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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

Acylated LprG binds lipoglycans in Mtb.SDS-PAGE analysis of proteins and co-purifying molecules isolated from Mtb H37Ra, M. smegmatis or E. coli. (A) Western blot with monoclonal anti-hexahistidine (anti-His6) to detect LprG or LprA. Mycobacterial components associated with lipoproteins were detected using (B) monoclonal anti-LAM antibody CS-35 or (C and D) rabbit polyclonal anti-Mtb antibody that detects both LAM and LM. Blots are representative of at least three independent experiments.
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ppat-1004471-g001: Acylated LprG binds lipoglycans in Mtb.SDS-PAGE analysis of proteins and co-purifying molecules isolated from Mtb H37Ra, M. smegmatis or E. coli. (A) Western blot with monoclonal anti-hexahistidine (anti-His6) to detect LprG or LprA. Mycobacterial components associated with lipoproteins were detected using (B) monoclonal anti-LAM antibody CS-35 or (C and D) rabbit polyclonal anti-Mtb antibody that detects both LAM and LM. Blots are representative of at least three independent experiments.

Mentions: In order to study the association of lipoglycans and glycolipids with LprG in Mtb, the lipoprotein was expressed with a hexahistidine tag and isolated from Mtb lysate by Ni affinity and anion exchange chromatography. These studies used acylated LprG, rather than non-acylated LprG (NA-LprG), which was used in prior studies, as acylation state is likely to affect subcellular localization and intersection with specific glycolipid and lipoglycan species (acylation is linked with translocation across the plasma membrane). SDS-PAGE was used to analyze LprG and any co-purifying molecules. Silver stain and Western blot (Fig. 1A) with anti-hexahistidine antibody showed a single band at ∼25 kDa, corresponding to the approximate molecular weight of LprG. Western blot with CS-35 anti-LAM monoclonal antibody revealed a characteristic diffuse band at ∼37 kDa that was associated with acylated Mtb LprG expressed in both Mtb and M. smegmatis (Fig. 1B). A polyclonal anti-Mtb antibody that detects LAM, LM and PIMs revealed bands at the expected positions for these lipoglycans (∼37 kDa, ∼18 kDa and ∼10 kDa; corresponding to positions of these lipoglycan/glycolipid standards on the gel) in association with Mtb LprG expressed in Mtb and M. smegmatis but not E. coli (Fig. 1C and 1D). The detection of LAM was more intense than the detection of LM and PIMs, and the association of all three lipoglycans/glycolipids was greater when LprG was expressed in Mtb relative to M. smegmatis (Fig. 1B, C). These lipoglycans/glycolipids were not associated with Mtb-expressed acylated LprA, a lipoprotein that is homologous to LprG (hexahistidine-tagged LprA was expressed and purified using the same protocol as for LprG, see Materials and Methods); this control demonstrates the specificity of lipoglycan/glycolipid binding to LprG. These studies establish that acylated LprG binds LAM and LM in Mtb.


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)

Acylated LprG binds lipoglycans in Mtb.SDS-PAGE analysis of proteins and co-purifying molecules isolated from Mtb H37Ra, M. smegmatis or E. coli. (A) Western blot with monoclonal anti-hexahistidine (anti-His6) to detect LprG or LprA. Mycobacterial components associated with lipoproteins were detected using (B) monoclonal anti-LAM antibody CS-35 or (C and D) rabbit polyclonal anti-Mtb antibody that detects both LAM and LM. Blots are representative of at least three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004471-g001: Acylated LprG binds lipoglycans in Mtb.SDS-PAGE analysis of proteins and co-purifying molecules isolated from Mtb H37Ra, M. smegmatis or E. coli. (A) Western blot with monoclonal anti-hexahistidine (anti-His6) to detect LprG or LprA. Mycobacterial components associated with lipoproteins were detected using (B) monoclonal anti-LAM antibody CS-35 or (C and D) rabbit polyclonal anti-Mtb antibody that detects both LAM and LM. Blots are representative of at least three independent experiments.
Mentions: In order to study the association of lipoglycans and glycolipids with LprG in Mtb, the lipoprotein was expressed with a hexahistidine tag and isolated from Mtb lysate by Ni affinity and anion exchange chromatography. These studies used acylated LprG, rather than non-acylated LprG (NA-LprG), which was used in prior studies, as acylation state is likely to affect subcellular localization and intersection with specific glycolipid and lipoglycan species (acylation is linked with translocation across the plasma membrane). SDS-PAGE was used to analyze LprG and any co-purifying molecules. Silver stain and Western blot (Fig. 1A) with anti-hexahistidine antibody showed a single band at ∼25 kDa, corresponding to the approximate molecular weight of LprG. Western blot with CS-35 anti-LAM monoclonal antibody revealed a characteristic diffuse band at ∼37 kDa that was associated with acylated Mtb LprG expressed in both Mtb and M. smegmatis (Fig. 1B). A polyclonal anti-Mtb antibody that detects LAM, LM and PIMs revealed bands at the expected positions for these lipoglycans (∼37 kDa, ∼18 kDa and ∼10 kDa; corresponding to positions of these lipoglycan/glycolipid standards on the gel) in association with Mtb LprG expressed in Mtb and M. smegmatis but not E. coli (Fig. 1C and 1D). The detection of LAM was more intense than the detection of LM and PIMs, and the association of all three lipoglycans/glycolipids was greater when LprG was expressed in Mtb relative to M. smegmatis (Fig. 1B, C). These lipoglycans/glycolipids were not associated with Mtb-expressed acylated LprA, a lipoprotein that is homologous to LprG (hexahistidine-tagged LprA was expressed and purified using the same protocol as for LprG, see Materials and Methods); this control demonstrates the specificity of lipoglycan/glycolipid binding to LprG. These studies establish that acylated LprG binds LAM and LM in Mtb.

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