table_1.doc

<p>Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), which mainly causes pulmonary injury and tubercles. Although macrophages are generally considered to harbor the main cells of M. tuberculosis, new evidence suggests that neutrophils are rapidly recruited to the infected lung. M. tuberculosis itself, or its early secreted antigenic target protein 6 (ESAT-6), can induce formation of neutrophil extracellular traps (NETs). However, NETs trap mycobacteria but are unable to kill them. The role of NETs’ formation in the pathogenesis of mycobacteria remains unclear. Here, we report a new M. tuberculosis extracellular factor, bifunctional enzyme Rv0888, with both nuclease and sphingomyelinase activities. Rv0888 sphingomyelinase activity can induce NETs’ formation in vitro and in the lung of the mice and enhance the colonization ability of Mycobacterium smegmatis in the lungs of mice. Mice infected by M. smegmatis harboring Rv0888 sphingomyelinase induced pathological injury and inflammation of the lung, which was mainly mediated by NETs, induced by Rv0888 sphingomyelinase, associated protein (myeloperoxidase) triggered caspase-3. In summary, the study sheds new light on the pathogenesis of mycobacteria and reveals a novel target for TB treatment.</p

CD spectra of the Rv0045c protein at different pH and temperatures.

<p>The CD measurements were made in the presence of various pH (A) at pH 2.0 (black), pH 3.0 (red), pH 4.0 (yellow), pH 6.0 (blue), pH 7.0 (purple), pH 8.0 (pink), pH 9.0 (green), pH 10.0 (gray), pH 11.0 (coral) and pH 12.0 (light green) at room temperature, and different temperatures (B) at 10°C (black), 20°C (gray), 30°C (yellow), 40°C (green), 50°C (blue), 60°C (purple) and 70°C (red) at pH 7.5, respectively. Values represent the mean ± SD of three analyses. The concentration of the Rv0045c protein was fixed at 0.35 mg/mL (20 mM Tris, pH 7.5).</p

SDS-PAGE analysis for expression and affinity chromatography of the Rv0045c protein.

<p>Lane 1, culture pellet (uninduced); Lane 2, culture pellet (induced with 0.3 mM IPTG at 16°C); Lane 3, the supernatant of induced cells after sonication; Lane 4, fluid through Ni²⁺-affinity chromatography column; Lane 5 and 7: purified Rv0045c protein eluted by 20 mM Tris, 150 mM NaCl, 200 mM Imidazole, pH 7.5; Lane 6 and 8: purified Rv0045c protein eluted by 20 mM Tris, 150 mM NaCl, 500 mM Imidazole, pH 7.5; Lane M: molecular mass markers.</p

MALDI-TOF peptide mass fingerprint (PMF) spectrometry of the Rv0045c protein.

<p>The PMF analysis was made from fragments of purified Rv0045c protein derived through trypsin digestion. The expected tryptic masses clearly matched, with 1 Da tolerance, the calculated values. The sequence coverage of these fragments was shown in bold red.</p

Relative enzyme activity of the Rv0045c protein toward <i>p</i>-nitrophenyl derivatives at pH 7.0 and 37°C.

<p><i>ND</i>: Not detectable</p>a<p>The specific activity toward <i>p</i>-nitrophenyl caproate (C₆) corresponding to 3.5 U/mg protein/min was defined as 100%. And one hydrolase unit is the quantity of enzyme required to increase absorbance by 0.01 units at 405 nm per min.</p

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<p>Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), which mainly causes pulmonary injury and tubercles. Although macrophages are generally considered to harbor the main cells of M. tuberculosis, new evidence suggests that neutrophils are rapidly recruited to the infected lung. M. tuberculosis itself, or its early secreted antigenic target protein 6 (ESAT-6), can induce formation of neutrophil extracellular traps (NETs). However, NETs trap mycobacteria but are unable to kill them. The role of NETs’ formation in the pathogenesis of mycobacteria remains unclear. Here, we report a new M. tuberculosis extracellular factor, bifunctional enzyme Rv0888, with both nuclease and sphingomyelinase activities. Rv0888 sphingomyelinase activity can induce NETs’ formation in vitro and in the lung of the mice and enhance the colonization ability of Mycobacterium smegmatis in the lungs of mice. Mice infected by M. smegmatis harboring Rv0888 sphingomyelinase induced pathological injury and inflammation of the lung, which was mainly mediated by NETs, induced by Rv0888 sphingomyelinase, associated protein (myeloperoxidase) triggered caspase-3. In summary, the study sheds new light on the pathogenesis of mycobacteria and reveals a novel target for TB treatment.</p

Purification of the Rv0045c protein by ion exchange chromatography and gel filtration chromatography.

<p>The Rv0045c protein was purified by anion exchange chromatography (A), cation exchange chromatography (B), and gel filtration chromatography (C). The purity was checked by SDS-PAGE analysis after each purification procedure.</p

Effects of temperature and pH on enzyme activity of the Rv0045c protein.

<p>The enzyme activities were measured using <i>p-butyrate caprylate (C₆)</i> as substrate in the presence of mild temperatures (36°C–40°C) at pH 6.0 (red), pH 7.0 (green) and pH 8.0 (blue). Values represent the mean ± SD of five analyses. The concentration of the Rvoo45c protein was fixed at 0.2 mg/mL (20 mM Tris, pH 7.5). The enzyme activities were expressed as units hydrolase/mg protein/min (one hydrolase unit is the quantity of enzyme required to increase absorbance by 0.01 units at 405 nm per min).</p

Enzymes Identified as Structurally Homologous to Rv0045c through DALI.

a<p>NRES = No. of aligned residues.</p>b<p>RN = Reference Number.</p

Sequence alignment of Rv0045c with other structurally homologous enzymes.

<p>Included enzymes are E-2AMS hydrolase (PDB ID: 3KXP), methylesterase PME-1 (PDB ID: 3C5V), hydrolase YP_496220.1 (PDB ID: 3BWX), CarC enzyme (PDB ID: 1J1I), esterase ybfF (PDB ID: 3BF7) and soluble epoxide hydrolase (PDB ID: 1EHY). Secondary structural elements and every the tenth residue of Rv0045c are indicated above the alignment. The “nucleophile elbow” of G-X-S-X-G sequence motif is marked with green stars and the nucleophilic serine residue Ser with a blue circle. The other catalytic residue is labeled using a purple triangle. Strictly conserved residues with the identity of >80% are highlighted by pink front.</p