Biology subjects

Cippa, V. L.

Publications and source records attributed to Cippa, V. L..

2 recordsLinked to original sources

bioRxiv2021

Minimalistic mycoplasmas harbor different functional toxin-antitoxin systems

Mycoplasmas are minute bacteria controlled by very small genomes ranging from 0.6 to 1.4 Mbp. They lack a cell wall and have been suggested to have progressed through reductive evolution from phylogenetically closely related Clostridia. They are known to colonize the respiratory tract or the urogenital tract among other organs and can cause chronic and subclinical diseases associated with long persistence of the causative agent. Toxin-antitoxin systems (TAS) are genetic elements that have been described for several respiratory and urogenital pathogens as well as for Clostridia, but never for pathogenic mycoplasmas. Here we describe for the first-time different types of TAS in a Mycoplasma pathogen, namely M. mycoides subsp. capri. We identified candidate TAS in silico via TASmania database. Two candidate TAS identified in silico and another candidate TAS suggested in a minimal cell based on transposon mutagenesis were systematically tested for their functionality in hosts with different phylogenetic distance using heterologous expression. Phylogenetic distance of the host used for heterologous expression influenced the outcome of the functional testing. We corroborated functionality of the three candidate TAS in Mycoplasma capricolum subsp. capricolum. Moreover, we confirmed transcription and translation of molecules of the TAS investigated during in vitro growth. We sequence analyzed 15 genomes of M. mycoides subsp. capri and revealed an unequal distribution of the TAS studied pointing towards dynamic gain and loss of TAS within the species. Author summaryMycoplasmas have a minimal genome and have never been shown to possess TAS. In this work we showed the presence of different functional TAS systems in Mycoplasma mycoides subsp. capri, a caprine pathogen for the first time. Sequence analysis of a number of Mycoplasma mycoides subsp. capri strains revealed a plasticity of the genome with respect to TAS carriage. This work paves the way to investigate the biological role of TAS (e.g. persistence, stress tolerance) during infection using mycoplasmas as a simple model organism. Since most mycoplasmas lack classical virulence factors such as exotoxins and go into a kind of stealth mode to evade the immune system, TAS are likely to contribute to the parasitic lifestyle of mycoplasmas and should be investigated in that respect. The availability of synthetic genomics tools to modify a range of Mycoplasma pathogens and well-established challenge models for the latter mycoplasmas will foster future research on TAS in mycoplasmas.

microbiology↗

bioRxiv2019

Removal of a subset of non-essential genes fully attenuates a highly virulent Mycoplasma strain

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 ten percent 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 nonessential 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 ten percent 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 reaching endpoint criteria within seven 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.\n\nIMPORTANCEMembers of the Mycoplasma mycoides cluster cause important animal plaques in Africa and Asia, which impact animal welfare, provision of food and the life of thousands of small-scale farmers. We applied synthetic biology tools to Mycoplasma mycoides in order to design and create a fully attenuated Mycoplasma strain that was subsequently confirmed in vivo using a novel caprine infection model. This is the first time that a Mycoplasma mutant developed by applying synthetic biology tools has been tested in vivo to pin point candidate virulence traits. The mutant strain is similar to \"apathogenic E. coli K12\" strains that boosted the research on host-pathogen interactions for the genus Escherichia and other bacterial genera.

microbiology↗