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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

Mannan competition with Mtb lipoglycans for binding to LprG implicates a role for polysaccharide components of lipoglycans in LprG binding.(A) Binding of Mannan to LprG was assessed as in Fig. 2 at the indicated concentrations of mannan. (B) The ability of LM to compete with mannan for binding to LprG was assessed by SPR with sequential injection of LM (“1st injection”) for 3 min followed by buffer for ∼10 min as the instrument prepared for injection of mannan (“2nd injection”). This injection order was chosen due to the fast dissociation rate for mannan. The dissociation rate for LM (shown here) is slower than for LAM, but experiments with LAM provided qualitatively similar results. Results for mannan binding in panels A and B are directly comparable except for the absence (panel A) or presence (panel B) of prior LM injection. Results for panels A and B are representative of two independent experiments. (C) Mannan inhibited LAM binding to LprG in a solid phase competitive binding assay. LprG was incubated with mannan at the indicated concentrations for 30 min and added to wells of plates coated with ManLAM. After incubation and washing, the plates were incubated with anti-LprG antibody followed by an HRP-linked secondary antibody to detect LprG binding to ManLAM (see Materials and Methods). Data are expressed as the means from three independent experiments.
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ppat-1004471-g005: Mannan competition with Mtb lipoglycans for binding to LprG implicates a role for polysaccharide components of lipoglycans in LprG binding.(A) Binding of Mannan to LprG was assessed as in Fig. 2 at the indicated concentrations of mannan. (B) The ability of LM to compete with mannan for binding to LprG was assessed by SPR with sequential injection of LM (“1st injection”) for 3 min followed by buffer for ∼10 min as the instrument prepared for injection of mannan (“2nd injection”). This injection order was chosen due to the fast dissociation rate for mannan. The dissociation rate for LM (shown here) is slower than for LAM, but experiments with LAM provided qualitatively similar results. Results for mannan binding in panels A and B are directly comparable except for the absence (panel A) or presence (panel B) of prior LM injection. Results for panels A and B are representative of two independent experiments. (C) Mannan inhibited LAM binding to LprG in a solid phase competitive binding assay. LprG was incubated with mannan at the indicated concentrations for 30 min and added to wells of plates coated with ManLAM. After incubation and washing, the plates were incubated with anti-LprG antibody followed by an HRP-linked secondary antibody to detect LprG binding to ManLAM (see Materials and Methods). Data are expressed as the means from three independent experiments.

Mentions: To investigate whether mannan components of LAM and LM contribute to lipoglycan binding to LprG, we tested the ability of S. cerevisiae mannan to bind to LprG. S. cerevisiae mannan was selected since its structure is comparable to the polysaccharide components of Mtb lipoglycans, except for the absence of the phosphatidyl-myo-inositol lipid anchor present in Mtb lipoglycans. SPR assays revealed mannan binding to LprG in a dose dependent manner, although with a lower affinity compared to LAM and LM (KD = 8.8×10−5 M, Fig. 5A and Table 3, compare to Table 1). Next, to test whether mannan and Mtb lipoglycans have overlapping binding site(s) on LprG, we assessed their ability to compete for binding to LprG in SPR assays. The potential approach to saturate immobilized LprG with mannan in a first injection and subsequently inject LAM or LM was not feasible due to the rapid dissociation rate of mannan (4.5×10−2 sec−1), which allowed dissociation of pre-bound mannan before injection of LAM or LM could be completed. Accordingly, we first injected the lipoglycan (e.g. LM, 2.5 µM), which bound to LprG (Fig. 5B, “first injection”); a subsequent injection of mannan (2.5 µM; Fig. 5B, “second injection”) did not reveal mannan binding in contrast to the ability of mannan to bind to LprG in the absence of LM (compare Fig. 5A and 5B). Similar results were obtained with LAM and mannan. These results indicate that mannan and Mtb lipoglycans compete for binding to LprG. To confirm this conclusion with a different approach, we used a solid phase binding assay that measured LprG binding to plate-immobilized ManLAM. LprG bound to plate-immobilized ManLAM in a dose-dependent fashion. When LprG was incubated with mannan, however, the binding of LprG to ManLAM was inhibited by the presence of mannan in a dose-dependent manner (Fig. 5C). These data further indicate that mannan and Mtb lipoglycans compete for binding to LprG. Taken together, these results support our other evidence that mannose residues contribute to the binding of Mtb lipoglycans to LprG, in addition to contributions of acyl chain interactions.


