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| Author(s): |
Maysa Santos Barbosa
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI) |
| Defense date: | 2021-11-19 |
| Examining board members: |
Jorge Timenetsky;
Aline Teixeira Amorim;
Silvana Beutinger Marchioro;
Enrique Mario Boccardo Pierulivo
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| Advisor: | Jorge Timenetsky |
| Abstract | |
Contagious agalaxia (CA) affects small ruminants and is classified by the World Organization for Animal Health (OIE) as a notifiable disease due to its significant economic impact on livestock. Mycoplasma agalactiae is the main cause of this disease and despite the progress in studies on diagnostic, therapeutic and prevention methods, strategies still remain ineffective in controlling CA. The present study aimed to functionally characterize M. agalactiae membrane proteins as adhesins and evaluate their interaction with host molecules and cells. Three M. agalactiae proteins (P40 - positive control -, MAG_1560 and MAG_6130), characterized in silico as antigenic membrane proteins, were expressed in Escherichia coli BL21 StarTM (DE3) One Shot and purified on affinity column. Subsequently, rabbits were immunized with recombinant proteins to produce polyclonal antibodies. The location of proteins in the microorganism was evaluated by colony blotting and western blotting after fractionation of the TX-114 phase. To assess whether the recombinant proteins are adhesins, adhesion assays to HeLa cells and sheep primary mammary stromal cells (MSC) were performed. The results obtained confirmed MAG_1560 and MAG_6130 as lipoproteins, such as the cytoadhesin P40. Proteins P40, MAG_1560 and MAG_6130 also demonstrated cytotoxicity by decreasing cell viability and increasing the expression of genes related to DNA repair pathways and pro-apoptotic genes. In order to assess whether these proteins interact with host molecules, adhesion to plasminogen, fibrinogen, lactoferrin and fibronectin was evaluated. Furthermore, this study demonstrated that MAG_6130 and MAG_1560 proteins have the ability to adhere to eukaryotic cells and the adhesion of these proteins to cells can be inhibited by specific antiserum using at least one methodology. P40 interacts significantly with plasminogen, and in general, P40, MAG_6130 and MAG_1560 exhibited significant binding to lactoferrin, fibrinogen and fibronectin, a feature that could potentially support the pathogen in host colonization, tissue migration and immune evasion. However, more studies are needed to more clearly establish the relationship between P40, MAG_1560 and MAG_6130 and their effects on hosts. Thus, it is shown that these proteins are involved in cytoadhesion and contribute to the bacterial pathogenicity of host cells, however, it is still unclear how these proteins activate the immune system and what is the induced DNA repair pathway. With the results of this study, we hope to better understand the function of surface molecules in mycoplasmas and their role during infection. (AU) | |
| FAPESP's process: | 16/23306-6 - Functional characterization and evaluation of the antigenicity and immunogenicity of recombinant proteins of Mycoplasma agalactiae |
| Grantee: | Maysa Santos Barbosa |
| Support Opportunities: | Scholarships in Brazil - Doctorate |