The biological role and structure function analyses of O-antigen modifying enzymes in the bacterial pathogen Salmonella
Lead Research Organisation:
University of York
Department Name: Biology
Abstract
Background: Many Gram negative bacteria modify their cell surface structures including the lipopolysaccharide (LPS) to facilitate immune evasion or directly enhance virulence. In the important foodborne human and livestock pathogen Salmonella enterica we identified a new group of LPS O-antigen modification proteins, in addition to ones known including OafA is a known LPS acetyltransferase. The biochemistry of the larger FamAc3 family of membrane proteins that OafA and other O-antigen acetyltransferases belong to family is poorly understood, and very little is known about the biochemistry and molecular biology leading to acetylation of the O-antigen. The only role attributed to date to O-antigen acetylation is seroconversion and phage sensitivity.
The overall objective is to elucidate the role and function of O-antigen acetyltransferases in Salmonella. This will be addressed in three Specific Aims.
1. Identify domains and motifs that are essential for protein function by generating a panel of mutants (evolved, directed), and assessing protein function using a suite of in vitro and in vivo assays.
2. Establish the significance of this O-antigen modification for virulence in a tissue culture model.
3. Examine the mechanism of action by purifying and solubilising representative members of these membrane proteins (or domains there of), and use these to identify the donor for the acetyl moiety. Attempt crystallography using traditional as well as lipidic cubic phase technology.
Novelty and Timeliness: This work will feed into a current and ongoing international effort to develop Salmonella LPS based vaccines, and local expertise on Salmonella and membrane protein biochemistry. We have assays for functionality and have cloned and expressed both proteins, facilitating progress. The student will receive broad training across disciplines and use a wide range of techniques. In general, membrane proteins are under-studied, and the project will further York's expertise and leadership in this area, will establish new collaborations, and will benefit from the novel lipidic cubic phase setup in YSBL.
Key Manuscript underpinning this project: Kintz et al, Mol Microbiol. 2015 Apr;96(2):263-75. doi: 10.1111/mmi.12933.
The overall objective is to elucidate the role and function of O-antigen acetyltransferases in Salmonella. This will be addressed in three Specific Aims.
1. Identify domains and motifs that are essential for protein function by generating a panel of mutants (evolved, directed), and assessing protein function using a suite of in vitro and in vivo assays.
2. Establish the significance of this O-antigen modification for virulence in a tissue culture model.
3. Examine the mechanism of action by purifying and solubilising representative members of these membrane proteins (or domains there of), and use these to identify the donor for the acetyl moiety. Attempt crystallography using traditional as well as lipidic cubic phase technology.
Novelty and Timeliness: This work will feed into a current and ongoing international effort to develop Salmonella LPS based vaccines, and local expertise on Salmonella and membrane protein biochemistry. We have assays for functionality and have cloned and expressed both proteins, facilitating progress. The student will receive broad training across disciplines and use a wide range of techniques. In general, membrane proteins are under-studied, and the project will further York's expertise and leadership in this area, will establish new collaborations, and will benefit from the novel lipidic cubic phase setup in YSBL.
Key Manuscript underpinning this project: Kintz et al, Mol Microbiol. 2015 Apr;96(2):263-75. doi: 10.1111/mmi.12933.
Organisations
Publications
Pearson C
(2022)
Diverse functions for acyltransferase-3 proteins in the modification of bacterial cell surfaces.
in Microbiology (Reading, England)
Studentship Projects
| Project Reference | Relationship | Related To | Start | End | Student Name |
|---|---|---|---|---|---|
| BB/M011151/1 | 01/10/2015 | 30/09/2023 | |||
| 1643733 | Studentship | BB/M011151/1 | 01/10/2015 | 30/09/2019 |
| Description | This work has made significant steps towards characterising the mechanism of action of O-antigen modifying enzymes in Salmonella. Its has infomed a refined hypothesis of the steps involved in donor substrate interaction and transfer of acyl groups to the acceptor substrate. Optimisation of in situ functional assays for these enzmes has greatly improved the scope for further characterisation of these proteins which are found accross a range of gram positive and gram negative bacteria and play roles in diverse processes, from antigenic vairation to initiation of symbiosis. |
| Exploitation Route | The experimental techniques optimised during this research, including optimisation of in situ functional assays, provide a basis for further characterisation of other related proteins. |
| Sectors | Manufacturing, including Industrial Biotechology |
| Description | Pint of Science - Showcase reasearch to the local community |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Public/other audiences |
| Results and Impact | I was involved in identifying and recruiting inspirational speakers and supporting them in the lead up to the Pint of Science event around the topic of 'Our Body'. I was responsible for hosting the speakers during the event, resourcing and providing engaging activities to entertain the audience during session breaks as well as promotion and administration before the event. This sparked interest from the audience about the research that is going on at the university of york and how it relates to the real world. |
| Year(s) Of Engagement Activity | 2018 |
| URL | https://pintofscience.co.uk/ |
| Description | Zombies in York - Infectious disease related outreach activity |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Public/other audiences |
| Results and Impact | Zombies in York is aimed at getting children to learn about infectious diseases by 'curing' a zombie outbreak. They study case files to identify the source of the zombie outbreak, the symptoms shown by infected zombies, and analyse organs and blood samples following a 'live' autopsy to determine the cause of the disease. The objective of the workshop is to teach the difference between viral and bacterial infections, how quickly they can be spread and different routes of transmission, and when antibiotics should and shouldn't be used. Three sessions were run throughout 2017 13 & 14 June and 30 October with an average of 20 children per session. The workshops proved a success in teaching children about infectious disease as they were able to select the correct treatment to cure the zombie outbreak at the end of the workshop. There were also many enthusiastic questions about how to track spread of infection and how it should be contained. |
| Year(s) Of Engagement Activity | 2017 |