Table_2_Large-Scale Analysis of the Mycoplasma bovis Genome Identified Non-essential, Adhesion- and Virulence-Related Genes.XLSX

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.

牛分支杆菌（Mycoplasma bovis）是牛的重要病原体，可引起牛分支杆菌病。临床症状多样，其中肺炎、乳腺炎和关节炎病例较为常见。目前尚无有效的疫苗可用，抗生素治疗也并非总是令人满意。设计新的、有效的预防性和治疗性策略需要更深入地理解导致牛分支杆菌致病性的分子机制。随机转座子诱变在支原体属中被广泛用于识别潜在的基因功能。此方法也可用于基因组筛选，寻找生长所必需和非必需的基因。在本研究中，我们构建了包含大约4000个独立插入位点的大约4000个独立插入位点的牛分支杆菌菌株JF4278的随机转座子突变库。随后，我们将该突变库的高通量筛选与转座子测序和生物信息学分析相结合，以识别牛分支杆菌的非必需、粘附和致病性相关基因。在富含培养基中，352个牛分支杆菌基因被确定为生长所必需。在剩余的非必需基因中，随后鉴定了潜在的致病性相关因子。通过使用原代牛乳腺上皮细胞对完整的突变库进行粘附筛选，鉴定出具有潜在粘附相关功能的条件性必需基因，这些基因对于生长是非必需的，但对于宿主细胞粘附却是必需的。通过单独评估六个选定的突变体的粘附能力，发现两种先前未知的因子和粘附素TrmFO与粘附表型降低相关。总体而言，我们的研究（i）揭示了新的、潜在的致病性相关基因；（ii）提供了一组潜在的粘附相关因子；（iii）为疫苗设计以及探索牛分支杆菌的生物学、发病机制和宿主相互作用提供了宝贵信息。