Charaterisation [sic] of the Shigella flexneri O antigen polymerase Wzy
| dc.contributor.advisor | Morona, Renato | |
| dc.contributor.author | Nath, Pratiti | |
| dc.contributor.school | School of Biological Sciences | en |
| dc.date.issued | 2015 | |
| dc.description.abstract | Shigella flexneri is the major causative agent of shigellosis that account for ~14000 deaths annually in Asia. The O antigen (Oag) component of S. flexneri lipopolysaccharide (LPS) is important for virulence and a protective antigen. It is synthesised by a Wzy-dependent mechanism. S. flexneri Wzy (Wzyᵴᵳ) has 12 transmembrane (TM) segments and two large periplasmic loops (PL). The modal chain length of the Oag is determined by chromosomally encoded Wzzᵴᵳ and pHS-2 plasmid encoded WzzpHS2 [pHS2 subscript]. Although Wzyᵴᵳ was identified 20 years ago, there is a lack of knowledge about its functional amino acid residues as Wzyᵴᵳ has low expression and poor detection. Wzyᵴᵳ is thought to interact with Wzzᵴᵳ however, there is no direct evidence on how these two proteins are associated. A wzyᵴᵳ-gfp expression construct (pWaldo-wzyᵴᵳ-TEV-GFP or pRMPN1) was made; it successfully expressed Wzyᵴᵳ-GFP and complemented a wzyᵴᵳ mutant (∆wzy). To identify functionally important amino acid residues in Wzyᵴᵳ, random mutagenesis was performed on the wzyᵴᵳ in pRMPN1, followed by screening with colicin E2. Analysis of the LPS conferred by mutated Wzyᵴᵳ proteins in the ∆wzy strain identified 4 different mutant classes, with mutations found in PL1, 2, 3, and 6; TM2, 4, 5, 7, 8, and 9, and cytoplasmic loop (CL) 1 and CL5. The association of Wzyᵴᵳ and Wzzᵴᵳ was investigated by transforming these mutated wzyᵴᵳ plasmids into a wzyᵴᵳ and wzzᵴᵳ deficient (∆wzy ∆wzz) strain. Comparison of the LPS profiles in the ∆wzy and ∆wzy ∆wzz backgrounds identified Wzyᵴᵳ mutants whose polymerisation activities were Wzzᵴᵳ dependent. Colicin E2 and bacteriophage Sf6c sensitivities were consistent with the LPS profiles. Analysis of the expression levels of the Wzyᵴᵳ-GFP mutants in the ∆wzy and ∆wzy ∆wzz backgrounds identified a role for Wzzᵴᵳ in Wzyᵴᵳ stability. Hence, in addition to its role in regulating Oag modal chain length, Wzzᵴᵳ also affects Wzyᵴᵳ activity and stability. Site-directed mutagenesis was performed on wzyᵴᵳ in pRMPN1 to alter Arg residues in Wzyᵴᵳ’s two large PLs (3 and 5) to Ala. Analysis of the LPS profiles conferred by mutated Wzyᵴᵳ proteins in the ∆wzy strain identified residues that affect Wzyᵴᵳ activity. The importance of the guanidium group of the Arg residues was investigated by altering the Arg residues to Lys and Glu, which generated Wzyᵴᵳ mutants conferring altered LPS Oag modal chain lengths. The dependence of these Wzyᵴᵳ mutants on Wzzᵴᵳ was investigated by expressing them in the ∆wzy ∆wzz strain. Comparison of the LPS profiles identified a role for the Arg residues in the association of Wzyᵴᵳ and Wzzᵴᵳ. Comparison of the expression levels of different mutant Wzyᵴᵳ-GFPs with the wild-type Wzyᵴᵳ-GFP showed that certain Arg residues affected production levels of Wzyᵴᵳ in a Wzzᵴᵳ-dependent manner. Wzyᵴᵳ-GFP-His8 was purified by affinity chromatography and I propose that Wzyᵴᵳ may form dimers. The negative dominance study suggested that the dimer formation may be not essential for functioning of Wzyᵴᵳ. In vivo crosslinking was performed in a ∆wzy strain carrying plasmids encoding His-tagged Wzyᵴᵳ and untagged Wzzᵴᵳ. In vivo crosslinking was followed by affinity purification of Wzyᵴᵳ, and Western immunoblotting with Wzzᵴᵳ antibody detected the co-purification of Wzzᵴᵳ. This was also supported by mass spectrometry analysis and provided the first report of complex formation between Wzyᵴᵳ and Wzzᵴᵳ. The Wzyᵴᵳ mutants (WzyᵴᵳR164A, WzyᵴᵳV92M, WzyᵴᵳY137H, and WzyᵴᵳR250K) having Wzz dependent activity were still able to form complexes with Wzzᵴᵳ which suggested that although their activity is Wzz-dependent, the mutational alterations do not affect the interaction of Wzyᵴᵳ with Wzzᵴᵳ. Thus the interaction may involve many regions of Wzyᵴᵳ. This thesis identified and characterised functionally important amino acid residues of Wzyᵴᵳ, identified several novel LPS phenotypes conferred by the Wzyᵴᵳ mutants, and found that Wzzᵴᵳ affects the functioning and stability of Wzyᵴᵳ, both positively and negatively. The work also first time identified direct physical interaction of Wzzᵴᵳ and Wzyᵴᵳ, and developed a purification method for Wzyᵴᵳ. Finally, I proposed a model of Wzy-dependent Oag polymerisation through the interaction of Wzy with Wzz. | en |
| dc.description.dissertation | Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, School of Biological Sciences, 2015. | en |
| dc.identifier.uri | http://hdl.handle.net/2440/101621 | |
| dc.provenance | This electronic version is made publicly available by the University of Adelaide in accordance with its open access policy for student theses. Copyright in this thesis remains with the author. This thesis may incorporate third party material which has been used by the author pursuant to Fair Dealing exceptions. If you are the owner of any included third party copyright material you wish to be removed from this electronic version, please complete the take down form located at: http://www.adelaide.edu.au/legals | en |
| dc.subject | Shigella flexneri | en |
| dc.subject | lipopolysaccharide (LPS) | en |
| dc.subject | O antigen | en |
| dc.subject | Wzy | en |
| dc.subject | Wzz | en |
| dc.subject | protein purification | en |
| dc.subject | mutagenesis | en |
| dc.subject | cross-linking | en |
| dc.subject | colicin | en |
| dc.subject | bacteriophage | en |
| dc.title | Charaterisation [sic] of the Shigella flexneri O antigen polymerase Wzy | en |
| dc.type | Theses | en |
Files
Original bundle
1 - 3 of 3
No Thumbnail Available
- Name:
- Restricted
- Size:
- 81.33 MB
- Format:
- Adobe Portable Document Format
- Description:
- Library staff access only