Trisaccharides of Phenolic Glycolipids Confer Advantages to Pathogenic Mycobacteria through Manipulation of Host-Cell Pattern-Recognition Receptors

Despite mycobacterial pathogens continue to be a threat to public health, the mechanisms that allow them to persist by modulating the host immune response are poorly understood. Among the factors suspected to play a role are phenolic glycolipids (PGLs), produced notably by the major pathogenic species such as <i>Mycobacterium tuberculosis</i> and <i>Mycobacterium leprae</i>. Here, we report an original strategy combining genetic reprogramming of the PGL pathway in <i>Mycobacterium bovis</i> BCG and chemical synthesis to examine whether sugar variations in the species-specific PGLs have an impact on pattern recognition receptors (PRRs) and the overall response of infected cells. We identified two distinct properties associated with the trisaccharide domains found in the PGLs from <i>M. leprae</i> and <i>M. tuberculosis</i>. First, the sugar moiety of PGL-1 from <i>M. leprae</i> is unique in its capacity to bind the lectin domain of complement receptor 3 (CR3) for efficient invasion of human macrophages. Second, the trisaccharide domain of the PGLs from <i>M. tuberculosis</i> and <i>M. leprae</i> share the capacity to inhibit Toll-like receptor 2 (TLR2)-triggered NF-κB activation, and thus the production of inflammatory cytokines. Consistently, PGL-1 was found to also bind isolated TLR2. By contrast, the simpler sugar domains of PGLs from <i>M. bovis</i> and <i>Mycobacterium ulcerans</i> did not exhibit such activities. In conclusion, the production of extended saccharide domains on PGLs dictates their recognition by host PRRs to enhance mycobacterial infectivity and subvert the host immune response

Structure of PGL-tb and of <i>p</i>-HBAD produced by <i>M. tuberculosis</i>.

<p>The genes involved in the synthesis of the carbohydrate moiety of PGL-tb are indicated. m = 15–17; n, n’ = 16–18; p, p’ = 2–4.</p

Biochemical analysis of products D, E and F.

<p>(<b>A</b>) MALDI-TOF mass spectra of purified product D from the PMM116:pPET1 mutant strain and of purified products E and F from the PMM122:pPET1:pRS26 mutant strain. (<b>B</b>) NMR analysis of products E and F. 1D ¹H-NMR spectra (3.0–6.0 ppm) of native glycolipids E (bottom) and F (upper) (600 MHz, in CDCl₃). The structures of the analyzed compounds are shown below the spectrum and the protons corresponding to the main signals are indicated. The table summarizes the assignments of resonances on the basis of chemical shift correlations deduced from the 2D-COSY spectra of native and per-<i>O</i>-acetylated compound E. Proton resonances shifted by acetylation are written in bold (grey cells). The first column of the table indicates the sugar residue: I, first rhamnosyl residue; II, second rhamnosyl residue; III, terminal fucosyl residue.</p

Effect of PptT depletion on the growth of <i>M. bovis</i> BCG and of <i>M. tuberculosis in vitro.</i>

<p><b>A. </b><i>M. tuberculosis</i> H37Rv wild-type (WT) and the PMM168 mutant strain were grown in 7H9 containing ADC (with Km, Hyg and ATc for PMM168) at 37°C and streaked onto 7H11 plates supplemented with OADC with or without ATc (300 ng/ml). Plates were incubated for 20 days at 37°C. <b>B.</b> The <i>M. tuberculosis</i> PMM168 mutant was grown in 7H9 containing or not containing ATc at 37°C. Numbers of CFU in cultures with ATc (squares) were determined by plating dilutions of theses cultures onto 7H11 plates supplemented with ATc on days 0, 4, 8 and 12. CFU counts in cultures lacking ATc were determined by plating dilutions on 7H11 plates supplemented with ATc (closed circles) or without ATc (open circles) to estimate the number of ATc-independent CFU. <b>C.</b> Bactericidal effect of PptT depletion. The <i>M. bovis</i> BCG PMM99 (left panel) and <i>M. tuberculosis</i> PMM168 (right panel) mutants were grown in 7H9 containing (+) or not containing (−) ATc at 37°C. Numbers of CFU in cultures were determined by plating dilutions of theses cultures onto 7H11 plates supplemented with ATc on days 0 (D0) and 4 (D4). Values are means ± standard deviations (error bars) of CFU counts for three independent experiments. <b>D.</b> PMM99 was grown in media supplemented with Tween-80 with (100, 1, 0.3, 0.1 ng/ml) and without ATc and bacterial growth was monitored by measuring the optical density at 600 nm (OD₆₀₀) (left panel). <i>M. bovis</i> BCG wild-type strain was grown in 7H9 supplemented with Tween-80. Data are representative of two independent experiments. Western blot visualization of PptT in crude cell lysates of PMM99 (5 µg/lane) cultivated for 6 days in a medium with ATc (100, 1, 0.3 ng/ml) and in a crude cell lysate of <i>M. bovis</i> BCG wild-type strain (5, 2, 1, 0.5 µg/lane) (right panel). The control lane was loaded with 100 ng of recombinant PptT fused to a poly-histidine tag produced in <i>E. coli</i>.</p

Biochemical analysis of products B and C from the PMM126:pPET1 mutant.

