Attenuation of a Pathogenic Mycoplasma Strain by Modification of the obg Gene by Using Synthetic Biology Approaches

Abstract

Mycoplasma 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 obg was modified directly on the Mycoplasma mycoides subsp. capri 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 M. mycoides subsp. capri mutants was characterized. Single-point obg mutations did not result in a strong TS+ phenotype in M. mycoides subsp. capri, but a clone presenting three obg mutations was shown to grow with difficulty at temperatures of ≥40°C. This particular mutant was then tested in a caprine septicemia model of M. mycoides subsp. capri 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 obg mutant, demonstrating an attenuation of virulence in the mutant. Moreover, the strain isolated from the euthanized animal in the group infected with the obg mutant was shown to carry a reversion in the obg 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. IMPORTANCE 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 obg gene of Mycoplasma mycoides subsp. capri. Some of the resulting mutants exhibited a marked temperature-sensitive phenotype. The virulence of one of the obg mutants was evaluated in a caprine septicemia model and found to be strongly reduced. Although the obg 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.

Minimalistic mycoplasmas harbor different functional toxin-antitoxin systems

“…Especially the T 133 subtilisin-like serine protease has been confirmed to be promising element for a kill switch in an engineered mycoplasma chassis, in case alternative inducible promotors are at hand. Recently, we engineered an obg -based temperature kill switch, that was activated during elevated temperatures and induced growth arrest [ 48 ]. In summary, the functional testing of mycoplasma candidate TA pairs was most informative when Mcap was used as host for ectopic expression.…”

Contagious Bovine and Caprine Pleuropneumonia: a research community’s recommendations for the development of better vaccines

“…capri, in order to decipher virulence traits in Mycoplasma mycoides and test them in vivo. These studies have used synthetic genomics techniques, which allow unprecedented precise genome engineering of mycoplasmas [34][35][36][37][38][39] and a caprine challenge model [40][41][42] . These experiments have provided in vivo evidence that capsular polysaccharide is a virulence trait in Mycoplasma mycoides 40 , as suggested more than 40 years ago 43 .…”

Biological Importance of Microbes in Agriculture, Food and Pharmaceutical Industry: A Review

“…The preparation of mostly attenuated vaccines is done from viruses and bacteria. These are the vaccines mainly used against typhoid, mumps, measles, yellow fever, and rubella [36].…”