Attenuation of a Pathogenic <i>Mycoplasma</i> Strain by Modification of the <i>obg</i> Gene by Using Synthetic Biology Approaches

Abstract

<i>Mycoplasma</i> species are responsible for several economically significant livestock diseases for which there is a need for new and improved vaccines. Most of the existing mycoplasma vaccines are attenuated strains that have been empirically obtained by serial passages or by chemical mutagenesis. The recent development of synthetic biology approaches has opened the way for the engineering of live mycoplasma vaccines. Using these tools, the essential GTPase-encoding gene <i>obg</i> was modified directly on the <i>Mycoplasma mycoides</i> subsp. <i>capri</i> genome cloned in yeast, reproducing mutations suspected to induce a temperature-sensitive (TS⁺) phenotype. After transplantation of modified genomes into a recipient cell, the phenotype of the resulting <i>M. mycoides</i> subsp. <i>capri</i> mutants was characterized. Single-point <i>obg</i> mutations did not result in a strong TS⁺ phenotype in <i>M. mycoides</i> subsp. <i>capri</i>, but a clone presenting three <i>obg</i> mutations was shown to grow with difficulty at temperatures of ≥40°C. This particular mutant was then tested in a caprine septicemia model of <i>M. mycoides</i> subsp. <i>capri</i> infection. Five out of eight goats infected with the parental strain had to be euthanized, in contrast to one out of eight goats infected with the <i>obg</i> mutant, demonstrating an attenuation of virulence in the mutant. Moreover, the strain isolated from the euthanized animal in the group infected with the <i>obg</i> mutant was shown to carry a reversion in the <i>obg</i> gene associated with the loss of the TS⁺ phenotype. This study demonstrates the feasibility of building attenuated strains of mycoplasma that could contribute to the design of novel vaccines with improved safety.<b>IMPORTANCE</b> Animal diseases due to mycoplasmas are a major cause of morbidity and mortality associated with economic losses for farmers all over the world. Currently used mycoplasma vaccines exhibit several drawbacks, including low efficacy, short time of protection, adverse reactions, and difficulty in differentiating infected from vaccinated animals. Therefore, there is a need for improved vaccines to control animal mycoplasmoses. Here, we used genome engineering tools derived from synthetic biology approaches to produce targeted mutations in the essential GTPase-encoding <i>obg</i> gene of <i>Mycoplasma mycoides</i> subsp. <i>capri</i> Some of the resulting mutants exhibited a marked temperature-sensitive phenotype. The virulence of one of the <i>obg</i> mutants was evaluated in a caprine septicemia model and found to be strongly reduced. Although the <i>obg</i> mutant reverted to a virulent phenotype in one infected animal, we believe that these results contribute to a strategy that should help in building new vaccines against animal mycoplasmoses.