Abstract
Mycobacterium avium subsp. paratuberculosis (MAP) is a causative agent of Johne's disease in ruminants and a potential zoonotic agent linked with Crohn's disease in humans. Despite the possible risk to public health, few studies have focused on the virulence of MAP against human macrophages. Therefore, a functional analysis of mycobacterial genes associated with virulence, especially the intracellular survival of MAP, was performed after infection of MAP CRISPR interference (CRISPRi) mutants in the human THP-1 macrophages. MAP mutants were targeted to four genes (mdh, pknG, MAP1981c, and icl). The optimal concentration of anhydrotetracycline (ATc) was determined to be 5 µg/mL by measuring the survival of the cells and the downregulation of gene expression levels in the cells up to Day 3. The clump formation and intracellular survival of MAP were investigated using transmission electron microscopy and the colony-forming units, respectively. The clump formation of MAP mutants induced by CRISPRi was decreased in THP-1 macrophages at 24 and 72 h post-infection. The survival rates of the MAP mutants significantly decreased with increasing ATc concentration and time course of infection in MAP-mdhKD, MAP1981cKD, and MAP-iclKD. Conversely, the survival rate of THP-1 macrophages increased with increasing ATc concentration. Our results suggest that these genes might be closely related to MAP virulence along with intracellular survival in THP-1 macrophages. These data can provide novel insights into the utilization of CRISPRi in further research on MAP virulence by exploring intracellular survival using mycobacterial genes related to the virulence of MAP during host infection. IMPORTANCE: Johne's disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP) is a worldwide issue in the dairy industry and has a possible connection to Crohn's disease (CD) in humans. Despite its potential contribution to the etiology of CD, there have been few studies focusing on the virulence of MAP against human macrophages. In the current study, we investigated MAP virulence along with intracellular survival in human THP-1 macrophages using functional analysis of MAP CRISPR interference (CRISPRi) mutants at the knockdown of genes associated with mycobacterial virulence. The identified potential genes represent novel candidate classes that could be necessary for MAP virulence by exploring intracellular survival during host infection and could provide novel insights for future studies on the utilization of CRISPRi.