Removal of a Subset of Non-essential Genes Fully Attenuates a Highly Virulent Mycoplasma Strain

Abstract

Mycoplasmas are the smallest free-living organisms and cause a number of economically important diseases affecting humans, animals, insects, and plants. Here, we demonstrate that highly virulent <i>Mycoplasma mycoides</i> subspecies <i>capri</i> (<i>Mmc</i>) can be fully attenuated <i>via</i> targeted deletion of non-essential genes encoding, among others, potential virulence traits. Five genomic regions, representing approximately 10% of the original <i>Mmc</i> genome, were successively deleted using <i>Saccharomyces cerevisiae</i> as an engineering platform. Specifically, a total of 68 genes out of the 432 genes verified to be individually non-essential in the JCVI-Syn3.0 minimal cell, were excised from the genome. <i>In vitro</i> characterization showed that this mutant was similar to its parental strain in terms of its doubling time, even though 10% of the genome content were removed. A novel <i>in vivo</i> challenge model in goats revealed that the wild-type parental strain caused marked necrotizing inflammation at the site of inoculation, septicemia and all animals reached endpoint criteria within 6 days after experimental infection. This is in contrast to the mutant strain, which caused no clinical signs nor pathomorphological lesions. These results highlight, for the first time, the rational design, construction and complete attenuation of a <i>Mycoplasma</i> strain via synthetic genomics tools. Trait addition using the yeast-based genome engineering platform and subsequent <i>in vitro</i> or <i>in vivo</i> trials employing the <i>Mycoplasma</i> chassis will allow us to dissect the role of individual candidate <i>Mycoplasma</i> virulence factors and lead the way for the development of an attenuated designer vaccine.