Surface-exposed Glycopeptidolipids of Mycobacterium smegmatis Specifically Inhibit the Phagocytosis of Mycobacteria by Human Macrophages

Abstract

Phagocytosis by macrophages represents the early step of the mycobacterial infection. It is governed both by the nature of the host receptors used and the ligands exposed on the bacteria. The outermost molecules of the nonpathogenic Mycobacterium smegmatis were extracted by a mechanical treatment and found to specifically and dose dependently inhibit the phagocytosis of both M. smegmatis and the opportunistic pathogen M. kansasii by human macrophages derived from monocytes. The inhibitory activity was attributed to surface lipids because it is extracted by chloroform and reduced by alkaline hydrolysis but not by protease treatment. Fractionation of surface lipids by adsorption chromatography indicated that the major inhibitory compounds consisted of phospholipids and glycopeptidolipids (GPLs). Mass spectrometry and nuclear magnetic resonance spectroscopy analyses, combined with chemical degradation methods, demonstrated the existence of a novel family of GPLs that consists of a core composed of the long-chain tripeptidyl amino-alcohol with a di-O-acetyl-6-deoxytalosyl unit substituting the allothreoninyl residue and a 2-succinyl-3,4-di-O-CH 3 -rhamnosyl unit linked to the alaninol end of the molecules. These compounds, as well as diglycosylated GPLs at the alaninol end and de-O-acylated GPLs, but not the non-serovar-specific di-O-acetylated GPLs, inhibited the phagocytosis of M. smegmatis and M. avium by human macrophages at a few nanomolar concentration without affecting the rate of zymosan internalization. At micromolar concentrations, the native GPLs also inhibit the uptake of both M. tuberculosis and M. kansasii. De-O-acylation experiments established the critical roles of both the succinyl and acetyl substituents. Collectively, these data provide evidence that surface-exposed mycobacterial glycoconjugates are efficient competitors of the interaction between macrophages and mycobacteria and, as such, could represent pharmacological tools for the control of mycobacterial infections.

The cell envelope structure and properties of Mycobacterium smegmatis mc2155: is there a clue for the unique transformability of the strain?

“…); the organic phases were washed with water and evaporated to dryness to yield crude lipid extracts. The mycolate-containing lipids were separated from the other types of lipids by precipitation with methanol (Villeneuve et al, 2003). Both kinds of lipids were further fractionated on a Florisil column irrigated with chloroform and then with a stepwise gradient of increasing concentrations of methanol and water in chloroform (Ortalo-Magné et al, 1996).…”

“…receptors for mycobacteriophages (Furuchi & Tokunaga, 1972;Goren et al, 1972), colony morphology Billman-Jacobe et al, 1999) or binding to human monocytederived macrophages (Villeneuve et al, 2003). They are also involved in the sliding motility of mc 2 155, as shown by the non-motility of GPL-deficient mutants (Martinez et al, 1999;Etienne et al, 2002).…”

“…GPLs are among the characteristic lipids of mycobacteria (Daffé & Lemassu 2000) and share a common lipopeptidyl core consisting of a mixture of 3-hydroxy and 3-methoxy C 26-34 fatty acids amidated by the tripeptide D-Phe-D-allo-Thr-D-Ala and terminated by L-alaninol (Daffé et al, 1983b). The diversity of GPLs has been recently extended in M. smegmatis (Ojha et al, 2002;Villeneuve et al, 2003). The major and first-described GPL of M. smegmatis (GPL I) is composed of, in addition to the core, a di-O-acetyl-6-deoxytalosyl residue linked to allo-threoninyl and a tri-Omethylrhamnosyl unit attached to the alaninol residue (Brennan, 1988).…”

“…The major and first-described GPL of M. smegmatis (GPL I) is composed of, in addition to the core, a di-O-acetyl-6-deoxytalosyl residue linked to allo-threoninyl and a tri-Omethylrhamnosyl unit attached to the alaninol residue (Brennan, 1988). Other components of this family include the deacetylated GPLs I (or GPLs IIa) and the hyperglycosylated GPLs I (or GPLs IIb), which possess a second di-Omethylrhamnosyl unit linked to alaninol (Ojha et al, 2002;Villeneuve et al, 2003). The succinylated GPLs (or GPLs IIIa and IIIb, with one and two rhamnosyl units, respectively) carry a succinyl substituent on the terminal di-O-methylrhamnosyl unit linked to the alaninol end (Villeneuve et al, 2003).…”

“…Other components of this family include the deacetylated GPLs I (or GPLs IIa) and the hyperglycosylated GPLs I (or GPLs IIb), which possess a second di-Omethylrhamnosyl unit linked to alaninol (Ojha et al, 2002;Villeneuve et al, 2003). The succinylated GPLs (or GPLs IIIa and IIIb, with one and two rhamnosyl units, respectively) carry a succinyl substituent on the terminal di-O-methylrhamnosyl unit linked to the alaninol end (Villeneuve et al, 2003). Crude cellular GPLs were prepared by methanol precipitation of the total cellular lipids and analysed by TLC (Fig.…”