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 Mycoplasma mycoides subspecies capri (Mmc) can be fully attenuated via targeted deletion of non-essential genes encoding, among others, potential virulence traits. Five genomic regions, representing approximately 10% of the original Mmc genome, were successively deleted using Saccharomyces cerevisiae 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. In vitro 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 in vivo 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 Mycoplasma strain via synthetic genomics tools. Trait addition using the yeast-based genome engineering platform and subsequent in vitro or in vivo trials employing the Mycoplasma chassis will allow us to dissect the role of individual candidate Mycoplasma virulence factors and lead the way for the development of an attenuated designer vaccine.

Keywords: Mycoplasma mycoides subsp. capri; attenuation; genome engineering; in vivo challenge; virulence traits.

FIGURE 1

Design of mutant GM12::YCpMmyc1.1-Δ68. Cartoon displaying the genomes of the parental strain GM12::YCpMmyc1.1 and its derivative, the deletion mutant GM12::YCpMmyc1.1-Δ68.

FIGURE 2

In vitro characteristics of the parental strains GM12, GM12::YCpMmyc1.1 and its deletion mutant GM12::YCpMmyc1.1-Δ68: (A) morphology revealed by scanning electron microscopy. The white size bar displays 500 nm; (B) in vitro doubling time; (C) production of hydrogen peroxide in the presence of glycerol; bars display the standard deviations in (B) and the SEM in (C).

FIGURE 3

In vitro and in vivo ability of the Mmc MIB-MIP system to degrade caprine IgG. (A) Cartoon displaying the proteolytic cleavage between the conserved C_H1 and the variable V_H domain of the IgG heavy chain. (B) Immunoblot showing the in vitro ability of GM12 (lane 1), GM12::YCpMmyc1.1 (lane 2) to degrade IgG in comparison to GM12::YCpMmyc1.1-Δ68 (lane 3). (C) Immunoblot showing the in vivo IgG degradation present in serum of septicaemic goat CK51 (GM12 group) after infection in contrast to the healthy goat CK45 (GM12::YCpMmyc1.1-Δ68 group): 4-caprine IgG, 5-goat CK51 at –1 dpi, 6-goat CK51 at 4 dpi, 7-goat CK45 at –1 dpi, 8-goat CK45 at 4 dpi; the black asterisk marks the 44 kDa cleaved fragment.

FIGURE 4

Comparison of the clinical parameters monitored during the in vivo challenge trial between the animals that received the GM12 and its derivative GM12::YCpMmyc1.1-Δ68. (A) Kaplan–Meier survival curve based on animals reaching endpoint criteria. (B) Average body temperatures during experimental infection. Values were generated using daily rectal temperatures from the two groups. (C) Average weight gain/loss during experimental infection. Values were generated using interval measures from the two groups. The standard deviations are displayed as bars in (B,C).

FIGURE 5

Composite figure displaying representative histological results from tracheal tissue at the inoculation site. Tissues were stained with hematoxylin and eosin. (A,C) Low (40×) and high (200×) magnification of trachea of a goat inoculated with Mmc GM12::YCP1.1-Δ68 depicting unaffected epithelium (dot), submucosa (diamond) and cartilage (asterisk). (B,D) Low (40×) and high (200×) magnification of trachea of a goat inoculated with Mmc GM12, depicting ulceration of the epithelium in (B) (thick arrow), massive extension of the submucosa (diamond) due to extensive areas of necrosis (arrowhead) and infiltration with large numbers of degenerate neutrophilic granulocytes (arrow). Size standards are displayed in each picture. Scale bars: (A,B) 500 μm; (C,D) 100 μm.