Abstract
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.
Highlights
Mycobacterium tuberculosis (M. tuberculosis) is a Gram-positive bacterium that causes tuberculosis (TB), which remains one of the world’s deadliest communicable diseases
The SignalP server revealed a signal sequence (1 to 31 residues) in the Rv0888 amino acid sequence and, as this hydrophobic sequence might increase the difficulty of expression, the fragment of rv0888 from M. tuberculosis H37Rv was cloned without the predicted signal sequence
A variety of studies characterizing extracellular nucleases have focused on the proteins that derived from Streptococcus agalactiae[6], Streptococcus pyogenes[18] and Staphylococcus aureus[6,19]
Summary
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. M. tuberculosis can proliferate within these immune cells, eventually escaping from the phagosome and migrating to draining lymph nodes to spread the infection[10,11,12]. We identified and characterized Rv0888, the first extracellular nuclease to be reported from M. tuberculosis. We demonstrate that this enzyme is highly active in www.nature.com/scientificreports/. We confirmed that Rv0888 is relevant to mycobacterial pathogenicity
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