The MnuA Nuclease and 5'-Nucleotidase Impact Mycoplasma bovis Fitness in Mastitis

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英语. Background: Nucleases as well as 5' nucleotidases play an essential role in central pathways of cell biology and are often associated with bacterial virulence. In Mycoplasma spp., possessing limited metabolic capacities, the role of such enzymes is of great value. Therefore, we hypothesized that the major membrane nuclease MnuA (MBOVPG45_0215) and 5'-nucleotidase (MBOVPG45_0690) are putative virulence factors of Mycoplasma bovis essential for its survival strategies and infectious potential and might contribute to mammary virulence of this pathogen. Objective: To investigate the potential role of the MnuA and 5' nucleotidase in M. bovis mastitis. Methods: Genome-wide transposon mutagenesis of M. bovis type strain PG45 was performed by using pMT85 plasmid and mutants of the interest were evaluated using in vitro and in vivo model systems. Results: The disruption of mnuA resulted in decreased nuclease activity, measured by PicoGreen assay, while disruption of MBOVPG45_0690 led to slower growth rate and smaller colony size. Using murine mastitis model, we compared the disease elicited by M. bovis WT and the ΔmnuA or Δ0690 mutants. The inactivation of the mnuA or MBOVPG45_0690 genes reduced the bacterial fitness of M. bovis in the mammary gland as shown by the significant reduction of post challenge mammary bacterial counts. However, mammary inflammation, measured by the relative expression of major inflammatory genes, was significantly reduced only with Δ0690. Whole genome transcriptome analysis of mouse mammary glands challenged with WT, ΔmnuA or Δ0690 mutants displays differences in activation of immune genes and pathways involved in immune cells recruitment and function, among them myeloperoxidase and neutrophil extracellular traps (NETs) formation pathway. Advanced image analysis demonstrated differences in in-vivo NET formation between the WT and the ΔmnuA mutant. In addition, pleiotropic alterations in cellular physiology, including fatty and amino acids metabolisms were identified in mammary glands of mice challenged with ΔmnuA or Δ0690 mutants. Conclusion: Collectively, these results suggest that both enzymes play an important role in the pathogenesis of M. bovis mastitis.