The role of sequence elements and protein interactions in focal subcellular localization of Sortase A in Enterococcus faecalis

Sortase A (SrtA) is a transmembrane protein responsible for covalently anchoring several virulence factors and adhesins to the cell wall of Gram-positive organisms, including Enterococci. In E. faecalis, SrtA localizes to single foci at the septum during early division phases and reorients to multip...

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Main Author: Mitra, Sumitra Debina
Other Authors: Kimberly Kline
Format: Theses and Dissertations
Language:English
Published: 2018
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Online Access:http://hdl.handle.net/10356/73628
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Institution: Nanyang Technological University
Language: English
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spelling sg-ntu-dr.10356-736282023-02-28T18:32:22Z The role of sequence elements and protein interactions in focal subcellular localization of Sortase A in Enterococcus faecalis Mitra, Sumitra Debina Kimberly Kline School of Biological Sciences DRNTU::Science::Biological sciences Sortase A (SrtA) is a transmembrane protein responsible for covalently anchoring several virulence factors and adhesins to the cell wall of Gram-positive organisms, including Enterococci. In E. faecalis, SrtA localizes to single foci at the septum during early division phases and reorients to multiple foci at sites of nascent cell division during later stages of the cell cycle. Structurally, SrtA consists of an N-terminal positively charged cytoplasmic tail, a single transmembrane helix (TMH), and a C-terminal catalytic domain. In this thesis, we identify factors that govern the localization of SrtA in E. faecalis. We carried out alanine scan mutagenesis to identify important residues on the cytoplasmic tail and TMH region that are important for focal localization (fused to GST) and identified seven individual mutations that resulted in mislocalization of GST. While these amino acids, individually, did not perturb localization in full-length SrtA, the Asn residue at position 31 on the TMH showed a defect in SrtA substrate attachment to the cell wall. We also identified novel interacting partners of SrtA through crosslinking and co-immunoprecipitation. Mass spectrometry analysis revealed putative interacting proteins including chaperone proteins DnaK, GroEL, and HtrA; cell division protein DivIVA; signal recognition particle receptor FtsY; and cell wall machinery protein FtsI. We validated the interactions in vivo and identified DnaK and FtsY to be the strongest interacting partners of SrtA. We further demonstrate that in a DnaK mutant background, SrtA is mislocalized to one half of the cell in the mid-division growth phase suggesting that DnaK governs SrtA in a cell cycle dependent manner. Together these findings suggest that SrtA localization to distinct foci in E. faecalis may be governed, independently or in conjunction, by sequence elements and multiple protein-protein interactions at the septum of the cell. ​Doctor of Philosophy (SBS) 2018-04-02T06:57:05Z 2018-04-02T06:57:05Z 2018 Thesis Mitra, S. D. (2018). The role of sequence elements and protein interactions in focal subcellular localization of Sortase A in Enterococcus faecalis. Doctoral thesis, Nanyang Technological University, Singapore. http://hdl.handle.net/10356/73628 10.32657/10356/73628 en 112 p. application/pdf
institution Nanyang Technological University
building NTU Library
continent Asia
country Singapore
Singapore
content_provider NTU Library
collection DR-NTU
language English
topic DRNTU::Science::Biological sciences
spellingShingle DRNTU::Science::Biological sciences
Mitra, Sumitra Debina
The role of sequence elements and protein interactions in focal subcellular localization of Sortase A in Enterococcus faecalis
description Sortase A (SrtA) is a transmembrane protein responsible for covalently anchoring several virulence factors and adhesins to the cell wall of Gram-positive organisms, including Enterococci. In E. faecalis, SrtA localizes to single foci at the septum during early division phases and reorients to multiple foci at sites of nascent cell division during later stages of the cell cycle. Structurally, SrtA consists of an N-terminal positively charged cytoplasmic tail, a single transmembrane helix (TMH), and a C-terminal catalytic domain. In this thesis, we identify factors that govern the localization of SrtA in E. faecalis. We carried out alanine scan mutagenesis to identify important residues on the cytoplasmic tail and TMH region that are important for focal localization (fused to GST) and identified seven individual mutations that resulted in mislocalization of GST. While these amino acids, individually, did not perturb localization in full-length SrtA, the Asn residue at position 31 on the TMH showed a defect in SrtA substrate attachment to the cell wall. We also identified novel interacting partners of SrtA through crosslinking and co-immunoprecipitation. Mass spectrometry analysis revealed putative interacting proteins including chaperone proteins DnaK, GroEL, and HtrA; cell division protein DivIVA; signal recognition particle receptor FtsY; and cell wall machinery protein FtsI. We validated the interactions in vivo and identified DnaK and FtsY to be the strongest interacting partners of SrtA. We further demonstrate that in a DnaK mutant background, SrtA is mislocalized to one half of the cell in the mid-division growth phase suggesting that DnaK governs SrtA in a cell cycle dependent manner. Together these findings suggest that SrtA localization to distinct foci in E. faecalis may be governed, independently or in conjunction, by sequence elements and multiple protein-protein interactions at the septum of the cell.
author2 Kimberly Kline
author_facet Kimberly Kline
Mitra, Sumitra Debina
format Theses and Dissertations
author Mitra, Sumitra Debina
author_sort Mitra, Sumitra Debina
title The role of sequence elements and protein interactions in focal subcellular localization of Sortase A in Enterococcus faecalis
title_short The role of sequence elements and protein interactions in focal subcellular localization of Sortase A in Enterococcus faecalis
title_full The role of sequence elements and protein interactions in focal subcellular localization of Sortase A in Enterococcus faecalis
title_fullStr The role of sequence elements and protein interactions in focal subcellular localization of Sortase A in Enterococcus faecalis
title_full_unstemmed The role of sequence elements and protein interactions in focal subcellular localization of Sortase A in Enterococcus faecalis
title_sort role of sequence elements and protein interactions in focal subcellular localization of sortase a in enterococcus faecalis
publishDate 2018
url http://hdl.handle.net/10356/73628
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