Limits...
Mycobacterium tuberculosis lipoprotein LprG binds lipoarabinomannan and determines its cell envelope localization to control phagolysosomal fusion.

Shukla S, Richardson ET, Athman JJ, Shi L, Wearsch PA, McDonald D, Banaei N, Boom WH, Jackson M, Harding CV - PLoS Pathog. (2014)

Bottom Line: We report that LprG expressed in Mtb binds to lipoglycans, such as lipoarabinomannan (LAM), that mediate Mtb immune evasion.Lipoglycan binding to LprG was dependent on both insertion of lipoglycan acyl chains into a hydrophobic pocket on LprG and a novel contribution of lipoglycan polysaccharide components outside of this pocket.An lprG mutant (Mtb ΔlprG) had lower levels of surface-exposed LAM, revealing a novel role for LprG in determining the distribution of components in the Mtb cell envelope.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Case Western Reserve University/University Hospitals Case Medical Center, Cleveland, Ohio, United States of America.

ABSTRACT
Mycobacterium tuberculosis (Mtb) virulence is decreased by genetic deletion of the lipoprotein LprG, but the function of LprG remains unclear. We report that LprG expressed in Mtb binds to lipoglycans, such as lipoarabinomannan (LAM), that mediate Mtb immune evasion. Lipoglycan binding to LprG was dependent on both insertion of lipoglycan acyl chains into a hydrophobic pocket on LprG and a novel contribution of lipoglycan polysaccharide components outside of this pocket. An lprG mutant (Mtb ΔlprG) had lower levels of surface-exposed LAM, revealing a novel role for LprG in determining the distribution of components in the Mtb cell envelope. Furthermore, this mutant failed to inhibit phagosome-lysosome fusion, an immune evasion strategy mediated by LAM. We propose that LprG binding to LAM facilitates its transfer from the plasma membrane into the cell envelope, increasing surface-exposed LAM, enhancing cell envelope integrity, allowing inhibition of phagosome-lysosome fusion and enhancing Mtb survival in macrophages.

No MeSH data available.


Related in: MedlinePlus

SPR analysis of substrate binding to LprG.(A) Schematic representation of mycobacterial glycolipids and lipoglycans used in this study. (B–F) Substrate binding to LprG was assessed by SPR. LprG was immobilized on a CM5 sensor chip. Sensograms were obtained by injecting increasing concentrations of (B) PIM2 (C) PIM6 (D) LM (E) ManLAM and (F) PI-LAM. Binding was measured as response units (RU). Binding curves were calculated with BIA evaluation 3.1 software with subtraction of non-specific binding of the substrates to the sensor chip control cells without immobilized LprG. Results are representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4214796&req=5

ppat-1004471-g002: SPR analysis of substrate binding to LprG.(A) Schematic representation of mycobacterial glycolipids and lipoglycans used in this study. (B–F) Substrate binding to LprG was assessed by SPR. LprG was immobilized on a CM5 sensor chip. Sensograms were obtained by injecting increasing concentrations of (B) PIM2 (C) PIM6 (D) LM (E) ManLAM and (F) PI-LAM. Binding was measured as response units (RU). Binding curves were calculated with BIA evaluation 3.1 software with subtraction of non-specific binding of the substrates to the sensor chip control cells without immobilized LprG. Results are representative of three independent experiments.

Mentions: To explore relationships between glycolipid structure and LprG-binding properties, we studied a panel of mycobacterial glycolipids and lipoglycans, including PIM2, PIM6, LM, ManLAM and PI-LAM, all of which share a mannosyl-phosphatidyl-myo-inositol domain with additional specific structural features (Fig. 2A). All of these molecules are expressed by Mtb except for PI-LAM, which is expressed by non-pathogenic mycobacteria. PIM2 (a precursor of PIM6, LM and LAM) has mannose residues at positions 2 and 6 of the myo-inositol ring of PI (Fig. 2A). PIM2 bound to LprG in a dose-dependent manner (Fig. 2B) but with relatively low affinity (KD = 1.09×10−6 M, Table 1). PIM6, LM, ManLAM and PI-LAM all bound to LprG (Fig. 2C–F). Of these glycolipids, LM, which consists of a long linear mannan chain extending from position 6 of the myo-inositol ring, bound to LprG with the highest affinity (KD = 1.58×10−9 M, Fig. 2D, Table 1). PIM6, which consists of a much shorter mannoside motif attached to position 6 of the myo-inositol ring, bound to LprG with the second highest affinity (KD = 4.5×10−8 M, Fig. 2C, Table 1). LAM is generated from LM by the addition of a branched arabinan domain with species-specific terminal caps (ManLAM for Mtb, PI-LAM for non-pathogenic mycobacteria, Fig. 2A). ManLAM and PI-LAM bound to LprG with lower affinities than LM and PIM6 (KD of 1.09×10−7 M for ManLAM and 1.6×10−6 M for PI-LAM, Fig. 2E–F, Table 1). Despite sharing the common acylated core structure that was previously implicated in binding to the hydrophobic pocket of LprG, these glycolipids and lipoglycans displayed distinct kinetics for LprG binding, indicating a previously unknown contribution of polysaccharide structures to LprG.


