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

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

doi:10.3389/fmicb.2019.02085

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.

Highlights

Mycoplasma bovis is one of the major causative agents of bovine mycoplasmosis
The most investigated ones are the variable surface lipoproteins (Vsps) that were shown to be involved in adhesion, antigenic variation, and immune evasion (Sachse et al, 1993, 1996, 2000; Rosengarten et al, 1994; Thomas et al, 2003; Buchenau et al, 2010)
The transposon sequencing reads were aligned to the M. bovis strain JF4278 genome retrieved from NCBI

Summary

Introduction

Mycoplasma bovis is one of the major causative agents of bovine mycoplasmosis. This bacterium is emerging in industrialized countries, where it leads to high economic losses in the dairy and beef industries (Nicholas, 2011; Bürki et al, 2015). The most investigated ones are the variable surface lipoproteins (Vsps) that were shown to be involved in adhesion, antigenic variation, and immune evasion (Sachse et al, 1993, 1996, 2000; Rosengarten et al, 1994; Thomas et al, 2003; Buchenau et al, 2010). Two nucleases, the homologs MBOVPG45_0215 and MnuA, and the secretory nuclease MBOV_RS02825, were shown to be associated with cytotoxicity or with the degradation of neutrophil extracellular traps (Sharma et al, 2015; Zhang et al, 2016; Mitiku et al, 2018)

Methods

Results

Conclusion