Molecular Characterization of EspB: A Phospholipid Binding Mycobacterium tuberculosis Virulence Factor
| dc.contributor.advisor | Chen, Jeffrey | |
| dc.contributor.committeeMember | Chelico, Linda | |
| dc.contributor.committeeMember | Stone, Scot | |
| dc.contributor.committeeMember | Bull, Harold | |
| dc.contributor.committeeMember | Koester, Wolfgang | |
| dc.creator | Chung, Beatrice | |
| dc.creator.orcid | 0000-0001-7590-0378 | |
| dc.date.accessioned | 2021-08-20T17:40:57Z | |
| dc.date.available | 2021-08-20T17:40:57Z | |
| dc.date.created | 2021-11 | |
| dc.date.issued | 2021-08-20 | |
| dc.date.submitted | November 2021 | |
| dc.date.updated | 2021-08-20T17:40:57Z | |
| dc.description.abstract | Mycobacterium tuberculosis (M. tb) is the causative agent of tuberculosis (TB), a severe lung infection that causes 1.4 million deaths per year. During infection, M. tb is able to subvert host immune responses and survive within the macrophage by using its type-7 early secreted antigenic target (ESAT-6) system 1 (ESX-1) to secrete virulence protein effectors including EspB. A previously published study showed that EspB has a virulence function distinct from other ESX-1 secreted proteins that may be dependent on phospholipid binding. The objective of this thesis project is to determine the molecular basis of EspB-phospholipid interactions and whether oligomerization of EspB is critical for its interaction with phospholipids and consequently important for its virulence function. Amino acids in EspB predicted to be critical for phospholipid binding (R17, H223, F159, and I246) or oligomerization (W176, K259, and K267) were replaced. Recombinant EspB variants (EspB_R17A/H223A, EspB_F159R/I246R, EspB_W176R, and EspB_K259A/K267A) were produced in Escherichia coli (E. coli) and were assessed for oligomerization and phospholipid binding. Two EspB variants, EspB_R17A/H223A and EspB_F159R/I246R were shown to form hyper-oligomers in native polyacrylamide gel electrophoresis (native-PAGE), dynamic light scattering, and transmission electron microscopy analyses. EspB_W176R and EspB_K259A/K267A did not appear to have significant differences in oligomerization compared to EspBWT. Replacement of the residues R17 and H223 in EspB_R17A/H223A abolished its phospholipid binding ability, whereas the replacement of residues in F159 and I246 in EspB_F159R/I246R appeared to enhance phospholipid binding. EspB_W176R was able to bind phospholipids similarly to EspB_WT and EspB_K259A/K267A was shown to have reduced binding. Addition of purified EspB_R17A/H223A to the M. tb 5’Tn::pe35 strain was able to enhance the induction of THP-1 macrophage cytotoxicity similarly to EspB_WT. The residues R17 and H223 appear to be important for phospholipid binding. However, mutation in predicted critical oligomeric sites of EspB did not have a significant impact on its phospholipid binding ability. Although phospholipid binding ability was predicted to affect the virulence function of EspB, purified EspB_R17A/H223A was still able to induce cytotoxicity in THP-1 macrophages similarly to EspB_WT, suggesting that phospholipid binding may not be the sole factor in EspB virulence. | |
| dc.format.mimetype | application/pdf | |
| dc.identifier.uri | https://hdl.handle.net/10388/13535 | |
| dc.subject | Mycobacterium tuberculosis | |
| dc.subject | bacterial secretion systems | |
| dc.subject | secreted virulence factors | |
| dc.title | Molecular Characterization of EspB: A Phospholipid Binding Mycobacterium tuberculosis Virulence Factor | |
| dc.type | Thesis | |
| dc.type.material | text | |
| thesis.degree.department | Microbiology and Immunology | |
| thesis.degree.discipline | Microbiology and Immunology | |
| thesis.degree.grantor | University of Saskatchewan | |
| thesis.degree.level | Masters | |
| thesis.degree.name | Master of Science (M.Sc.) |