In vivo role of capsular polysaccharide in Mycoplasma mycoides

Jores, Joerg; Schieck, Elise; Liljander, Anne; Sacchini, Flavio; Posthaus, Horst; Lartigue, Carole; Blanchard, Alain; Labroussaa, Fabien; Vashee, Sanjay

doi:10.1093/infdis/jiy713

Cited by 21 publications

(29 citation statements)

References 12 publications

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“…The second aim was to verify this attenuation in vivo using the native host, since no rodent animal models for highly virulent M. mycoides exist (Jores et al, 2013). Many candidate virulence factors of M. mycoides have been suggested but, none except the capsular polysaccharide (Jores et al, 2018) have ever been confirmed according to Falkow’s postulates in vivo (Falkow, 1988).…”

Section: Discussionmentioning

confidence: 99%

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

Jores

Stolzmann

et al. 2019

Front. Microbiol.

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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.

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Section: Discussionmentioning

confidence: 99%

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

Jores

Stolzmann

et al. 2019

Front. Microbiol.

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“…In the control group, 5 goats out of 8 met the endpoint criteria before the envisaged end of the trial (i.e., 35 dpi) and were euthanized in the course of the experiment, whereas only 1 goat (animal CM135) out of 8 met the same criteria in the obg group (Fig. 4A) (24). M.…”

Section: Resultsmentioning

confidence: 99%

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

Lartigue

Timana

Labroussaa

et al. 2019

mSphere

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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.

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“…The second aim was to verify this attenuation in vivo using the native host, since no rodent animal models for highly virulent M. mycoides exist (3). Many candidate virulence factors of Mycoplasma mycoides have been suggested but, all except capsular polysaccharide (19) have never been confirmed according to Falkow’s postulates in vivo (20).…”

Section: Discussionmentioning

confidence: 99%

Removal of a subset of non-essential genes fully attenuates a highly virulentMycoplasmastrain

Jores

Stolzmann³

et al. 2019

Preprint

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IMPORTANCE 43Members of the Mycoplasma mycoides cluster cause important animal plaques in Africa and 44 Asia, which impact animal welfare, provision of food and the life of thousands of small-scale 45 farmers. We applied synthetic biology tools to Mycoplasma mycoides in order to design and 46 create a fully attenuated Mycoplasma strain that was subsequently confirmed in vivo using a 47 65 pathogenicity. Due to their regressive evolution by gene loss , mycoplasmas appear to lack 66 many of the common bacterial effectors and toxins used to interact with their hosts or to escape 67 the hosts' immune systems (6, 7). Lipoproteins have been proposed to be involved in both 68 aspects by using their cytoadherent properties and allowing antigenic variability through phase 69 or sequence variation (8). Other candidate virulence traits, such as the Mycoplasma Ig binding 70 protein-Mycoplasma Ig protease (MIB-MIP) system (9) and the hydrogen peroxide production 71 system (10) have been suggested, but not confirmed in vivo. 72

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In vivo role of capsular polysaccharide in Mycoplasma mycoides

Cited by 21 publications

References 12 publications

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

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

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

Removal of a subset of non-essential genes fully attenuates a highly virulentMycoplasmastrain

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