Table 1.
Primers used in this study.
Table 2.
Primers used for PCR of 10 Lip family genes from M. tuberculosis.
Fig 1.
(A) The total lipase activities were assayed using cell lysate supernatants from the 10 recombinant M. smegmatis strains. The p-NP palmitate (C16) was used as the substrate for the lipase activities analysis. The values represent the means ± standard deviations (SD) of three independent experiments. (B) Lipase activity of LipL towards p-NP esters with various chain lengths (C2, acetate; C4, butyrate; C6, caproate; C8, caprylate; C12, laurate; C14, myristate; C16, palmitate; and C18, stearate). The values represent the means ± SD of three independent experiments. (C) The effect of pH on lipase activity of LipL. (D) The effect of temperature on lipase activity of LipL.
Fig 2.
The effect of detergents on lipase activity of LipL.
(A) Addition of Tween 20 at concentration ranging from 0%–1% in the LipL lipase activity assays. (B) Addition of Tween 80 at concentration ranging from 0%–1%. (C) Addition of SDS at concentration ranging from 0 mM–5 mM. (D) Addition of PMSF at concentration ranging from 0 mM–5 mM. Data represent the means ± SD of three independent experiments.
Fig 3.
The analysis of the lipase activities of the mutants.
The six residue substitutions of site-directed mutagenesis were G49A, G50A, G51A, S88A, K91A, and S361A. (A) A schematic of the LipL protein sequence with predicted catalytic motif and the mutations. (B) The experiment was performed in the standard lipase activity assays. The values represent the means ± SD of three independent experiments.
Fig 4.
Homology model of LipL (template: EstA carboxylesterase, PDB code: 3ZYT).
(A) Ribbon view of LipL model which consisted of several α helices and β sheets. The predicted active residues are shown in different colors. (B) Enlarged view of GGG motif (residues G49 and G51 are shown in blue; residue G50 is shown in red). (C) The close-up view of the S-x-x-K motif (residue S88 is shown in red; residue K91 is shown in blue). (D) Enlarged view of the G-x-S-x-G motif (residue S361 is shown in purple). (E) The partially transparent surface representation of LipL. The GGG motif is highlighted shown in blue and red colors. It is noteworthy that the GGG motif is formed a catalytic pocket surface structure. (F) The close-up view of the surface representation of the GGG motif.
Fig 5.
Subcellular localization, the humoral and cell-mediated immune responses of LipL.
(A) Subcellular localization of LipL in M. smegmatis. Bacteria were lysed and fractionated to separate the cytoplasm (Cy) from the cell wall (CW). Equal amounts of protein (20 μg) from each fraction were subjected to SDS-PAGE, transferred onto a nitrocellulose membrane, and probed with either monoclonal anti-His antibodies (top) or rabbit anti-KatG antiserum (bottom). (B and C) The humoral responses induced by the recombinant LipLMs (B) and LipLE (C) proteins in TB patients compared with healthy donors. The plates were coated with purified LipLMs or LipLE. ELISA reactions of LipLMs and LipLE to the sera of either TB patients or healthy controls (HC) were assayed (n = 51 for patients; n = 45 for healthy controls). (D) ELISA reaction of LipLMs to the sera of either TB group 1 or group 2 patients were assayed (n = 29 for group 1; n = 22 for group 2). Student’s t test was used for analysis of statistical significance (P value). Only P values of < 0.05 were considered significant (* P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant). (E) Graphical representation of spleen CD3+ CD8+ T cell percentages from LipLMs and PBS immunized mice. (F) Graphical representation of spleen CD3+ CD4+ T cell percentages from LipLMs and PBS immunized mice. The data represent the means ± standard errors of the means (SEM) of three independent experiments, and the statistically significant differences were revealed using unpaired Student’s t test (* P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant).