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Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages
Published on: December 10, 2013
Functional analysis of the intracellular survival of Mycobacterium avium subsp. paratuberculosis in THP-1 cells using
Jun Ho Lee1,2, Eun-Seo Lee1,2, Su Min Kyung1,3
1Department of Infectious Disease, College of Veterinary Medicine, Seoul National University, Seoul, Republic of Korea.
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.
Insights
Mycobacterium avium subsp. paratuberculosis (MAP) virulence was studied in human macrophages using CRISPR interference (CRISPRi) mutants. Knockdown of specific genes reduced MAP survival, suggesting their role in virulence and potential for therapeutic targets.
Area of Science:
- Microbiology
- Immunology
- Genetics
Background:
- Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne's disease in ruminants and is a potential zoonotic agent linked to Crohn's disease.
- Limited research exists on MAP virulence against human macrophages, despite public health implications.
Purpose of the Study:
- To functionally analyze genes associated with MAP virulence and intracellular survival in human THP-1 macrophages.
- To investigate the efficacy of CRISPR interference (CRISPRi) for studying MAP virulence.
Main Methods:
- CRISPR interference (CRISPRi) was used to create MAP mutants targeting four specific genes (mdh, pknG, MAP1981c, icl).
- MAP mutants were infected into human THP-1 macrophages, and their intracellular survival and clump formation were assessed.
- Gene expression and cell viability were measured to determine optimal conditions for CRISPRi (5 µg/mL ATc).
Main Results:
- CRISPRi-induced knockdown of MAP genes (mdh, MAP1981c, icl) significantly decreased MAP clump formation and intracellular survival in THP-1 macrophages.
- MAP survival decreased with increasing anhydrotetracycline (ATc) concentration and infection time.
- THP-1 macrophage survival increased with higher ATc concentrations, indicating reduced host cell damage by MAP mutants.
Conclusions:
- The studied genes (mdh, MAP1981c, icl) are crucial for MAP virulence and intracellular survival in human macrophages.
- CRISPRi is a valuable tool for functional analysis of MAP virulence genes.
- Findings offer insights into MAP-host interactions and potential therapeutic strategies against MAP infections.
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