation

Please use this url to cite or link to this publication:

MLA
Vanhauteghem, Donna, et al. “Glycine and Its N-Methylated Analogues Cause PH-Dependent Membrane Damage to Enterotoxigenic Escherichia Coli.” Cytométrie/Cytometry 2011 : Livre Des Résumés, Association française de Cytométrie (AFC), 2011.
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), Paris, France.
Chicago author-date
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).
Chicago author-date (all authors)
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).
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. In: Cytométrie/Cytometry 2011 : livre des résumés. Pont-Evêque, France: Association française de Cytométrie (AFC); 2011.
IEEE
[1]
D. Vanhauteghem et al., “Glycine and its N-methylated analogues cause pH-dependent membrane damage to enterotoxigenic Escherichia coli,” in Cytométrie/Cytometry 2011 : livre des résumés, Paris, France, 2011.
@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 <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    = {{Cytométrie/Cytometry 2011 : livre des résumés}},
  keywords     = {{Escherichia coli,glycine,membrane permeability,flow cytometry,bacterial viability,sarcosine,DMG,betaine}},
  language     = {{eng}},
  location     = {{Paris, France}},
  publisher    = {{Association française de Cytométrie (AFC)}},
  title        = {{Glycine and its N-methylated analogues cause pH-dependent membrane damage to enterotoxigenic Escherichia coli}},
  year         = {{2011}},
}