Mycobacterium tuberculosis lipoprotein LprG (Rv1411c) binds triacylated glycolipid agonists of Toll-like receptor 2

Abstract

Knockout of lprG results in decreased virulence of Mycobacterium tuberculosis (MTB) in mice. MTB lipoprotein LprG has TLR2 agonist activity, which is thought to be dependent on its N-terminal triacylation. Unexpectedly, here we find that nonacylated LprG retains TLR2 activity. Moreover, we show LprG association with triacylated glycolipid TLR2 agonists lipoarabinomannan, lipomannan and phosphatidylinositol mannosides (which share core structures). Binding of triacylated species was specific to LprG (not LprA) and increased LprG TLR2 agonist activity; conversely, association of glycolipids with LprG enhanced their recognition by TLR2. The crystal structure of LprG in complex with phosphatidylinositol mannoside revealed a hydrophobic pocket that accommodates the three alkyl chains of the ligand. In conclusion, we demonstrate a glycolipid binding function of LprG that enhances recognition of triacylated MTB glycolipids by TLR2 and may affect glycolipid assembly or transport for bacterial cell wall biogenesis.

Fig. 1. NA-LprG carries a mycobacterial TLR2 agonist

(A) HEK293.TLR2/CD14 cells show a dose-dependent IL-8 response to LprA, LprG, and NA-LprG, but no response to NA-LprA. Control HEK293 cells lacking TLR2 and CD14 failed to respond to all four proteins (data not shown). Absence of CD14 (HEK293.TLR2 cells) reduced the apparent potency of NA-LprG but not acylated LprG or LprA (Supplemental Fig. S-1). (B, C) NA-LprG can acquire TLR2 agonist activity from mycobacterial lysates. NA-LprG and NA-LprA were expressed in E. coli, purified by Ni-affinity and anion-exchange chromatography, incubated with control buffer or a lysate of M. smegmatis (B), Mtb H37Ra (C) or Mtb H37Rv (C), repurified by Ni-affinity (B) or Ni-affinity and ion exchange chromatography (C), and incubated with HEK293.TLR2 cells for 12 h. Minor technical differences between the panels resulted in different plateau IL-8 levels, but this was not due to intrinsic differences in activities of materials from M. smegmatis vs. Mtb (Fig. 3 and data not shown). HEK293.TLR2/CD14 cells used in panel A give higher IL-8 secretion responses than HEK293.TLR2 cells used in panels B and C (see Supplemental Fig. S-1). For all data panels, IL-8 production was quantified by ELISA, and data are reported as the mean +/− SD of triplicate HEK293.TLR2 assays. Results are representative of at least 3 independent experiments.

Fig. 2. Crystal structure of NA-LprG reveals a hydrophobic pocket with the potential to carry a TLR2 agonist

(A) LprG structure viewed in ribbons. (B) LprG hydrophobic surface slab view clipped to cavity center (carbon in white, oxygen in red, nitrogen in blue and sulfur in yellow). (C) Electrostatic surface view of the LprG cavity entrance (negative in red, positive in blue, neutral in white). In order to show the entrance, LprG is rotated 90 to the right and 30 down relative to the other views. The cavity entrance is located between β-sheet and α-helices.

Fig. 3. Site-directed mutagenesis to alter the structure of the NA-LprG pocket and binding of TLR2 agonists

(A) Superimposition ribbon view of NA-LprG (blue, V91 in orange) and mutant NA-LprG-V91W (tan, W91 in yellow). In addition to the V91W mutation, NA-LprG and NA-LprG-V91W constructs used for crystallization included minor differences unrelated to the pocket structure (e.g. NA-LprG-V91W has a longer C-terminal coil). Otherwise, overall structures were similar with differences due to the V91W mutation localized to the cavity and entrance. Movement of β3, β4 and the loop between β3 and β4 affects cavity and entrance size. (B) Hydrophobic view of the NA-LprG entrance (white, carbon; red, oxygen; blue, nitrogen; yellow, V91). (C) Hydrophobic view of NA-LprG-V91W (yellow, W91). The extra C-terminal coil of NA-LprG-V91W was removed for better surface comparison. The V91W mutation causes the cavity wall to shift by 3.5 Ǻ. (D) View revealing hydrophobic surface of the NA-LprG cavity. (E) NA-LprG-V91W cavity. The V91W mutation narrows the pocket to reduce space of ligand binding. (F) TLR2 activity of NA-LprG and NA-LprG-V91W expressed in M. smegmatis and tested on HEK293.TLR2 cells. (G) TLR2 activity of NA-LprG and NA-LprG-V91W expressed in E. coli, purified, incubated with Mtb H37Ra or H37Rv lysate and repurified. Data are reported as mean +/− SD of triplicate HEK293.TLR2 assays.