<p>(<b>A</b>) MALDI-TOF mass spectra of purified product B (left panel) and of purified product C (right panel) from the PMM126:pPET1 mutant strain<b>.</b> (<b>B</b>) NMR analysis of products B and C. 1D ¹H-NMR spectra (1.8–5.6 ppm) of native (bottom) or per-<i>O</i>-acetylated (upper) glycolipid B (600 MHz, in CDCl₃). The structures of the analyzed compounds are shown below the spectrum and the protons corresponding to the main signals are indicated. The tables summarize the assignments of resonances on the basis of chemical shift correlations deduced from the 2D-COSY spectra of native and per-<i>O</i>-acetylated compounds B and C. Proton resonances shifted by acetylation are written in bold (grey cells). The first column of each table indicates the sugar residue: I, first rhamnosyl residue; II, second rhamnosyl residue; III, terminal fucosyl residue.</p

Role of PptT in <i>M. tuberculosis</i>.

<p><b>A.</b> Enzymatic reaction catalyzed by PPTases. CP: carrier protein. <b>B.</b> Schematic diagram of the role of PptT in the biosynthesis pathways for mycolic acids, polyketide-derived lipids and siderophores in <i>M. tuberculosis</i>. DIM: phthiocerol dimycocerosates, PGL: phenolglycolipids, PAT: polyacyltrehaloses, SL: sulfolipids.</p

Localization of <i>Rv2954c</i>, <i>Rv2955c</i>, and <i>Rv2956</i> within the DIM+PGL locus and multiple alignment of the amino acid sequences predicted to be encoded by these genes.

<p>(<b>A</b>) Genetic organisation of the DIM+PGL locus in <i>M. tuberculosis</i> from <i>Rv2953</i> to <i>Rv2962c</i>. Genes belonging to the PGL-tb synthesis pathway are represented by solid black arrows and <i>Rv2954c</i>, <i>Rv2955c</i>, <i>Rv2956</i> by solid gray arrows. <i>Rv2960c</i> and <i>Rv2961</i> (open arrows) encode a hypothetical unknown protein and a putative transposase, respectively, and are probably not involved in PGL biosynthesis. (<b>B</b>) Multiple sequence alignment based on the primary sequences of Rv2954c (residues 1–241), Rv2955c (residues 87–321) and Rv2956 (residues 1–243). Alignment was performed using the CLUSTALW algorithm (EMBnet, <a href="http://www.ch.embnet.org" target="_blank">http://www.ch.embnet.org</a>). Motifs I and II found in SAM-methyltransferases are boxed.</p

Strains and plasmids.

<p>Strains and plasmids.</p

TLC analyses of glycolipids extracted from the wild-type, mutant, and complemented mutant strains of <i>M. tuberculosis.</i>

<p>Lipids extracted from the <i>Rv2954c</i> (<b>A</b>), <i>Rv2955c</i> (<b>B</b>) and <i>Rv2956</i> (<b>C, left panel</b>) <i>M. tuberculosis</i> recombinant strains complemented with pPET1 and from the complemented <i>M. tuberculosis</i> mutant strains were spotted on silica gel G60 plates and separated with CHCl₃/CH₃OH (95∶5, v/v). Glycolipids were visualized by spraying the plates with a 0.2% anthrone solution (w/v) in concentrated H₂SO₄, followed by heating. The positions of PGL-tb and of compounds A, B, C, D, E, F are indicated. Glycolipids from PMM122:pPET1:pRS26 were separated using a Sep-Pak Florisil cartridge and further purified by preparative chromatography on Silica Gel G60. Purified glycoconjugates were spotted on a silica gel G60 plate and separated with CHCl₃/CH₃OH (90∶10, v/v) before visualization with anthrone (<b>C, right panel</b>).</p

<i>In vitro</i> activity of PptT.

<p><b>A.</b> Diagrammatic representation of the domain organization of PKS13 and of ACP and ACPb domains. ACP: Acyl Carrier protein, KS: ketosynthase, AT: acyltransferase, TE: thioesterase. <b>B.</b> ACP activation with CoA and acetyl-CoA. The <i>apo</i>-ACP module was incubated with (+) or without (−) PptT in the presence of either CoA (left panel) or acetyl-CoA (right panel). <i>apo</i>- (a) and <i>holo</i>-ACP (h) forms were separated on urea polyacrylamide gels and stained with Coomassie blue (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003097#s4" target="_blank">materials and methods</a>). M: PageRuler prestained protein ladder plus (Fermentas). <b>C.</b> ACP activation with CoA analogs. PptT or Sfp were incubated with the <i>apo</i>-ACP domain in the presence of either fluorescent CoA analogs (CoA488 or CoA547) or CoA-biotin. Fluorescent <i>holo</i>-ACP forms (h) were resolved by SDS-PAGE and visualized by fluorescence scanning using a Typhoon scanner (GE Healthcare) (upper panel). Biotin-labeled ACP was detected by spotting 5 µl of the reaction mix onto the nitrocellulose membrane and incubation with streptavidin peroxidase followed by enhanced chemiluminescence detection (lower panel). The dashed-line circle shows the drop zone for the PptT reaction.</p