Dependence of Mycoplasma bovis on a novel nucleoside transporter for survival in association with host cells

Transport systems play a vital role in metabolism in mycoplasmas by facilitating the uptake of essential nutrients from host cells, on which mycoplasmas are highly dependent. Co-culture of Mycoplasma bovis with Madin-Darby bovine kidney cells has shown that the gene MBOVPG45_0748 is essential for the survival of M. bovis in association with host cells. To characterize the function of this protein, intracellular and extracellular metabolomic profiles of a mutant strain with a transposon inserted into the MBOVPG45_0748 gene (∆MBOVPG45_0748) were compared to those of the parent strain, PG45. The ∆MBOVPG45_0748 mutant contained lower levels of metabolites involved in purine, pyrimidine, and glycerol metabolism and elevated levels of intermediates in the pentose phosphate and glycolysis pathways. Depletion of nucleosides from the culture supernatant occurred at a significantly reduced rate in the mutant, suggesting a role of the product of MBOVPG45_0748 in nucleoside uptake. This was further supported by isotope labeling studies with 13 C 5 -thymidine, which confirmed that nucleoside uptake and interconversion were disrupted in ∆MBOVPG45_0748 and suggested a compensatory strategy for the maintenance of nucleic acid biosynthesis in the mutant. The poorer survival of ∆MBOVPG45_0748 in medium with DNA supplementation showed that the MBOVPG45_0748 protein was involved in the efficient import of nucleosides derived from degradation of extracellular DNA. Overall, these findings suggest that the MBOVPG45_0748 protein is a nucleoside transporter involved in the uptake of purines and pyrimidines and maintenance of their intracellular balance, which is essential for the survival of M. bovis in association with host cells. IMPORTANCE Mycoplasma bovis causes pneumonia, mastitis, arthritis, keratoconjunctivitis, and reproductive tract disease in cattle, compromising animal welfare and imposing economic losses on farmers. Development of effective control strategies against M. bovis requires a better understanding of the host-microbe interactions involved in the pathogenesis of the diseases it causes. Mycoplasmas are dependent on transport systems to acquire nutrients from host cells. Thus, these systems play an important role in their survival and virulence. We used a combination of metabolomic techniques to establish that a transporter gene that is essential for the survival of M. bovis in association with host cells plays a role in nucleoside uptake. These results highlight the importance of purine and pyrimidine metabolism in the interactions between M. bovis and host cells.