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Identification of carbohydrate structures expressed by Mycoplasma hyopneunomiae and M.hyorhinis as potential vaccine antigens


Project Description

The proposed project will advance our understanding of Mycoplasma spp interaction with porcine host cells in the initial step of an infection, and how this knowledge can be used to potentially design carbohydrate-based vaccines. The proposal fits well with the BBSRC priority area “Agriculture and Food Security”. It will underpin long term commercial aims for improved therapeutic and preventative methods to reduce respiratory infectious disease in pigs. Cells in the respiratory tract sense the microenvironment through several types of receptors. Here, C-type lectins receptors (CLRs) recognize and internalize specific carbohydrate antigens. The targeting of these receptors is becoming an efficient strategy for pathogens in the first attachment and subsequent invasion of host cells. This provides a new opportunity to develop carbohydrate-based vaccines, which allow potentially for this first step to be inhibited. Whereas the field of carbohydrate-based vaccines is currently expanding in human medicine, information regarding the presence of CLRs in farm animals is scarce. Understanding the presence of porcine CLRs will enable the development of C-type lectin binding assays to assess the presence of carbohydrates in the membrane of Mycoplasma spp. methods can be established once a carbohydrate recognition domain-Fc library has been developed.
Interestingly, there is strong evidence from publications in the 1970’s that Mycoplasma ssp. express a variety of carbohydrate structures (1-3), containing galactose and glucose units. Such structures have been found in Mycoplasma (M.) pneumoniae, M.mycoides and capri, M.pulmonis, M.gallinarum, and M.gallisepticum, binding for example to surfactant protein A (SP-A), one of the many soluble CLR. (4). Thus, there is a good chance that such structures are also present in the porcine pathogens M.hyorhinis and M.hyopneumoniae, the main causative agents of enzootic pneumonia (EP), which is normally detected at slaughter in 30-80% of pigs. Infections with these pathogens result in significant losses to the pig industry through reduced weight gain, decreased feed efficiency and increased days to slaughter (5). They form together with swine influenza the respiratory disease complex, which is considered to be the most economically important disease complex for the pig industry.
We therefore hypothesise that we will be able to identify porcine CLR that will bind to molecules expressed within both Mycoplasma spp.. Our aim is to identify this interaction, and this will be achieved through the following objectives:
Objective 1) Data mine the porcine genome for porcine CLR using comparative genomic analysis with the human and bovine genome (currently funded through BBSRC BB/P008461/1).
Objective 2) Create a porcine specific CLR-Fc library using established technologies.
Objective 3) Assess binding of these fusion proteins to the two mentioned Mycoplasma strains. As the sugar binding spectrum of these CLR is well described, it provides us with informations regarding the sugar-components expressed on the surface of Mycoplasma strains, and thus potential targets for carbohydrate-based vaccines.
Objective 4) Assess presence of the corresponding receptors in target tissues by qRT-PCR and IHC. Givien that that M.hyopneunomiae mainly affects the cranioventral lobules of the lung, and M.hyorhinis mainly the joints, we will attempt to assess whether the C-type lectin receptor expression spectrum varies in these two compartments, potentially explaining the differences in bacterial localisation. To achieve this, we will use either precision cut lung slices or microtome cut tissue from joints. Expertise in the generation of these tissue preparations is already present within the Werling group and RVC.
We believe that such identification will result in new vaccine antigens to be identified, which can subsequently be used for mucosal vaccination, similar as described for other pathogens (6).

Applications

Applications must be complete, including both references, by 24th January at 5pm

Funding Notes

Fully funded place including home (UK) tuition fees and a tax-free stipend in the region of £17,009. Students from the EU are welcome to submit an application for funding, any offers will be subject to BBSRC approval and criteria.

References

1. Kahane I, Tully JG. 1976. Binding of plant lectins to mycoplasma cells and membranes. J Bacteriol 128: 1-7
2. Schiefer HG, Gerhardt U, Brunner H. 1974. Proceedings: Immunological studies on the localization of phosphatidylglycerol in Mycoplasma membranes. Hoppe Seylers Z Physiol Chem 355: 1248
3. Schiefer HG, Gerhardt U, Brunner H, Krupe M. 1974. Studies with lectins on the surface carbohydrate structures of mycoplasma membranes. J Bacteriol 120: 81-8
4. Chiba H, Pattanajitvilai S, Evans AJ, Harbeck RJ, Voelker DR. 2002. Human surfactant protein D (SP-D) binds Mycoplasma pneumoniae by high affinity interactions with lipids. Journal of Biological Chemistry 277: 20379-85
5. Brewster VR, Maiti HC, Tucker AW, Nevel M. 2017. Associations between EP-like lesions and pleuritis and post trimming carcass weights of finishing pigs in England. Livestock Science 201: 1-4
6. Nishat S, Andreana PR. 2016. Entirely Carbohydrate-Based Vaccines: An Emerging Field for Specific and Selective Immune Responses. Vaccines (Basel) 4

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