Comparative Genomics of Mycoplasma bovis Strains Reveals That Decreased Virulence with Increasing Passages Might Correlate with Potential Virulence-Related Factors

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.

Gap analysis ofMycoplasma bovisdisease, diagnosis and control: An aid to identify future development requirements

“…Completely assembled genome sequences have been determined for twelve M. bovis isolates (including four derived from highly passaged isolates from strain HB0801 [Rasheed et al., ]), with draft genomes available for 20 additional strains (NCBI database on 24 January 2018). To date, detailed comparative genomic analyses have only been reported on the first three complete M. bovis genomes to be sequenced (Li et al., ; Qi et al., ; Wise et al., ) and for the high‐passage derivatives of HB0801 (Rasheed et al., ). Although the overall genomic framework and gene synteny are largely conserved, these are punctuated by incursions of integrative conjugative elements (ICE), variable copy numbers and integration sites of insertion sequences (IS) and a large chromosomal inversion in the PG45 type strain (generated by recombination between two IS units).…”

“…To date, only one study has been reported in which it was possible to correlate specific genes with pathogenicity. By comparing 115‐, 150‐ and 180‐passage isolates (with decreasing pathogenicity) to the parental HB0801 isolate, ten metabolism‐related genes together with one variable surface protein‐encoding gene were identified as germane to the observed attenuation (Rasheed et al., ). However, no gene has been unambiguously demonstrated to influence the virulence profile of any M. bovis isolate in vivo .…”

“…() serially passaged a strain of M. bovis and showed the loss of a potential pathogenic factor linked to hydrogen peroxide production. These lineages should be tested in animals to determine whether they are attenuated, as has been established recently for a series of highly passaged M. bovis strains (derived from the sequenced strain HB0801), which also exhibited reduced hydrogen peroxide elaboration (Rasheed et al., ). Sequence analysis of the latter strains disclosed mutations in genes that had no obvious link to peroxide production.…”

Multi-locus sequence typing of Mycoplasma bovis to assess its genetic diversity from 2009-2018 in Ningxia Hui Autonomous Region, China

“…The M. bovis strains were cultured in PPLO broth BD DifcoTM, US California [4,20]. M. bovis genomic DNA was extracted using a bacterial DNA extraction kit (Tiangen, Beijing, China) [21].…”

Two dimensional gel electrophoresis (2-DE) for high-throughput proteome analyses of Mycoplasma bovis

“…Comprehensive investigation of pathogenesis of M. bovis was sought and began in 2008 in China with the identification and isolation of M. bovis strain HB0801 from calf lung lesions in the Hubei province (Lei et al, 2008). Consequently, complete genome sequencing of the HB0801 strain and its comparison with the genomes of other strains and Mycoplasmas species laid down a foundation for the clarification of pathogenesis of M. bovis (Qi et al, 2012;Rasheed et al, 2017).…”