Spatial and temporal localization of cell wall associated pili in Enterococcus Faecalis

Enterococcus faecalis relies upon a number of cell wall-associated proteins for virulence. One sortase-assembled virulence factor is the endocarditis and biofilm associated pilus (Ebp), an important factor for biofilm formation. The current paradigm for virulence factor assembly in Gram-positive bac...

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Main Author: Choo, Pei Yi
Other Authors: Kimberly Kline
Format: Thesis-Master by Research
Language:English
Published: Nanyang Technological University 2021
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Online Access:https://hdl.handle.net/10356/145650
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spelling sg-ntu-dr.10356-1456502023-02-28T18:51:32Z Spatial and temporal localization of cell wall associated pili in Enterococcus Faecalis Choo, Pei Yi Kimberly Kline School of Biological Sciences Singapore Centre for Environmental Life Sciences and Engineering (SCELSE) KKline@ntu.edu.sg Science::Biological sciences Enterococcus faecalis relies upon a number of cell wall-associated proteins for virulence. One sortase-assembled virulence factor is the endocarditis and biofilm associated pilus (Ebp), an important factor for biofilm formation. The current paradigm for virulence factor assembly in Gram-positive bacteria is that Sortase A recognizes and cleaves at the LPXTG motif within its substrates and covalently attaches them to the growing cell wall at sites of new cell wall synthesis. While the cell wall anchoring mechanism and polymerization of Ebp is well characterized, less is known about the spatial and temporal deposition of this protein on the cell surface. We followed the distribution of Ebp and peptidoglycan (PG) at different growth stages of E. faecalis via immunofluorescence, along with fluorescent D-amino acids (FDAA) staining. Surprisingly, cell surface Ebp did not co-localize with newly synthesized PG. Instead, surface-anchored Ebp was localized to the cell hemisphere but never at the septum where new cell wall is deposited. In addition, the older hemisphere of mid-division cells was completely saturated with Ebp, while at the newer hemisphere, Ebp appeared as two foci directly adjacent to the newly synthesized PG. When cell wall synthesis was inhibited by ramoplanin, an antibiotic that inhibits lipid II, new Ebp was still deposited at the cell surface. Based on these data, we propose an alternative paradigm for sortase substrate deposition in E. faecalis, in which Ebp can be anchored directly onto uncross-linked cell wall, independent of cell wall synthesis. Master of Science 2021-01-03T11:25:58Z 2021-01-03T11:25:58Z 2020 Thesis-Master by Research Choo, P. Y. (2020). Spatial and temporal localization of cell wall associated pili in Enterococcus Faecalis. Master's thesis, Nanyang Technological University, Singapore. https://hdl.handle.net/10356/145650 10.32657/10356/145650 en This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0). application/pdf Nanyang Technological University
institution Nanyang Technological University
building NTU Library
continent Asia
country Singapore
Singapore
content_provider NTU Library
collection DR-NTU
language English
topic Science::Biological sciences
spellingShingle Science::Biological sciences
Choo, Pei Yi
Spatial and temporal localization of cell wall associated pili in Enterococcus Faecalis
description Enterococcus faecalis relies upon a number of cell wall-associated proteins for virulence. One sortase-assembled virulence factor is the endocarditis and biofilm associated pilus (Ebp), an important factor for biofilm formation. The current paradigm for virulence factor assembly in Gram-positive bacteria is that Sortase A recognizes and cleaves at the LPXTG motif within its substrates and covalently attaches them to the growing cell wall at sites of new cell wall synthesis. While the cell wall anchoring mechanism and polymerization of Ebp is well characterized, less is known about the spatial and temporal deposition of this protein on the cell surface. We followed the distribution of Ebp and peptidoglycan (PG) at different growth stages of E. faecalis via immunofluorescence, along with fluorescent D-amino acids (FDAA) staining. Surprisingly, cell surface Ebp did not co-localize with newly synthesized PG. Instead, surface-anchored Ebp was localized to the cell hemisphere but never at the septum where new cell wall is deposited. In addition, the older hemisphere of mid-division cells was completely saturated with Ebp, while at the newer hemisphere, Ebp appeared as two foci directly adjacent to the newly synthesized PG. When cell wall synthesis was inhibited by ramoplanin, an antibiotic that inhibits lipid II, new Ebp was still deposited at the cell surface. Based on these data, we propose an alternative paradigm for sortase substrate deposition in E. faecalis, in which Ebp can be anchored directly onto uncross-linked cell wall, independent of cell wall synthesis.
author2 Kimberly Kline
author_facet Kimberly Kline
Choo, Pei Yi
format Thesis-Master by Research
author Choo, Pei Yi
author_sort Choo, Pei Yi
title Spatial and temporal localization of cell wall associated pili in Enterococcus Faecalis
title_short Spatial and temporal localization of cell wall associated pili in Enterococcus Faecalis
title_full Spatial and temporal localization of cell wall associated pili in Enterococcus Faecalis
title_fullStr Spatial and temporal localization of cell wall associated pili in Enterococcus Faecalis
title_full_unstemmed Spatial and temporal localization of cell wall associated pili in Enterococcus Faecalis
title_sort spatial and temporal localization of cell wall associated pili in enterococcus faecalis
publisher Nanyang Technological University
publishDate 2021
url https://hdl.handle.net/10356/145650
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