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Mycoplasmas are bacteria belonging to the Mollicutes class. They infect a wide range of organisms, among which humans, farm animals, herd animals and pets. Infections by mycoplasmas are currently treated with antibiotics, although many are ineffective due to the lack of cell wall of these bacteria. Moreover, this strategy leads to the development of antibiotics-resistant bacteria. The use of vaccines against mycoplasmas may be a solution for preventing wide spreading of the chronic infections they cause.The human pathogen Mycoplasma pneumoniae, causative agent of atypical pneumonia, can be used as a universal chassis to be deployed as a single- or multi-vaccine in a range of animal hosts. However, due to its reduced genome and dearth of many biosynthetic pathways, this bacterium depends on rich, undefined medium for growth, which makes the large-scale production of the vaccine challenging and expensive. For this reason, a genome-scale, constraint-based metabolic model was deployed to design a serum-free medium that supports growth of Mycoplasma pneumoniae with a rate comparable to the one obtained in rich medium. Model simulations highlighted, among the other components, the importance of two key lipids, part of Mycoplasma pneumoniae’s membrane. The serum-free medium MCMyco was then developed including these components and tested in vitro, showing robust growth of a range of mycoplasmas. Another strategy to increase Mycoplasma pneumoniae’s growth consisted in the design of a fatty acid-prototrophic strain of Mycoplasma. Indeed, despite the inability of synthetizing fatty acids, Mycoplasma pneumoniae needs them to construct its membrane lipids. The study is performed through a combination of DNA engineering, genome-scale modelling and gap-filling algorithm approaches, revealing a bottleneck related to NADPH, one the cofactor mostly used in the fatty acid biosynthesis pathway. As a result, the computationally-designed strain implements a pathway synthetizing fatty acids, which are then integrated as acyl chains into the membrane lipids, and genetic modifications leading to a more efficient NADPH regeneration.The deployment of Mycoplasma pneumoniae as a universal chassis for vaccination might lead to issues related to the causative link of the bacterium to post-infectious Guillain-Barré-Stohl syndrome, a neurodegenerative disorder. Galactocerebroside, a glycolipid synthetized by Mycoplasma, has been identified as the compound that triggers the autoimmune reactions causing the syndrome. The biosynthesis pathway of this lipid was therefore investigated, with the aim of finding those enzymes of Mycoplasma pneumoniae to target to prevent the synthesis. The computational analysis, performed through genomic comparison, functional annotation and Hidden Markov Models, was extended to all mycoplasmas, revealing not all these bacteria have the potential of synthetizing galactocerebroside. In consequence, such group of mycoplasmas could be considered safe for application. A protein motif that, when found in the enzyme synthetizing the glycolipid, could be an indication of synthesis capability in mycoplasmas was built. This motif can be used it as a signature to investigate on the potentiality of galactocerebroside synthesis in microorganisms.A universal Mycoplasma pneumoniae-based vaccine is a genetically-modified organism produced at large-scale and deployed in open environment. Therefore, biocontainment strategies must be applied to prevent its proliferation outside the desired operating conditions. Several Ordinary Differential Equations models were developed to sustain the experimental design of biosafety circuits to insert into the Mycoplasma pneumoniae’s genome. The model yielded robust results with a killing switch inserted in the bacterial genome in double copy, which showed to be resistant to inactivating mutations over several strain passages.The dissertation ends with a discussion on the computational approaches used, a recapitulation of the status of the vaccine and its future perspectives. Moreover, the importance of serum-free media in growth cultures is highlighted, with a special focus on their model-driven design. A reflection on the safety of live attenuated vaccine is proposed, and, finally, safety-by-design is suggested as a strategy to be implemented in the developmental phase of therapeutic and pharmaceutical products. |
