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Role of glycolipids in the pathogenesis of Enterococcus faecalis urinary tract infection.

Diederich AK, Wobser D, Spiess M, Sava IG, Huebner J, Sakιnç T - PLoS ONE (2014)

Bottom Line: We also demonstrated that these mutants showed a more than three-fold increased binding to human bladder carcinoma cells line T24 compared to the wild-type strain.Lipoteichoic acid (LTA), wall-teichoic acid (WTA), and glycosaminoglycans (GAGs) were not significantly involved in binding of E. faecalis to the bladder epithelial cell line.Further experiments will be needed to clarify the exact mechanism underlying the adhesion through glycolipids and their cognate receptors.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases, University Hospital Freiburg, Freiburg, Germany.

ABSTRACT

Background: After uropathogenic Escherichia coli (UPEC), Enterococcus faecalis is the second most common pathogen causing urinary tract infections. Monoglucosyl-diacylglycerol (MGlcDAG) and diglucosyl-diacylglycerol (DGlcDAG) are the main glycolipids of the E. faecalis cell membrane. Examination of two mutants in genes bgsB and bgsA (both glycosyltransferases) showed that these genes are involved in cell membrane glycolipid biosynthesis, and that their inactivation leads to loss of glycolipids DGlcDAG (bgsA) or both MGlcDAG and DGlcDAG (bgsB). Here we investigate the function of bgsB and bgsA regarding their role in the pathogenesis in a mouse model of urinary tract infection and in bacterial adhesion to T24 bladder epithelial cells.

Results: In a mouse model of urinary tract infection, we showed that E. faecalis 12030ΔbgsB and E. faecalis 12030ΔbgsA mutants, colonize uroepithelial surfaces more efficiently than wild-type bacteria. We also demonstrated that these mutants showed a more than three-fold increased binding to human bladder carcinoma cells line T24 compared to the wild-type strain. Bacterial binding could be specifically inhibited by purified glycolipids. Lipoteichoic acid (LTA), wall-teichoic acid (WTA), and glycosaminoglycans (GAGs) were not significantly involved in binding of E. faecalis to the bladder epithelial cell line.

Conclusions: Our data show that the deletion of bgsB and bgsA and the absence of the major glycolipid diglucosyl-diacylglycerol increases colonization and binding to uroepithelial cells. We hypothesize that secreted diglucosyl-diacylglycerol blocks host binding sites, thereby preventing bacterial adhesion. Further experiments will be needed to clarify the exact mechanism underlying the adhesion through glycolipids and their cognate receptors.

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Attachment to T24 cells.(A) E. faecalis 12030 wild-type (white bar), E. faecalis 12030ΔbgsB mutant (grey bar), reconstituted E. faecalis 12030ΔbgsB mutant (grey squared bar), E. faecalis 12030ΔbgsA mutant (black bar) and reconstituted E. faecalis 12030ΔbgsA mutant (black squared bar) were tested for their ability to bind to T24 cells. T24 cells were incubated for 2 hours with bacteria grown to mid-log phase at a bacteria-to-cell ratio of 100∶1. Total cell-associated bacteria include surface-adherent and intracellular bacteria. Both mutants showed clearly increased binding compared to the wild-type strain and the reconstituted mutant showed reduce binding compared to the mutants. Data represent the means with standard error of the mean (SEM). B) Inhibition of bacterial attachment to T24 cells using glycolipids. E. faecalis 12030 wild-type (a, white bar), E. faecalis 12030ΔbgsB mutant (b, grey bar) and E. faecalis 12030ΔbgsA mutant (c, black bar) were tested. T24 cells were incubated with glycolipids (M: MGlyDAG, D: DGlyDAG and DM: mixture of MGlyDAG and DGlyDAG) for 30 min before addition of bacteria to assess attachment. Each glycolipid alone and the mixture of both significantly reduced bacterial binding using ANOVA with Bonferroni's multiple comparison test. For all inhibitions using glycolipids, differences were statistically significant at p<0.001. Bars represent average ± S.E.
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pone-0096295-g001: Attachment to T24 cells.(A) E. faecalis 12030 wild-type (white bar), E. faecalis 12030ΔbgsB mutant (grey bar), reconstituted E. faecalis 12030ΔbgsB mutant (grey squared bar), E. faecalis 12030ΔbgsA mutant (black bar) and reconstituted E. faecalis 12030ΔbgsA mutant (black squared bar) were tested for their ability to bind to T24 cells. T24 cells were incubated for 2 hours with bacteria grown to mid-log phase at a bacteria-to-cell ratio of 100∶1. Total cell-associated bacteria include surface-adherent and intracellular bacteria. Both mutants showed clearly increased binding compared to the wild-type strain and the reconstituted mutant showed reduce binding compared to the mutants. Data represent the means with standard error of the mean (SEM). B) Inhibition of bacterial attachment to T24 cells using glycolipids. E. faecalis 12030 wild-type (a, white bar), E. faecalis 12030ΔbgsB mutant (b, grey bar) and E. faecalis 12030ΔbgsA mutant (c, black bar) were tested. T24 cells were incubated with glycolipids (M: MGlyDAG, D: DGlyDAG and DM: mixture of MGlyDAG and DGlyDAG) for 30 min before addition of bacteria to assess attachment. Each glycolipid alone and the mixture of both significantly reduced bacterial binding using ANOVA with Bonferroni's multiple comparison test. For all inhibitions using glycolipids, differences were statistically significant at p<0.001. Bars represent average ± S.E.

