Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's disease, a chronic enteritis in ruminants, and is capable of persisting within macrophages despite the activation of host immune defenses. Although this intracellular persistence is a key determinant of MAP pathogenicity, the bacterial factors and host responses that regulate this process remain poorly understood. In this study, we established the first CRISPR interference (CRISPRi) platform applied to bovine monocyte-derived macrophages (MDM) to evaluate the functions of MAP genes involved in intracellular survival and to perform an integrative analysis of host transcriptomic responses. MAP mutants were targeted to two genes (mdh and MAP1981c). The optimal concentration of anhydrotetracycline (ATc) was determined to be 2 μg/ml by measuring the survival of the cells and the downregulation of gene expression levels in the cells up to 72 h. The gene expression profiles and intracellular MAP levels were investigated using RNA-seq and colony-forming units, respectively. The survival rates of the MAP mutants significantly decreased with the time course of infection in MAP-mdhKD and MAP1981cKD (KD, knockdown). RNA-seq-based gene expression profiling suggested that target gene silencing in MAP mutants led to altered expression of host genes involved in lipid metabolism, T-cell activation reduction, and antimicrobial response in bovine MDM, contributing to reduced intracellular survival of MAP. Our study demonstrates that the downregulation of mdh and MAP1981c in MAP significantly alters the host transcriptomic landscape in bovine MDM, revealing their critical roles in subverting host immune defenses for intracellular persistence.
