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Phthiocerol dimycocerosate (DIM) is a major virulence factor of the pathogen Mycobacterium tuberculosis ( Mtb ). While this lipid promotes the entry of Mtb into macrophages, which occurs via phagocytosis, its molecular mechanism of action is unknown. Here, we combined biophysical, cell biology, and modeling approaches to reveal the molecular mechanism of DIM action on macrophage membranes leading to the first step of Mtb infection. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry showed that DIM molecules are transferred from the Mtb envelope to macrophage membranes during infection. Multiscale molecular modeling and 31 P-NMR experiments revealed that DIM adopts a conical shape in membranes and aggregates in the stalks formed between 2 opposing lipid bilayers. Infection of macrophages pretreated with lipids of various shapes uncovered a general role for conical lipids in promoting phagocytosis. Taken together, these results reveal how the molecular shape of a mycobacterial lipid can modulate the biological response of macrophages.

Mycobacterium tuberculosis (Mtb) synthesizes a variety of atypical lipids that are exposed at the cell surface and help the bacterium infect macrophages and escape elimination by the cell’s immune responses. In the present study, we investigate the mechanism of action of one family of hydrophobic lipids, the phthiocerol dimycocerosates (DIM/PDIM), major lipid virulence factors. DIM are transferred from the envelope of Mtb to host membranes during infection. Using the polarity-sensitive fluorophore C-Laurdan, we visualized that DIM increase the membrane polarity of a supported lipid bilayer put in contact with mycobacteria, even beyond the site of contact. We observed that DIM activate the complement receptor 3, a predominant receptor for phagocytosis of Mtb by macrophages. DIM also increased the activity of membrane-permeabilizing effectors of Mtb, among which the virulence factor EsxA. This is consistent with previous observations that DIM help Mtb disrupt host cell membranes. Taken together, our data show that transferred DIM spread within the target membrane, remodel lipid organization and increase the activity of host cell receptors and bacterial effectors, diverting in a nonspecific manner host cell functions. We therefore bring new insight into the molecular mechanisms by which DIM increase Mtb’s capability to escape the cell’s immune responses.

Autophagy is an important innate immune defense mechanism that controls Mycobacterium tuberculosis (Mtb) growth inside macrophages. Autophagy machinery targets Mtb-containing phagosomes via xenophagy after damage to the phagosomal membrane due to the Type VII secretion system Esx-1 or via LC3-associated phagocytosis without phagosomal damage. Conversely, Mtb restricts autophagy-related pathways via the production of various bacterial protein factors. Although bacterial lipids are known to play strategic functions in Mtb pathogenesis, their role in autophagy manipulation remains largely unexplored. Here, we report that the lipid virulence factors sulfoglycolipids (SLs) and phthiocerol dimycocerosates (DIMs) control autophagy-related pathways through distinct mechanisms in human macrophages. Using knock-out and knock-in mutants of Mtb and Mycobacterium bovis BCG (Bacille Calmette Guerin) and purified lipids, we found that (i) Mtb mutants with DIM and SL deficiencies promoted functional autophagy via an MyD88-dependent and phagosomal damage-independent pathway in human macrophages; (ii) SLs limited this pathway by acting as TLR2 antagonists; (iii) DIMs prevented phagosomal damage-independent autophagy while promoting Esx-1-dependent xenophagy; (iv) and DIMs, but not SLs, limited the acidification of LC3-positive Mtb compartments. In total, our study reveals an unexpected and intricate role for Mtb lipid virulence factors in controlling autophagy-related pathways in human macrophages, thus providing further insight into the autophagy manipulation tactics deployed by intracellular bacterial pathogens.

