Genome Engineering of the Fast-Growing Mycoplasma feriruminatoris toward a Live Vaccine Chassis

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作   者：

Talenton, Vincent;  Baby, Vincent;  Gourgues, Geraldine;  Mouden, Charlotte;  Claverol, Stephane;  Vashee, Sanjay;  Blanchard, Alain;  Labroussaa, Fabien;  Jores, Joerg;  Arfi, Yonathan;  Sirand-Pugnet, Pascal;  Lartigue, Carole; 

作者机构：

J Craig Venter Inst;  Univ Bordeaux;  Univ Bern; 

关键词：

TRANSFORMATION;  CELLS;  BACTERIA;  CReasPy-cloning;  contagious caprine pleuropneumonia;  GLYCEROL METABOLISM;  VIRULENCE;  Mycoplasma feriruminatoris;  TRANSPLANTATION;  synthetic biology;  vaccine;  ESSENTIAL GENES;  CLONING;  CRISPR-Cas9;  SYSTEMS;  bacterial chassis;  SYNTHETIC BIOLOGY;  genome transplantation;  genome engineering; 

期刊名称：

ACS Synthetic Biology

i s s n：

2161-5063

年卷期：

2022 年 11 卷 5 期

页   码：

1919-1930

页   码：

摘   要：

:development of a new generation of vaccines is a key challenge for the control of infectious diseases affecting bothhumans and animals. Synthetic biology methods offer new ways to engineer bacterial chassis that can be used as vectors to presentheterologous antigens and train the immune system against pathogens. Here, we describe the construction of a bacterial chassisbased on the fast-growingMycoplasma feriruminatoris, and thefirst steps toward its application as a live vaccine against contagiouscaprine pleuropneumonia (CCPP). To do so, theM. feriruminatorisgenome was cloned in yeast, modified by iterative cycles ofCas9-mediated deletion of loci encoding virulence factors, and transplanted back inMycoplasma capricolumsubsp.capricolumrecipient cells to produce the designedM. feriruminatorischassis. Deleted genes encoded the glycerol transport and metabolismsystems GtsABCD and GlpOKF and the Mycoplasma Ig binding protein-Mycoplasma Ig protease (MIB-MIP) immunoglobulincleavage system. Phenotypic assays of theM. feriruminatorischassis confirmed the corresponding loss of H2O2production and IgGcleavage activities, while growth remained unaltered. The resulting mycoplasma chassis was further evaluated as a platform for theexpression of heterologous surface proteins. A genome locus encoding an inactivated MIB-MIP system from the CCPP-causativeagentMycoplasma capricolumsubsp.capripneumoniaewas grafted in replacement of its homolog at the original locus in the chassisgenome. Both heterologous proteins were detected in the resulting strain using proteomics, confirming their expression. This studydemonstrates that advanced genome engineering methods are henceforth available for the fast-growingM. feriruminatoris, facilitatingthe development of novel vaccines, in particular against major mycoplasma diseases.