Physiological, genetical and morphological alterations in Mycobacterium avium subsp. paratuberculosis mutants generated with the CRISPRi system

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne’s disease (JD), a chronic granulomatous enteritis in ruminants. In humans, MAP has been implicated in Crohn’s disease for decades, without conclusive evidence of pathogenicity. Despite the existence of JD’s association with ruminants and humans, the mechanism that links pathogenicity of MAP to resistance to host defense using mycobacterial virulence is not fully understood. Therefore, we tried to confirm the phenotypic characteristics of genes related to mycobacterial virulence involved in pathogenicity by observing in vitro the pathophysiological characteristics of MAP mutants generated with the CRISPRi system. Four genes (pknG, icl, MAP1981c, and mdh) were targeted to generate MAP mutants. The optimal concentration of anhydrotetracycline was determined as 30 µg/ml to downregulate the gene expression with the CRISPRi system (P < 0.05). Growth and colony-forming ability of each mutant under stress conditions (nutrient starvation, oxidative stress, and acidic stress) were observed depending on ATc using CFU count and OD600 measurement. Morphological changes in colony and aggregation in broth culture, and the margin of the MAP of the mutants via field emission scanning electron microscopy were also observed. In the growth patterns of the mutants, the decrease in the growth of Δmdh upon the downregulation of the gene expression suggested the importance of the gene for the growth of MAP. The colony-forming ability of the Δicl and Δ1981c decreased under nutrient starvation and oxidative stress, respectively, and both in acidic stress. Morphological changes in colony and aggregation were observed for the ΔpknG and Δ1981c under nutrient starvation and oxidative stress conditions, respectively while Δicl was observed under nutrient starvation and acidic stress conditions. Morphological changes in the margins of the MAP of the mutants were observed. We investigated the phenotypic characteristics of genes related to growth and intracellular survival by observing the physiological, genetical, and morphological characteristics of MAP mutants generated with CRISPRi system in specific environmental conditions. These results suggest that those genes might be closely related to the pathogenicity of MAP in vitro and the CRISPRi system could be applied to further investigation related to virulence of MAP.