Fig. 4. Triacylated mycobacterial glycolipids are associated with NA-LprG

(A-D) SDS-PAGE analysis of proteins purified from M. smegmatis. Samples were visualized by silver stain (A), Pro-Q stain for carbohydrates (B), polyclonal anti-BCG Western blot (C), and monoclonal anti-His₆ [pink] and anti-LAM [green] (D). The 10-kDa molecular weight marker ran at the gel dye front. Arrows on right that are labeled LAM, LM and PIM represent the positions observed with glycolipid standards run in this gel system. Arrowheads within the gels indicate the expected positions for LAM, LM and PIM whether or not those species are present. See supplemental methods. (E) Negative mode electrospray ionization mass spectrometry of methanol-treated NA-LprG yielded ions corresponding in mass to mycobacterial phospholipids. Ions of m/z 851.5, 1013.5 and 1413.7 were subjected to negative mode collision induced dissociation mass spectrometry, yielding ions that corresponded to masses and fragments indicated in the insets and Fig. S-2. Ions detected near m/z 823.5 correspond to an alternately acylated form of phosphatidylinositol, and ions detected near m/z 949.9 correspond to an H₃PO₄ adduction of phosphatidylinositol. Ions detected near 1175.5 correspond to diacylated PIM2. (F-I) LC-MS analysis was carried out on 4.3 nanomoles of NA-LprG (pink), NA-LprG-V91W (blue), NA-LprA (green) and solvent blank (black). The total ion current trace shows that signals from non-lipidic components of protein preparations were detected at similar levels, serving as a control for equivalent loading of proteins onto the column (F). Mass chromatograms measured in narrow mass ranges corresponding to the masses of phosphatidylinositol (m/z 851.5) (G) diacyl PIM₁ (m/z 1013.6) (H), and triacyl Ac₁PIM₂ (m/z 1413.76) (I) are shown.

Fig. 5

Crystal structure of Ac₁PIM₂ bound to NA-LprG. (A) Ac₁PIM₂ (in cyan) bound to NA-LprG (stereo pair image). Residues (in yellow) in the cavity interact with Ac₁PIM₂. The cavity provides enough space for the tri-acyl chains of Ac₁PIM₂. (B) Structure of Ac₁PIM₂ from NA-LprG-Ac₁PIM₂ (in orange) superimposed onto the structure of NA- LprG-V91W (blue, W91 in yellow). Tryptophan 91 hinders a part of the binding pocket that accommodates one of the Ac₁PIM₂ acyl chains. (C) Electron density of 2F_o-F_c map at 1 sigma level for Ac₁PIM₂ as bound to NA-LprG. (D) View of the cavity entrance with a close-up (colored according to surface electrostatic potential with Ac₁PIM₂ in yellow). The sugar moieties of Ac₁PIM₂ are bound closely to a negatively charged site on the LprG surface.

Fig. 6. LprG binds purified mycobacterial glycolipids and facilitates their recognition by TLR2

(A, B) NA-LprG was purified from E. coli, incubated with LAM or LM from M. smegmatis, repurified and assessed for TLR2 activity using bone marrow-derived macrophages. TNF-alpha production was quantified by ELISA. “LAM:NA-LprG” represents NA-LprG that was incubated with LAM and repurified; “NA-LprG unloaded” represents NA-LprG that was sham-loaded with buffer and repurified; and “Naked LAM” represents glycolipid in the absence of NA-LprG. Data are reported as mean +/− SD of triplicate macrophage assays. (C) Acylated LprG, LprA, and LprG-V91W were purified from M. smegmatis and assessed for TLR2 agonist activity with HEK293.TLR2-CD14 as in Fig. 1. Data are reported as mean +/− SD of triplicate HEK293.TLR2-CD14 assays.