Construction of a bacterial chassis based on the fast-growing Mycoplasma feriruminatoris

International audience

Inglés. Background – Development of a new generation of vaccines is a key challenge for both human and animal medicine. Synthetic biology methods offer new ways to design and build engineered bacterial chassis that can be used as vectors to present heterologous antigens and raise immune systems against bacterial and viral pathogens. Here we present the construction of a bacterial chassis based on the fast-growing Mycoplasma feriruminatoris (Mferi) and further steps towards the production of a vaccine candidate against Contagious Caprine Pleuropneumoniae (CCPP). Methods – Mferi genome was cloned in yeast by Creaspy cloning, engineered using CRISPR/Cas9 and subsequently transplanted in Mycoplasma capricolum subsp. capricolum recipient cells. Engineered strains were genome sequenced and proteomes were determined by mass spectrometry. Results – The genome of Mferi strain G5847 was cloned in yeast. Iterative deletions of genome regions encoding the glycerol transporters gtsABCD and glpOKF and the MIB-MIP Ig cleavage system were performed to get an attenuated Mferi chassis. Genome sequencing and phenotypic assays confirmed deletions and loss of H2O2 production and Ig cleavage activities. Fast growing was conserved with a generation time of ~35 min. Then, four genome regions including 13 selected genes from the Mycoplasma capricolum subsp. capripneumoniae F38 vaccine strain were grafted in replacement of their homologs in the genome of the Mferi chassis. Conclusion – Synthetic biology methods were successfully developed in Mferi and genome regions potentially involved in virulence were removed. MCCP antigens were successfully grafted as a step forward the development of a novel vaccine strain against CCPP.