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The F1 chaperone:usher translocon of Yersinia pestis and potential applications

Othman, N. (2017) The F1 chaperone:usher translocon of Yersinia pestis and potential applications. PhD thesis, University of Reading

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Plague is a notifiable disease caused by Yersinia pestis for which there is still no reliable vaccine available. The fraction 1 (F1) antigen forms an immunogenic capsule around the cell surface that decreases efficiency of phagocytosis. Many E. coli strains and other Gramnegative bacteria coat themselves in similar, remarkably stable protein fibres assembled via specific, dedicated chaperone-usher (CU) pathways. Fibres are assembled through a pore within each usher protein, via a cycling of specific chaperone:subunit:usher interactions leading to subunit polymerisation and translocation to the cell surface. This project has used bioinformatics analysis and modelling to improve understanding of details of the Y. pestis F1 chaperone:usher translocon and has applied this knowledge to enhance export of heterologous epitopes. The number of members of the γ3 family of ushers, which assemble simple fibres of one or two subunits, has been expanded and includes for the first time a CU system more closely related to that of the Yersinia Caf system. This plasmid encoded usher from the commensal E. coli SE11 shares 70% identity with Caf1A. Caf1A usher was modelled using Intfold and ITASSER, based on the crystal structures of E. coli FimD and PapC usher in the closed state (PDB: 2VQI, 3.2Å). The complete Caf translocon was modelled based on the open FimD structure (PDB: 3RFZ, 2.8Å) with the Caf1M chaperone:Caf1:Caf1 subunit structure (PDB:1Z9S, 2.2Å) docked in the translocon. Based on the modelled Caf translocon and multiple sequence alignments of related ushers, conserved residues within the β-barrel facing the pore cavity or subunit; residues interacting between the plug and β-barrel in the closed pore and residues interacting between β-barrel and subunit in the ‘open’ translocating pores were identified. These were then tested by mutagenesis for impact on F1 assembly. No single point mutation within the usher abolished F1 assembly. The most profound effect was following mutation of Ser289 to Ala, the level of surface F1 decreased to 70.81% ± 3.0 of levels with wildtype Caf1A. The potential of F1 fibre to act as a carrier of epitopes was tested by replacing 4 loops within Caf1 with either Gly residues or charged residues. The model was used to understand and optimise limitations in export of modified fibres. One permissive site (loop 5) was identified as the optimum site for loop replacement and surface assembly of modified F1 was enhanced by mutation of a clashing Gln in the β-barrel wall. In addition, the study identified a critical role for a conserved Asn, Asn80, within loop 2 of Caf1 subunit that was modelled interacting with Tyr266 in the plug. The permissive loop 5 was also adapted to export a polyhistidine sequence. While 4 His residues were readily accommodated, F1::1His6 conferred toxicity, although there was still evidence of surface polymer. Thus, this study demonstrated flexibility of the F1 CU pathway for surface display of short peptides and the ability to use the translocon model to identify problem areas and repair export. As F1 is considered to be unique to Y. pestis, the presence of a Caf related CU locus in a commensal E. coli was of interest to understanding its phylogenetic relationship to F1 and also in diagnostic applications in plague detection. Bioinformatic analysis and recombinant expression of the E. coli SE11-P2 CU locus, combined with mass spectrometry analysis, confirmed surface assembly of polymers of both subunits from this locus. A purification strategy for isolation of recombinant SE11-P2 polymers was developed. Purified protein will be used in future studies to raise antibody for further study of this CU system and to test for any cross-reaction with Y. pestis F1 antigen, that might potentially interfere with serological tests for Y. pestis. F1 antigen remains a component of interest in both anti-plague vaccine design and plague detection. Results from this study have potential to improve reliability of both approaches to plague control.

Item Type:Thesis (PhD)
Thesis Supervisor:MacIntyre, S. and McGuffin, L.
Thesis/Report Department:School of Biological Sciences
Identification Number/DOI:
Divisions:Life Sciences > School of Biological Sciences > Biomedical Sciences
ID Code:78232


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