The highly contagious human pathogen Mycobacterium tuberculosis is responsible for an estimated 1.5 million deaths each year. A major feature of M. tuberculosis is its unique capacity to survive within the host for decades. In contrast, the evolutionary early-branching rare tubercle bacilli Mycobacterium canettii, which is thought to have originated from the same pool of ancestors as M. tuberculosis, rarely causes tuberculosis (TB) in humans and has a reduced capacity to persist and cause disease in the mouse model. Our objectives are to identify the genetic adaptations which allow persistence of TB bacilli within the host, and to characterise their functional consequences. Using an experimental evolution strategy, we have shown that recurrent in vivo passaging of a M. canettii strain (for more than 2 years) allows clones to emerge as “persistent mutants” that have a greater ability to survive in the host, and gain the characteristics of classical M. tuberculosis strains. Several “persistent mutants” were selected and the persistence phenotype was confirmed in several mouse genetic backgrounds. Next, these mutants were subjected to extensive phenotypic characterisation and were found to have enhanced resistance to stress encountered during infection, and increased virulence in human monocyte-derived macrophages. Currently, the mutations responsible for these phenotypes are being characterised. Comparing the genetic differences between the parental M. canettii strain, the “persistent” M. canettii clones and M. tuberculosis will help identify the molecular mechanisms driving evolution from putative generalist mycobacteria, towards professional human pathogen.

International audience; Although the bovine tuberculosis (TB) agent, Mycobacterium bovis, may infect humans and cause disease, long-term epidemiological data indicate that humans represent a spill-over host in which infection with M. bovis is not self-maintaining. Indeed, human-to-human transmission of M. bovis strains and other members of the animal lineage of the tubercle bacilli is very rare. Here, we report on three mutations affecting the two-component virulence regulation system PhoP/PhoR (PhoPR) in M. bovis and in the closely linked Mycobacterium africanum lineage 6 (L6) that likely account for this discrepancy. Genetic transfer of these mutations into the human TB agent, Mycobacterium tuberculosis, resulted in down-regulation of the PhoP regulon, with loss of biologically active lipids, reduced secretion of the 6-kDa early antigenic target (ESAT-6), and lower virulence. Remarkably, the deleterious effects of the phoPR mutations were partly compensated by a deletion, specific to the animal-adapted and M. africanum L6 lineages, that restores ESAT-6 secretion by a PhoPR-independent mechanism. Similarly, we also observed that insertion of an IS6110 element upstream of the phoPR locus may completely revert the phoPR-bovis-associated fitness loss, which is the case for an exceptional M. bovis human outbreak strain from Spain. Our findings ultimately explain the long-term epidemiological data, suggesting that M. bovis and related phoPR-mutated strains pose a lower risk for progression to overt human TB, with major impact on the evolutionary history of TB.

Several specific lipids of the cell envelope are implicated in the pathogenesis of M. tuberculosis (Mtb), including phthiocerol dimycocerosates (DIM) that have clearly been identified as virulence factors. Others, such as trehalose-derived lipids, sulfolipids (SL), diacyltrehaloses (DAT) and polyacyltrehaloses (PAT), are believed to be essential for Mtb virulence, but the details of their role remain unclear. We therefore investigated the respective contribution of DIM, DAT/PAT and SL to tuberculosis by studying a collection of mutants, each with impaired production of one or several lipids. We confirmed that among those with a single lipid deficiency, only strains lacking DIM were affected in their replication in lungs and spleen of mice in comparison to the WT Mtb strain. We found also that the additional loss of DAT/PAT, and to a lesser extent of SL, increased the attenuated phenotype of the DIM-less mutant. Importantly, the loss of DAT/PAT and SL in a DIM-less background also affected Mtb growth in human monocyte-derived macrophages (hMDMs). Fluorescence microscopy revealed that mutants lacking DIM or DAT/PAT were localized in an acid compartment and that bafilomycin A1, an inhibitor of phagosome acidification, rescued the growth defect of these mutants. These findings provide evidence for DIM being dominant virulence factors that mask the functions of lipids of other families, notably DAT/PAT and to a lesser extent of SL, which we showed for the first time to contribute to Mtb virulence.