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)

Mannan competition with Mtb lipoglycans for binding to LprG implicates a role for polysaccharide components of lipoglycans in LprG binding.(A) Binding of Mannan to LprG was assessed as in Fig. 2 at the indicated concentrations of mannan. (B) The ability of LM to compete with mannan for binding to LprG was assessed by SPR with sequential injection of LM (“1st injection”) for 3 min followed by buffer for ∼10 min as the instrument prepared for injection of mannan (“2nd injection”). This injection order was chosen due to the fast dissociation rate for mannan. The dissociation rate for LM (shown here) is slower than for LAM, but experiments with LAM provided qualitatively similar results. Results for mannan binding in panels A and B are directly comparable except for the absence (panel A) or presence (panel B) of prior LM injection. Results for panels A and B are representative of two independent experiments. (C) Mannan inhibited LAM binding to LprG in a solid phase competitive binding assay. LprG was incubated with mannan at the indicated concentrations for 30 min and added to wells of plates coated with ManLAM. After incubation and washing, the plates were incubated with anti-LprG antibody followed by an HRP-linked secondary antibody to detect LprG binding to ManLAM (see Materials and Methods). Data are expressed as the means from three independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4214796&req=5

ppat-1004471-g005: Mannan competition with Mtb lipoglycans for binding to LprG implicates a role for polysaccharide components of lipoglycans in LprG binding.(A) Binding of Mannan to LprG was assessed as in Fig. 2 at the indicated concentrations of mannan. (B) The ability of LM to compete with mannan for binding to LprG was assessed by SPR with sequential injection of LM (“1st injection”) for 3 min followed by buffer for ∼10 min as the instrument prepared for injection of mannan (“2nd injection”). This injection order was chosen due to the fast dissociation rate for mannan. The dissociation rate for LM (shown here) is slower than for LAM, but experiments with LAM provided qualitatively similar results. Results for mannan binding in panels A and B are directly comparable except for the absence (panel A) or presence (panel B) of prior LM injection. Results for panels A and B are representative of two independent experiments. (C) Mannan inhibited LAM binding to LprG in a solid phase competitive binding assay. LprG was incubated with mannan at the indicated concentrations for 30 min and added to wells of plates coated with ManLAM. After incubation and washing, the plates were incubated with anti-LprG antibody followed by an HRP-linked secondary antibody to detect LprG binding to ManLAM (see Materials and Methods). Data are expressed as the means from three independent experiments.
Mentions: To investigate whether mannan components of LAM and LM contribute to lipoglycan binding to LprG, we tested the ability of S. cerevisiae mannan to bind to LprG. S. cerevisiae mannan was selected since its structure is comparable to the polysaccharide components of Mtb lipoglycans, except for the absence of the phosphatidyl-myo-inositol lipid anchor present in Mtb lipoglycans. SPR assays revealed mannan binding to LprG in a dose dependent manner, although with a lower affinity compared to LAM and LM (KD = 8.8×10−5 M, Fig. 5A and Table 3, compare to Table 1). Next, to test whether mannan and Mtb lipoglycans have overlapping binding site(s) on LprG, we assessed their ability to compete for binding to LprG in SPR assays. The potential approach to saturate immobilized LprG with mannan in a first injection and subsequently inject LAM or LM was not feasible due to the rapid dissociation rate of mannan (4.5×10−2 sec−1), which allowed dissociation of pre-bound mannan before injection of LAM or LM could be completed. Accordingly, we first injected the lipoglycan (e.g. LM, 2.5 µM), which bound to LprG (Fig. 5B, “first injection”); a subsequent injection of mannan (2.5 µM; Fig. 5B, “second injection”) did not reveal mannan binding in contrast to the ability of mannan to bind to LprG in the absence of LM (compare Fig. 5A and 5B). Similar results were obtained with LAM and mannan. These results indicate that mannan and Mtb lipoglycans compete for binding to LprG. To confirm this conclusion with a different approach, we used a solid phase binding assay that measured LprG binding to plate-immobilized ManLAM. LprG bound to plate-immobilized ManLAM in a dose-dependent fashion. When LprG was incubated with mannan, however, the binding of LprG to ManLAM was inhibited by the presence of mannan in a dose-dependent manner (Fig. 5C). These data further indicate that mannan and Mtb lipoglycans compete for binding to LprG. Taken together, these results support our other evidence that mannose residues contribute to the binding of Mtb lipoglycans to LprG, in addition to contributions of acyl chain interactions.

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