Mentions: E. faecalis 12030ΔbgsB and E. faecalis 12030ΔbgsA mutants were investigated for their binding to T24 bladder carcinoma cell line. These mutants showed a more than three-fold increased binding (2.6×106 CFU/mL and 2.3×106 CFU/mL) to the T24 human bladder carcinoma cell line compared to wild-type bacteria (0.7×106 CFU/mL, Figure 1A). The increased attachment was significantly lower (more than 50%) with the complemented strains of both mutants E. faecalis 12030ΔbgsB rec and E. faecalis 12030ΔbgsA rec (1.1×106 CFU/mL and 0.8×106 CFU/mL, Figure 1A).


Role of glycolipids in the pathogenesis of Enterococcus faecalis urinary tract infection.

Diederich AK, Wobser D, Spiess M, Sava IG, Huebner J, Sakιnç T - PLoS ONE (2014)

Attachment to T24 cells.(A) E. faecalis 12030 wild-type (white bar), E. faecalis 12030ΔbgsB mutant (grey bar), reconstituted E. faecalis 12030ΔbgsB mutant (grey squared bar), E. faecalis 12030ΔbgsA mutant (black bar) and reconstituted E. faecalis 12030ΔbgsA mutant (black squared bar) were tested for their ability to bind to T24 cells. T24 cells were incubated for 2 hours with bacteria grown to mid-log phase at a bacteria-to-cell ratio of 100∶1. Total cell-associated bacteria include surface-adherent and intracellular bacteria. Both mutants showed clearly increased binding compared to the wild-type strain and the reconstituted mutant showed reduce binding compared to the mutants. Data represent the means with standard error of the mean (SEM). B) Inhibition of bacterial attachment to T24 cells using glycolipids. E. faecalis 12030 wild-type (a, white bar), E. faecalis 12030ΔbgsB mutant (b, grey bar) and E. faecalis 12030ΔbgsA mutant (c, black bar) were tested. T24 cells were incubated with glycolipids (M: MGlyDAG, D: DGlyDAG and DM: mixture of MGlyDAG and DGlyDAG) for 30 min before addition of bacteria to assess attachment. Each glycolipid alone and the mixture of both significantly reduced bacterial binding using ANOVA with Bonferroni's multiple comparison test. For all inhibitions using glycolipids, differences were statistically significant at p<0.001. Bars represent average ± S.E.
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pone-0096295-g001: Attachment to T24 cells.(A) E. faecalis 12030 wild-type (white bar), E. faecalis 12030ΔbgsB mutant (grey bar), reconstituted E. faecalis 12030ΔbgsB mutant (grey squared bar), E. faecalis 12030ΔbgsA mutant (black bar) and reconstituted E. faecalis 12030ΔbgsA mutant (black squared bar) were tested for their ability to bind to T24 cells. T24 cells were incubated for 2 hours with bacteria grown to mid-log phase at a bacteria-to-cell ratio of 100∶1. Total cell-associated bacteria include surface-adherent and intracellular bacteria. Both mutants showed clearly increased binding compared to the wild-type strain and the reconstituted mutant showed reduce binding compared to the mutants. Data represent the means with standard error of the mean (SEM). B) Inhibition of bacterial attachment to T24 cells using glycolipids. E. faecalis 12030 wild-type (a, white bar), E. faecalis 12030ΔbgsB mutant (b, grey bar) and E. faecalis 12030ΔbgsA mutant (c, black bar) were tested. T24 cells were incubated with glycolipids (M: MGlyDAG, D: DGlyDAG and DM: mixture of MGlyDAG and DGlyDAG) for 30 min before addition of bacteria to assess attachment. Each glycolipid alone and the mixture of both significantly reduced bacterial binding using ANOVA with Bonferroni's multiple comparison test. For all inhibitions using glycolipids, differences were statistically significant at p<0.001. Bars represent average ± S.E.
Mentions: E. faecalis 12030ΔbgsB and E. faecalis 12030ΔbgsA mutants were investigated for their binding to T24 bladder carcinoma cell line. These mutants showed a more than three-fold increased binding (2.6×106 CFU/mL and 2.3×106 CFU/mL) to the T24 human bladder carcinoma cell line compared to wild-type bacteria (0.7×106 CFU/mL, Figure 1A). The increased attachment was significantly lower (more than 50%) with the complemented strains of both mutants E. faecalis 12030ΔbgsB rec and E. faecalis 12030ΔbgsA rec (1.1×106 CFU/mL and 0.8×106 CFU/mL, Figure 1A).