International audience; Although phthiocerol dimycocerosates (DIM) are major virulence factors of Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, little is known about their mechanism of action. Localized in the outer membrane of mycobacterial pathogens, DIM are predicted to interact with host cell membranes. Interaction with eukaryotic membranes is a property shared with another virulence factor of Mtb, the early secretory antigenic target EsxA (also known as ESAT‐6). This small protein, which is secreted by the type VII secretion system ESX‐1 (T7SS/ESX‐1), is involved in phagosomal rupture and cell death induced by virulent mycobacteria inside host phagocytes. In this work, by the use of several knock‐out or knock‐in mutants of Mtb or Mycobacterium bovis BCG strains and different cell biological assays, we present conclusive evidence that ESX‐1 and DIM act in concert to induce phagosomal membrane damage and rupture in infected macrophages, ultimately leading to host cell apoptosis. These results identify an as yet unknown function for DIM in the infection process and open up a new research field for the study of the interaction of lipid and protein virulence factors of Mtb.

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 Mycobacterium tuberculosis and Mycobacterium leprae. Here, we report an original strategy combining genetic reprogramming of the PGL pathway in Mycobacterium bovis 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 M. leprae and M. tuberculosis. First, the sugar moiety of PGL-1 from M. leprae 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 M. tuberculosis and M. leprae 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 M. bovis and Mycobacterium ulcerans 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.

Several mycobacteria are remarkably successful human pathogens that cause wellknown infections such as tuberculosis and leprosy or emerging diseases such as Buruli ulcer. On encountering the virulent mycobacteria, the host develops a strong immune response which is dedicated to combat the invasion while bacilli have evolved several strategies to counteract and adapt to this hostile environment. Being positioned at the bacterial surface, mycobacterial lipids contribute to the interplay between host and pathogen. This review will briefly discuss some unusual mycobacterial lipids that have been identified, focusing on their immunomodulatory properties.

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 allo-threoninyl residue and a 2-succinyl-3,4-di-O-CH3-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.

Optic atrophy type 1 (OPA1, MIM 165500) is a dominantly inherited optic neuropathy occurring in 1 in 50,000 individuals that features progressive loss in visual acuity leading, in many cases, to legal blindness. Phenotypic variations and loss of retinal ganglion cells, as found in Leber hereditary optic neuropathy (LHON), have suggested possible mitochondrial impairment. The OPA1 gene has been localized to 3q28-q29 (refs 13-19). We describe here a nuclear gene, OPA1, that maps within the candidate region and encodes a dynamin-related protein localized to mitochondria. We found four different OPA1 mutations, including frameshift and missense mutations, to segregate with the disease, demonstrating a role for mitochondria in retinal ganglion cell pathophysiology.

Neutrophils contain several populations of secretory granules with characteristic sets of proteins. Granule proteins are sorted into their respective granule types by temporal regulation of their expression during cell differentiation and/or by specific targeting signals. We investigated the expression of some granule proteins in human promyelocytic NB4 cells. Like other myeloid cell lines which can be differentiated into neutrophils, NB4 cells lack the specific-granule population. We report here that, nevertheless, they express the specific-granule matrix protein lactoferrin, when differentiated with retinoic acid. Lactoferrin and the azurophil-granule protein beta-glucuronidase were simultaneously expressed, whereas myeloperoxidase expression had stopped, showing that azurophil-granule proteins are not all produced concomitantly. Cell fractionation by Percoll gradient revealed that while beta-glucuronidase co-fractionated with myeloperoxidase, lactoferrin was mostly contained in a vesicular compartment free of markers for azurophil granules, plasma membrane, and Golgi. This vesicular compartment was not implicated in regulated exocytosis since it was not mobilized by secretagogues, which, in parallel, induced the release of myeloperoxidase. Furthermore, the specific granule-membrane protein cytochrome b558 also became expressed during NB4-cell differentiation. However, it did not co-localize with lactoferrin but was present in the plasma-membrane fraction. Therefore, differentiation of NB4 cells with retinoic acid leads to the expression of specific- and azurophil-granule proteins and provides a unique cell line model to study the mechanisms involved in the sorting of azurophil- and specific-granule proteins.

