Bacterial extracellular nucleases play important roles in virulence, biofilm formation, utilization of extracellular DNA as a nutrient, and degradation of neutrophil DNA extracellular traps. However, there is no current data available for extracellular nucleases derived from M. tuberculosis. Herein, we have identified and characterized Rv0888, an extracellular nuclease in M. tuberculosis. The protein was overexpressed in E. coli, and the purified Rv0888 protein was found to require divalent cations for activity, with an optimal temperature and pH of 41 °C and 6.5, respectively. Further results demonstrated that Rv0888 nuclease activity could be inhibited by four Chinese medicine monomers. Based on sequence analysis, Rv0888 nuclease exhibited no homology with any known extracellular nucleases, indicating that Rv0888 is a novel nuclease. Site-directed mutagenesis studies revealed that the H353, D387, and D438 residues play catalytic roles in Rv0888. In vivo infection studies confirmed that Rv0888 is required for infection and is related to pathogenicity, as the persistent ability of recombinant Mycobacterium smegmatis (rMS) Rv0888NS/MS and Rv0888S/MS is significantly higher than pMV262/MS in the lung tissue, and the Rv0888NS/MS and Rv0888S/MS could produce pathological changes in the mice lung. These results show that Rv0888 is relevant to pathogenicity of M. tuberculosis. A variety of bacteria produce extracellular DNases/nucleases that are important for virulence, biofilm formation, utilization of extracellular DNA as a nutrient, and degradation of neutrophil DNA extracellular traps (NETs). Extracellular nucleases produced by Shewanella, Serratia marcescens, Staphylococcus aureus allow these bacteria to degrade extracellular DNA to its basic components: phosphate, carbon, and nitrogen 1-3 . A recent study by Seper et al. demonstrated that wild-type Vibrio cholerae rapidly degraded the DNA component of NETs through the combined activity of two extracellular nucleases, Dns and Xds 4 . In S. aureus, an extracellular nuclease also degraded NETs, triggering caspase-3-mediated death of immune cells 5 . Moreover, nuclease A (Gbs0661), an extracellular nuclease of Streptococcus agalactiae, is required for full virulence of the organism 6 and attacks NETs, which are a component of the host defense response that immobilizes microbes for subsequent clearance by innate immunity machinery, including phagocytosis by macrophages. A Mn 2+ -stimulated deoxyribonuclease (Nuc B) is released by Bacillus subtilis 168 during sporulation in glucose-deficient medium, which degrades structurally important nucleic acids, thereby controlling the development and dispersal of bacterial biofilms 7,8 . Mycobacterium tuberculosis (M. tuberculosis) is a Gram-positive bacterium that causes tuberculosis (TB), which remains one of the world's deadliest communicable diseases. In 2013, an estimated 9.0 million people contracted TB and a subsequent 1.5 million people died from the disease, of whom 360,000 were HIV-positive 9 . M. tuberculosis is phagocytosed by alveolar macrophages and dendritic cells after inhalation into the lung. However, M. tuberculosis can proliferate within these immune cells, eventually escaping from the phagosome and migrating to draining lymph nodes to spread the infection 10-12 . Rv0888, a protein that belongs to the large endonuclease/exonuclease/phosphatase family (Pfam family PF03372) 13 , has sphingomyelinase activity that has been detected in M. tuberculosis culture filtrates 14 . In this study, we identified and characterized Rv0888, the first extracellular nuclease to be reported from M. tuberculosis. We demonstrate that this enzyme is highly active in degrading different types of nucleic acids (chromosomal DNA, double-stranded PCR products, plasmid DNA, and baker's yeast RNA) utilizing divalent metal cations (Ca 2+ and Mn 2+ ). In addition, the nuclease activity of Rv0888 is inhibited by four Chinese medicine monomers (Oleuropein, 6-Gingerol, Corylifolinin, and Acteoside). Finally, we confirmed that Rv0888 is relevant to mycobacterial pathogenicity.
Output: Characterization of Rv0888, a Novel Extracellular Nuclease from Mycobacterium tuberculosis OPEN