Abstract
Mycoplasma bovis is an important cause of bovine respiratory disease worldwide. To understand its virulence mechanisms, we sequenced three attenuated M. bovis strains, P115, P150, and P180, which were passaged in vitro 115, 150, and 180 times, respectively, and exhibited progressively decreasing virulence. Comparative genomics was performed among the wild-type M. bovis HB0801 (P1) strain and the P115, P150, and P180 strains, and one 14.2-kb deleted region covering 14 genes was detected in the passaged strains. Additionally, 46 non-sense single-nucleotide polymorphisms and indels were detected, which confirmed that more passages result in more mutations. A subsequent collective bioinformatics analysis of paralogs, metabolic pathways, protein-protein interactions, secretory proteins, functionally conserved domains, and virulence-related factors identified 11 genes that likely contributed to the increased attenuation in the passaged strains. These genes encode ascorbate-specific phosphotransferase system enzyme IIB and IIA components, enolase, L-lactate dehydrogenase, pyruvate kinase, glycerol, and multiple sugar ATP-binding cassette transporters, ATP binding proteins, NADH dehydrogenase, phosphate acetyltransferase, transketolase, and a variable surface protein. Fifteen genes were shown to be enriched in 15 metabolic pathways, and they included the aforementioned genes encoding pyruvate kinase, transketolase, enolase, and L-lactate dehydrogenase. Hydrogen peroxide (H2O2) production in M. bovis strains representing seven passages from P1 to P180 decreased progressively with increasing numbers of passages and increased attenuation. However, eight mutants specific to eight individual genes within the 14.2-kb deleted region did not exhibit altered H2O2 production. These results enrich the M. bovis genomics database, and they increase our understanding of the mechanisms underlying M. bovis virulence.
Highlights
Mycoplasma bovis is a member of the Mycoplasmataceae family in the class of Mollicutes
All the strains were propagated in pleuropneumonialike organism (PPLO) medium supplemented with 10% horse serum (Thermo Fisher Scientific, Waltham, MA, USA) at 37◦C for 48–72 h as described previously (Zhang et al, 2014)
83,750,973 bases with 286,408 reads were obtained for strain P115, while 80,961,239 bases with 277,020 reads were obtained for strain P150, and 83,589,289 bases with 260,056 reads were obtained for strain P180, resulting in 85.7-fold (P115), 82.8-fold (P150), and 85.5fold (P180) depths of sequencing
Summary
Mycoplasma bovis is a member of the Mycoplasmataceae family in the class of Mollicutes It was first identified as a causative agent of bovine mastitis in 1961, and it was later recognized as an important pathogen of bovine respiratory disease in 1976 (Caswell and Archambault, 2007). Only a few, including one M. bovis secretory protein, have been discovered (Zhang et al, 2016) Secondary metabolites such as hydrogen peroxide (H2O2) are considered to play a significant role in the pathogenesis of some Mycoplasma species, including M. pneumoniae (Hames et al, 2009) and Mycoplasma mycoides subsp. Pathogenicity islands (PAIs) play a significant role in genome evolution and pathogenesis because many virulence-related factors are shared and acquired by PAIs. no PAIs and secretory systems have been detected in any Mycoplasma species (Guo and Wei, 2012). Using the virulence factors database (VFDB), some virulence genes were identified in the M. bovis genome (Parker et al, 2016), but their impact on M. bovis virulence remains to be investigated
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