As previously described for endothelin-3, platelet exposure to cyclic GMP-elevating agents such as sodium nitroprusside and M&B-22948 (2-o-propoxyphenyl-8-azapurin-6-one), a cGMP phosphodiesterase inhibitor, lowered Ca2+ mobilization in response to thrombin. Interestingly, when cGMP phosphodiesterases were blocked, endothelin-3 produced a dose-dependent cGMP accumulation (P < 0.001). Since endothelin-3 has been proposed to decrease the activity of Ca2+ accumulating pumps, we examined whether this latter effect could be mediated by a rise in cGMP content. Cyclic GMP decreased in a dose-dependent manner the initial rate and plateau value of the ATP-dependent 45Ca2+ uptake in platelet membrane vesicles (P = 0.006 for each). Furthermore, combined treatment with endothelin-3 and M&B-22948 or a moderate concentration of Na+-nitroprusside further reduced the thrombin-evoked Ca2+ discharge (P = 0.004 and 0.01, respectively), suggesting that endothelin-3 pre-exposure had reduced the amount of mobilizable Ca2+. We propose that the depletion of platelet Ca2+ stores and the reduction of Ca2+ release evoked by endothelin-3 could be due, at least in part, to the elevation of cGMP content and to a decrease in Ca2+ accumulating pump activity.

1. The present study demonstrates that endothelin-3 (ET-3), previously shown to attenuate thrombin-evoked aggregation of human platelets, delayed the dose-dependent aggregatory response to thapsigargin (Tg). As this Ca(2+)-ATPase inhibitor induces platelet activation in part through the depletion of internal Ca(2+)-stores, we examined the influence of ET-3 on Ca2+ discharge from internal pools. 2. Cytosolic Ca2+ concentration was evaluated with Fura-2 in the absence of Ca2+ influx. Platelet preincubation for 15 min with 5 x 10(-7) M ET-3 decreased the Ca2+ release evoked by thrombin and U46619, a thromboxane-mimetic. However, ET-3 did not affect Ca2+ movements induced by 1 microM ADP. Addition of Tg (0.5 to 5 microM) to resting platelets induced a cytosolic [Ca2+] rise with concentration-dependent increase of the initial rate and decrease of the time to reach the peak. ET-3 slowed down these dose-dependent effects with a more marked influence on the responses induced by low concentrations of Tg. 3. ET-3 did not modify the Ca2+ response to another Ca(2+)-ATPase inhibitor, 2,5-di-(tert-butyl)-1,4-benzohydroquinone(tBuBHQ). The thromboxane A2 receptor antagonist, SQ 29548, reduced by 53% the calcium signal evoked by 1 microM Tg, which became similar to that induced by 15 microM tBuBHQ. Under these conditions, the ET-3 effects were suppressed. A subsequent addition of thrombin induced a substantial further Ca2+ increase which was again sensitive to ET-3. 4. ET-3 attenuates Ca2+ mobilization from an internal pool dependent on the stimulation of thrombin and thromboxane A2 receptors and insensitive to the direct effect of Ca2+-ATPase inhibitors. The small but significant inhibitory effect of ET-3 leads us to propose that endothelin-3 acts as a modulator of platelet activation.

Chronic myelogenous leukemia (CML) is characterized by an initial chronic phase that invariably evolves to the more aggressive phase of blast crisis. Although the determinants of this transition are still unknown, it has been shown that the blast crisis is accompanied by genetic instability.The expression and activity of the error-prone DNA polymerase beta (pol beta) were investigated in blood samples from CML patients, by Western blotting and by an in vitro replication assay, respectively.Pol beta expression and activity were significantly higher in CML samples compared to those of healthy donors.Our results suggest that the excess of pol beta in CML could contribute to the genetic instability observed during the evolution of the disease from the chronic phase to blast crisis.