Mycobacterium tuberculosis lipoprotein LprG binds lipoarabinomannan and determines its cell envelope localization to control phagolysosomal fusion.

Shukla S, Richardson ET, Athman JJ, Shi L, Wearsch PA, McDonald D, Banaei N, Boom WH, Jackson M, Harding CV - PLoS Pathog. (2014)

SPR analysis of substrate binding to LprG.(A) Schematic representation of mycobacterial glycolipids and lipoglycans used in this study. (B–F) Substrate binding to LprG was assessed by SPR. LprG was immobilized on a CM5 sensor chip. Sensograms were obtained by injecting increasing concentrations of (B) PIM2 (C) PIM6 (D) LM (E) ManLAM and (F) PI-LAM. Binding was measured as response units (RU). Binding curves were calculated with BIA evaluation 3.1 software with subtraction of non-specific binding of the substrates to the sensor chip control cells without immobilized LprG. Results are representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4214796&req=5

ppat-1004471-g002: SPR analysis of substrate binding to LprG.(A) Schematic representation of mycobacterial glycolipids and lipoglycans used in this study. (B–F) Substrate binding to LprG was assessed by SPR. LprG was immobilized on a CM5 sensor chip. Sensograms were obtained by injecting increasing concentrations of (B) PIM2 (C) PIM6 (D) LM (E) ManLAM and (F) PI-LAM. Binding was measured as response units (RU). Binding curves were calculated with BIA evaluation 3.1 software with subtraction of non-specific binding of the substrates to the sensor chip control cells without immobilized LprG. Results are representative of three independent experiments.
Mentions: To explore relationships between glycolipid structure and LprG-binding properties, we studied a panel of mycobacterial glycolipids and lipoglycans, including PIM2, PIM6, LM, ManLAM and PI-LAM, all of which share a mannosyl-phosphatidyl-myo-inositol domain with additional specific structural features (Fig. 2A). All of these molecules are expressed by Mtb except for PI-LAM, which is expressed by non-pathogenic mycobacteria. PIM2 (a precursor of PIM6, LM and LAM) has mannose residues at positions 2 and 6 of the myo-inositol ring of PI (Fig. 2A). PIM2 bound to LprG in a dose-dependent manner (Fig. 2B) but with relatively low affinity (KD = 1.09×10−6 M, Table 1). PIM6, LM, ManLAM and PI-LAM all bound to LprG (Fig. 2C–F). Of these glycolipids, LM, which consists of a long linear mannan chain extending from position 6 of the myo-inositol ring, bound to LprG with the highest affinity (KD = 1.58×10−9 M, Fig. 2D, Table 1). PIM6, which consists of a much shorter mannoside motif attached to position 6 of the myo-inositol ring, bound to LprG with the second highest affinity (KD = 4.5×10−8 M, Fig. 2C, Table 1). LAM is generated from LM by the addition of a branched arabinan domain with species-specific terminal caps (ManLAM for Mtb, PI-LAM for non-pathogenic mycobacteria, Fig. 2A). ManLAM and PI-LAM bound to LprG with lower affinities than LM and PIM6 (KD of 1.09×10−7 M for ManLAM and 1.6×10−6 M for PI-LAM, Fig. 2E–F, Table 1). Despite sharing the common acylated core structure that was previously implicated in binding to the hydrophobic pocket of LprG, these glycolipids and lipoglycans displayed distinct kinetics for LprG binding, indicating a previously unknown contribution of polysaccharide structures to LprG.

Bottom Line: We report that LprG expressed in Mtb binds to lipoglycans, such as lipoarabinomannan (LAM), that mediate Mtb immune evasion.Lipoglycan binding to LprG was dependent on both insertion of lipoglycan acyl chains into a hydrophobic pocket on LprG and a novel contribution of lipoglycan polysaccharide components outside of this pocket.An lprG mutant (Mtb ΔlprG) had lower levels of surface-exposed LAM, revealing a novel role for LprG in determining the distribution of components in the Mtb cell envelope.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Case Western Reserve University/University Hospitals Case Medical Center, Cleveland, Ohio, United States of America.

ABSTRACT
Mycobacterium tuberculosis (Mtb) virulence is decreased by genetic deletion of the lipoprotein LprG, but the function of LprG remains unclear. We report that LprG expressed in Mtb binds to lipoglycans, such as lipoarabinomannan (LAM), that mediate Mtb immune evasion. Lipoglycan binding to LprG was dependent on both insertion of lipoglycan acyl chains into a hydrophobic pocket on LprG and a novel contribution of lipoglycan polysaccharide components outside of this pocket. An lprG mutant (Mtb ΔlprG) had lower levels of surface-exposed LAM, revealing a novel role for LprG in determining the distribution of components in the Mtb cell envelope. Furthermore, this mutant failed to inhibit phagosome-lysosome fusion, an immune evasion strategy mediated by LAM. We propose that LprG binding to LAM facilitates its transfer from the plasma membrane into the cell envelope, increasing surface-exposed LAM, enhancing cell envelope integrity, allowing inhibition of phagosome-lysosome fusion and enhancing Mtb survival in macrophages.

No MeSH data available.


Related in: MedlinePlus