Extracellular DNase MAP3916c attacks the neutrophil extracellular traps and is needed for Mycobacterium avium subsp....

Area of Science:

• Microbiology
• Immunology
• Bacterial Pathogenesis

Background:

• Extracellular DNases/nucleases are recognized virulence factors in bacterial pathogens.
• Mycobacterium avium subsp. paratuberculosis (MAP) lacks reported DNase/nuclease activity, despite its role in paratuberculosis.

Purpose of the Study:

• To identify and characterize a putative nuclease (MAP3916c) in MAP K-10.
• To investigate the role of MAP3916c in bacterial virulence, immune evasion, and host-pathogen interactions.

Main Methods:

• Genome analysis to identify the map3916c gene.
• Biochemical characterization of MAP3916c's DNase activity, including optimal conditions (temperature, pH) and cation requirements.
• Site-directed mutagenesis to identify key active site residues.
• In vitro and in vivo experiments to assess MAP3916c's effect on neutrophil extracellular traps (NETs), bacterial colonization, granuloma formation, and cytokine release in a mouse model.

Main Results:

• MAP3916c was confirmed as an extracellular, non-specific DNase requiring divalent cations, particularly Mg2+, with optimal activity at 41°C and pH 9.0.
• Histidine at position 125 was identified as crucial for MAP3916c's DNase activity.
• MAP3916c effectively degraded both in vitro-induced NETs and NETs generated during MAP K-10 infection.
• MAP3916c enhanced MAP K-10 colonization, promoted granuloma formation in mice, and increased the release of IL-1β, IL-6, and TNF-α.

Conclusions:

• MAP3916c is a functional extracellular DNase that plays a significant role in Mycobacterium avium subsp. paratuberculosis virulence.
• MAP3916c contributes to immune evasion by degrading neutrophil extracellular traps (NETs).
• The characterized nuclease activity of MAP3916c provides insights into MAP's pathogenicity and immune evasion mechanisms.