Protein tyrosine phosphatases from several microorganisms have been shown to play a role as virulence factors by modifying the phosphorylation/dephosphorylation equilibrium in cells of their host. Two tyrosine phosphatases, MptpA and MptpB, secreted by Mycobacterium tuberculosis, have been identified. Expression of MptpA is upregulated upon infection of monocytes, but its role in host cells has not been elucidated. A eukaryotic expression vector containing the mptpA cDNA has been transfected into macrophages. We report that MptpA reduced phagocytosis of mycobacteria, opsonized zymosan or zymosan, but had no effect on phagocytosis of IgG-coated particles. We also noted that the presence of F-actin at the surface of phagosomes containing opsonized zymosan was significantly increased in cells expressing MptpA. In the presence of recombinant MptpA, the process of actin polymerization at the surface of isolated phagosomes was increased; this was not the case in the presence of the phosphatase-dead mutant MptpAC11S. MptpA had no effect when IgG-coated particles were present inside isolated phagosomes. These results indicate that, like other tyrosine phosphatases of pathogens, MptpA plays a role in phagocytosis and actin polymerization. However, MptpA had no effect on IgG particles, suggesting that its putative substrate(s) is not linked to the signaling pathways of Fcγ receptors.  2005 Elsevier SAS. All rights reserved.

To analyze the mechanisms by which endothelin-3 (ET-3) attenuates agonist-mediated Ca2+ mobilization from an internal pool, we investigated ET-3 effects on 45Ca2+ uptake in platelet membrane vesicles. They were compared to those of thapsigargin (Tg), a specific inhibitor of the dense tubule Ca2+ pumps. In the absence of ATP, ET-3 up to 1 microM did not affect the amount of Ca2+ bound to membrane sites. In the presence of ATP, ET-3 dose-dependently reduced the initial rate and the extent of Ca2+ uptake (p < 0.001). In comparison, Tg dose-dependently inhibited both the ATP-independent Ca2+ binding (p < 0.001) and the ATP-dependent Ca2+ accumulation (p < 0.001), with half-maximal effects at 7 nM. Pretreatment with 1 microM ET-3 decreased the inhibitory effect of 10 nM Tg, but only on the initial rate of ATP-dependent Ca2+ uptake (p = 0.04; n = 6). These results indicate that ET-3 is functionally coupled to Ca(2+)-ATPases of the dense tubules. Its inhibitory effects are probably due to inhibition of the catalytic cycle of the Ca2+ pumps. Such inhibition could lead to a depletion of Ca2+ stores and therefore to reduced Ca2+ release in response to agonists.

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 allo-threoninyl residue and a 2-succinyl-3,4-di-O-CH3-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 mycobacterial lipoarabinomannans (LAMs) are glycosylphosphatidyl-myo-inositol-anchored lipoglycans with diverse biological activities. It has been shown that purified LAMs interact directly, or indirectly, through receptors with the plasma membrane receptors of target cells located in domains rich in glycosylphosphatidylinositol-anchored proteins that contain Src family protein tyrosine kinases. To examine whether LAMs could activate Src-related kinases, human neutrophils were exposed to mannosylated LAMs (ManLAMs) purified from the vaccinal strainMycobacterium bovisBCG and to phosphoinositol-capped LAMs (AraLAM or PILAM) obtained from the nonpathogenic speciesMycobacterium smegmatis. We report first that both ManLAMs and PILAMs activate Hck in a rapid and transient manner and second that complete deacylation of ManLAM abolished its effect on Hck activity, thereby demonstrating that acylation of LAM but not mannosylation is critical for Hck activation. These data indicate that Hck is involved in the signaling pathway of LAMs, molecules known for their ability to trigger several responses in eukaryotic cells.

