Membrane lipids of Enterococcus faecalis as microbial pathogens

Hochschulschrift

Zusammenfassung: Since Gram-positive have superseded Gram-negative bacteria as the most common cause for severe sepsis it is of very high concern to determine the substructures responsible for pathogenicity. Intra-peritoneal infection with an Enterococcus faecalis mutant, depleted of the glycosyltransferase BgsA resulting in accumulation of a monoglycosyl-diacylglycerol (MGlcDAG), was shown to substantially increase mortality in mice. The aim of the study was to analyze what was causing the phenotype. By purification of the accumulating MGlcDAG and subsequent structural analysis, it was found that the glycolipid had a similar structure as virulent glycolipids of other Gram-positive bacteria. However, in vivo experiments in accordance with previous results indicated that the glycolipid is not the substructure of interest. Hence, it was focused next on lipoproteins since they were debated lately to be a major pathogen-associated molecular pattern (PAMP) of Gram-positive bacteria. Indeed, it was observed that by inactivation of the prolipoprotein diacylglycerol transferase (lgt) in ΔbgsA, TNF-α induction in macrophages was abrogated. Here, the results show that oxidation of the thioether by hydrogen peroxide abolished release of TNF-α in macrophages after stimulation with lipoproteins. Additionally, proteomic analysis of the cell membrane surface proteome revealed that 13 of a total of 40 upregulated proteins of E. faecalis ΔbgsA were lipoproteins. Overall, the lipoprotein content was 35.8% in ΔbgsA compared to only 9.4% in wild type bacteria. Lipoprotein extracts from the supernatant and the cell membrane surface of ΔbgsA induced a 3-fold and 10-fold higher production of TNF-α, respectively, compared to the wild type. MS-shotgun analysis of the supernatant extract confirmed that TNF-α induction was caused by an overall upregulation of lipoproteins and not overexpression by a defined lipoprotein. However, stimulation with the purified E. faecalis lipoprotein EF1641 revealed that this lipoprotein is a strong stimulator of TNF-α and was even more potent than SitC, a well-studied lipoprotein from Staphylococcus aureus. As lipids of the cell membrane were shown to be involved in pathogenicity, it was focused next on the role of unsaturated fatty acid synthesis on growth and virulence in E. faecalis. The enzyme isomerase/dehydratase FabN catalyzes the first steps in unsaturated fatty acid synthesis. Deletion resulted in unsaturated fatty acid auxotrophy and could be restored by supplementation with oleic acid or sterile human serum. Nonetheless, in a preparation of MGlcDAG from the fabN mutant the not supplemented, unsaturated cis-vaccenic acid was detected, suggesting another enzyme with low activity was also capable of synthesizing unsaturated fatty acids. Inactivation of fabN in the E. faecalis ΔbgsA mutant slightly decreased induction of TNF-α by the supernatant, but it was still 3-fold more active than the wild type. This indicates that unsaturated fatty acids do not play an important role in the ΔbgsA phenotype. In this thesis, it is altogether shown that lipids of the cell membrane from E. faecalis 12030 play an important role in inflammation and pathogenicity

Object Details

• Location: Deutsche Nationalbibliothek Frankfurt am Main
• Extent: Online-Ressource
• Language: Englisch
• Notes: Albert-Ludwigs-Universität Freiburg, Dissertation, 2015

Classification and Topics

• Keyword: Streptococcus faecalis ; Lipide

Contributors, Places and Time

• Event: Veröffentlichung
• (where): Freiburg
• (who): Universität
• (when): 2016
• Creator: Diederich, Ann-Kristin
• Contributor: Hübner, Johannes

Further information

• DOI: 10.6094/UNIFR/10508
• URN: urn:nbn:de:bsz:25-freidok-105088
• Rights: Der Zugriff auf das Objekt ist unbeschränkt möglich.
• Last update: 14.08.2025, 11:02 AM CEST