Whole transcriptome analysis of Mycoplasma bovis-host interactions under in vitro and in vivo conditions

Mycoplasma bovis mastitis is becoming increasingly problematic for dairy cattle farming in different parts of the world. M. bovis is inherently resistant to several antimicrobial classes and there is no effective vaccine. The major constraints to developing effective control tools are limited knowledge of M. bovis virulence factors and the underlying pathogenic mechanisms. The objective of this study was to determine virulence-associated genes of M. bovis and host immune response genes expressed during the early stages of host-pathogen interactions. In this study, we conducted in vitro infection of mammary epithelial cell (MAC-T) lines and in vivo intramammary infection of dairy cows with M. bovis strain PG45 and evaluated whole transcriptome differential gene expression and pathway enrichment analysis. We found a total of 614 (304 up-regulated, 310 down-regulated) and 7161 (3935 up-regulated, 3226 down-regulated) genes of M. bovis and bovine host cells were differentially expressed, respectively. Of the up-regulated genes of M. bovis, insertion sequence (IS) genes that are involved in transposase activity such as ISMbov1, ISMbov2, ISMbov3, and ISMbov9 were significantly up-regulated, whereas protein translation-associated genes were significantly down-regulated. In MAC-T cells, genes involved in apoptosis pathways and proinflammatory cytokines were significantly up-regulated, whereas genes involved in cell cycle, ribosome biogenesis, and steroid biosynthesis were significantly down-regulated. Genes encoding formation of neutrophil extracellular traps and proinflammatory cytokines, were significantly up-regulated in the mammary gland of M. bovis challenged cows, whereas genes involved in steroid biosynthesis and metabolism were significantly down-regulated. In conclusion, there were increased expressions of IS elements which are involved in transposase activity during early stages of M. bovis infection. This observation warrants further detailed investigation to determine important specific potential targets involved in the transposition for intervention. Apoptosis might be in favor of progression of M. bovis infection but intervention that reduces apoptosis and enhances quick and effective polymorphonuclear neutrophilic recruitment with increased extracellular trap activity may improve M. bovis clearance. Our findings are key to determining important targets for immunity-based intervention. To our knowledge, this is the first whole transcriptome analysis in both M. bovis and the host under the same in vitro and in vivo conditions. Therefore, we recommend additional metatranscriptomic, metaproteomic, metabolomic and metagenomic evaluation of M. bovis and other potentially non-culturable microbes in the mammary glands during the pathogenesis of M. bovis mastitis in the bovine host to understand a complete picture of these interactions in maintaining healthy homeostatic state or mastitis. MAC-T (bovine mammary epithelial cells) and dairy cows were infected using Mycoplasma bovis to investigate early stage transcriptomic changes both in the pathogen and host. Mycoplasma bovis only, MAC-T only and uninfected cow controls were included in the study. All samples (5) were prepared in duplicate.