The DHH-DHHA1 domain phosphodiesterase of Mycoplasma bovis employs multiple strategies to modulate macrophage cellular processes.

The multifunctional DHH-DHHA1 phosphodiesterase plays critical roles in bacterial physiological regulation and host-pathogen interactions. In Mycoplasma bovis, the DhhP-type phosphodiesterase MbovP328 exhibits both phosphodiesterase and nanoRNase activities, with the H291 residue critical for phosphodiesterase function. Transposon insertion in Mbov_0328 inactivated both activities in the mutant T9.386. Complementation restored both activities in CNT9.386, while only nanoRNase activity was recovered in CNT9.386. The present study elucidates the effect of each activity on BoMac cells infected with M. bovis wild-type HB0801 and each mutant. RNA-sequencing revealed that phosphodiesterase activity altered the expression of 142 genes, predominantly linked to innate immune responses. Nine differentially expressed genes (DEGs) showed consistent expression trend in CNT9.386- and HB0801-infected cells. Overexpression and RNAi silencing assays confirmed CD70 and CST7 regulate IFN-β expression. Non-phosphodiesterase functions affected the expression of 104 genes, many of which enriched in signal transduction and immune pathways. DEGs ATP6V1E2, CXCL8, TNFRSF11A, and ACP5 were linked to rheumatoid arthritis, while TNFRSF11A, ACP5, and FCGR2A were associated with osteoclast differentiation, a process tied to bone metabolism. These findings demonstrate M. bovis DHH-DHHA1 domain phosphodiesterase regulates macrophages through dual mechanisms, offering insights into pathogen-host interactions and implicating its role in arthritis pathogenesis.