Bottom Line: We also demonstrated that these mutants showed a more than three-fold increased binding to human bladder carcinoma cells line T24 compared to the wild-type strain.Lipoteichoic acid (LTA), wall-teichoic acid (WTA), and glycosaminoglycans (GAGs) were not significantly involved in binding of E. faecalis to the bladder epithelial cell line.Further experiments will be needed to clarify the exact mechanism underlying the adhesion through glycolipids and their cognate receptors.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases, University Hospital Freiburg, Freiburg, Germany.

ABSTRACT

Background: After uropathogenic Escherichia coli (UPEC), Enterococcus faecalis is the second most common pathogen causing urinary tract infections. Monoglucosyl-diacylglycerol (MGlcDAG) and diglucosyl-diacylglycerol (DGlcDAG) are the main glycolipids of the E. faecalis cell membrane. Examination of two mutants in genes bgsB and bgsA (both glycosyltransferases) showed that these genes are involved in cell membrane glycolipid biosynthesis, and that their inactivation leads to loss of glycolipids DGlcDAG (bgsA) or both MGlcDAG and DGlcDAG (bgsB). Here we investigate the function of bgsB and bgsA regarding their role in the pathogenesis in a mouse model of urinary tract infection and in bacterial adhesion to T24 bladder epithelial cells.

Results: In a mouse model of urinary tract infection, we showed that E. faecalis 12030ΔbgsB and E. faecalis 12030ΔbgsA mutants, colonize uroepithelial surfaces more efficiently than wild-type bacteria. We also demonstrated that these mutants showed a more than three-fold increased binding to human bladder carcinoma cells line T24 compared to the wild-type strain. Bacterial binding could be specifically inhibited by purified glycolipids. Lipoteichoic acid (LTA), wall-teichoic acid (WTA), and glycosaminoglycans (GAGs) were not significantly involved in binding of E. faecalis to the bladder epithelial cell line.

Conclusions: Our data show that the deletion of bgsB and bgsA and the absence of the major glycolipid diglucosyl-diacylglycerol increases colonization and binding to uroepithelial cells. We hypothesize that secreted diglucosyl-diacylglycerol blocks host binding sites, thereby preventing bacterial adhesion. Further experiments will be needed to clarify the exact mechanism underlying the adhesion through glycolipids and their cognate receptors.

Show MeSH
Related in: MedlinePlus