Engineering Mycoplasma species for biotechnological and biomedical applications

Ariadna Montero Blay

Prof. Luis Serrano Pubul Dr. Carlos Piñero Lambea

CRG Pulmobiotics

Mycoplasmas are a group of bacteria characterized by minimized genomes, limited biosynthetic capacities, and simplified metabolic networks. In this work, we explored how Mycoplasma species can be exploited for biotechnological and therapeutic applications. In chapter two, we engineered M. pneumoniae transposons so that they could efficiently transform different Mycoplasma species. This fact allows the generation of essentiality studies in these species, a critical analysis to identify genes that could be deleted to create attenuated vaccination strains. Chapter three developed a new method that uses high-resolution transposon and proteomics data to infer active metabolic pathways within a cell at a specific moment. This information can be used when engineering attenuated strains. In chapter four, we explored the capacity of M. pneumoniae to express functional human biologics in vitro and in mice lungs. Chapter five used the protein design algorithm FoldX and ModelX to mutate a human interleukin to enhance its properties in terms of affinity to its receptors and increased bacterial expression. Chapter six identified the secretion signals and designed synthetic promoters in the recent-discovered fast-growing Mycoplasma feriruminatoris. Altogether this thesis develops the tools for exploiting Mycoplasmas for biotechnological purposes (M. agalactiae, M. feriruminatoris) and validates M. pneumoniae for human lung therapy immunomodulation.

In summary, this thesis firstly proposes genetic tools to modify bacteria to generate vaccines for livestock. In the second part of this thesis, the bacterium M. pneumoniae is exploited to treat lung diseases. Using this bacterial vehicle and modifying an anti-inflammatory interleukin to optimize its properties, it has been possible to completely eliminate lung inflammation caused by infection with another bacteria (work under patent and under review in PNAS). The work of this thesis, which deals with the exploitation of M. pneumoniae as an in vivo biological expression system, could be licensed to the spin-off company Pulmobiotics or others in the field of therapies mediated by viruses or bacteria. In addition, the work of engineering molecules per se (modifying cytokines to optimize their properties for therapeutic purposes) can be applied to other cytokines with structural similarities. It is intended that said work will be exploited through a new spin-off, of which Ariadna Montero will be one of the co-founders, being also a co-inventor in the different patents in progress or planned.

Mycoplasmas; Minimized Genomes; Biosynthetic Capacities; Simplified Metabolic Networks; Biotechnological Applications; Therapeutic Applications; M. pneumoniae Transposons; Efficient Transformation; Mycoplasma Species; Essentiality Studies; Attenuated Vaccination Strains; High-Resolution Transposon Data; Proteomics Data; Active Metabolic Pathways; Attenuated Strains; Functional Human Biologics; In Vitro Expression; Mice Lungs; Protein Design Algorithm; FoldX; ModelX; Human Interleukin; Affinity Enhancement; Receptor Affinity; Increased Bacterial Expression; Secretion Signals; Synthetic Promoters; Fast-Growing Mycoplasma feriruminatoris; M. agalactiae; Human Lung Therapy Immunomodulation.