Large-Scale Analysis of the Mycoplasma bovis Genome Identified Non-essential, Adhesion- and Virulence-Related Genes

Christoph Josi; Sibylle Bürki; Sara Vidal; Emilie Dordet-Frisoni; Christine Citti; Laurent Falquet; Paola Pilo

doi:10.3389/fmicb.2019.02085

Large-Scale Analysis of the Mycoplasma bovis Genome Identified Non-essential, Adhesion- and Virulence-Related Genes

Front Microbiol. 2019 Sep 13:10:2085. doi: 10.3389/fmicb.2019.02085. eCollection 2019.

Authors

Christoph Josi^{1

2}, Sibylle Bürki¹, Sara Vidal¹, Emilie Dordet-Frisoni³, Christine Citti³, Laurent Falquet⁴, Paola Pilo¹

Affiliations

¹ Department of Infectious Diseases and Pathobiology, Vetsuisse Faculty, Institute of Veterinary Bacteriology, University of Bern, Bern, Switzerland.
² Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland.
³ UMR 1225, IHAP, Université de Toulouse, INRA, ENVT, Toulouse, France.
⁴ Department of Biology, Faculty of Science and Medicine, Swiss Institute of Bioinformatics, University of Fribourg, Fribourg, Switzerland.

Abstract

Mycoplasma bovis is an important pathogen of cattle causing bovine mycoplasmosis. Clinical manifestations are numerous, but pneumonia, mastitis, and arthritis cases are mainly reported. Currently, no efficient vaccine is available and antibiotic treatments are not always satisfactory. The design of new, efficient prophylactic and therapeutic approaches requires a better understanding of the molecular mechanisms responsible for M. bovis pathogenicity. Random transposon mutagenesis has been widely used in Mycoplasma species to identify potential gene functions. Such an approach can also be used to screen genomes and search for essential and non-essential genes for growth. Here, we generated a random transposon mutant library of M. bovis strain JF4278 containing approximately 4000 independent insertion sites. We then coupled high-throughput screening of this mutant library to transposon sequencing and bioinformatic analysis to identify M. bovis non-essential, adhesion- and virulence-related genes. Three hundred and fifty-two genes of M. bovis were assigned as essential for growth in rich medium. Among the remaining non-essential genes, putative virulence-related factors were subsequently identified. The complete mutant library was screened for adhesion using primary bovine mammary gland epithelial cells. Data from this assay resulted in a list of conditional-essential genes with putative adhesion-related functions by identifying non-essential genes for growth that are essential for host cell-adhesion. By individually assessing the adhesion capacity of six selected mutants, two previously unknown factors and the adhesin TrmFO were associated with a reduced adhesion phenotype. Overall, our study (i) uncovers new, putative virulence-related genes; (ii) offers a list of putative adhesion-related factors; and (iii) provides valuable information for vaccine design and for exploring M. bovis biology, pathogenesis, and host-interaction.

Keywords: Mycoplasma bovis; adhesion; non-essential genes; random transposon mutagenesis; virulence.