The antiaggregatory properties of trimetazidine were investigated further by analyzing its effects on cytosolic calcium and proton concentrations, well-known regulators of platelet reactivity. Aggregatory responses of washed platelets were assessed by turbidometry, and cytosolic Ca2+ concentration ([Ca2+]i) and pH (pHi) were determined by their respective fluorescent probes: Fura-2 and BCECF. Preincubation with trimetazidine dose-dependently inhibited platelet aggregation induced by 0.05 U/ml thrombin (p0.001). At concentrationsor = 1 mM, trimetazidine did not affect the resting [Ca2+]i value but slightly alkalinized the cytosol by 0.05 +/- 0.03 pH units (p0.02, n = 11). In platelets stimulated by 0.05 U/ml thrombin, 0.1 mM trimetazidine did not modify pHi variations but decreased [Ca2+]i variations (p0.003, n = 16), blunting by 28 +/- 6% the transient peak of [Ca2+]i (p0.006) and decreasing by 6 +/- 2% the equilibrium value (p0.005). These inhibitory effects were inversely dependent on thrombin concentrations (p0.004, n = 21) and were abolished in the virtual absence of external Ca2+. Trimetazidine therefore attenuates the Ca2+ influx evoked by thrombin, thereby limiting Ca2+ accumulation in stimulated platelets. Such a protective effect may participate in the antiaggregatory properties of trimetazidine.

The species-specific phenolic glycolipid 1 (PGL-1) is suspected to play a critical role in the pathogenesis of leprosy, a chronic disease of the skin and peripheral nerves caused by Mycobacterium leprae. Based on studies using the purified compound, PGL-1 was proposed to mediate the tropism of M. leprae for the nervous system and to modulate host immune responses. However, deciphering the biological function of this glycolipid has been hampered by the inability to grow M. leprae in vitro and to genetically engineer this bacterium. Here, we identified the M. leprae genes required for the biosynthesis of the species-specific saccharidic domain of PGL-1 and reprogrammed seven enzymatic steps in M. bovis BCG to make it synthesize and display PGL-1 in the context of an M. leprae-like cell envelope. This recombinant strain provides us with a unique tool to address the key questions of the contribution of PGL-1 in the infection process and to study the underlying molecular mechanisms. We found that PGL-1 production endowed recombinant BCG with an increased capacity to exploit complement receptor 3 (CR3) for efficient invasion of human macrophages and evasion of inflammatory responses. PGL-1 production also promoted bacterial uptake by human dendritic cells and dampened their infection-induced maturation. Our results therefore suggest that M. leprae produces PGL-1 for immune-silent invasion of host phagocytic cells., Author Summary Mycobacterium leprae, the causative agent of leprosy, is a chronic human disease responsible for irreversible peripheral nerve damage and deformities. Lepromatous leprosy, the most severe form of the disease, is accompanied by T-cell unresponsiveness, suggesting that M. leprae has evolved strategies to modulate host immune responses. However, the molecular mechanisms of M. leprae infection remain poorly understood, mainly because this bacterium has been to date impossible to grow in vitro. The present study reports an innovative approach to study the contribution of a phenolic glycolipid (PGL-1) specific of M. leprae in the cross-talk of the pathogen with host cells. We reprogrammed a biosynthetic pathway in a surrogate host, M. bovis BCG, to make it synthesize and display PGL-1 in the context of a mycobacterial envelope. Using this novel microbial tool, we found that PGL-1 production enhances the cellular invasiveness of BCG and promotes the entry via complement receptor 3-mediated phagocytosis. Bacterial uptake via this route was associated with reduced inflammatory responses in infected human macrophages. In addition, we showed that PGL-1 production inhibited the infection-induced maturation of human dendritic cells. Our findings thus provide new insights into the contribution and molecular mechanisms of action of PGL-1 in leprosy pathogenesis.

Pathogenic mycobacteria parasitize macrophages and reside within phagosomes, which do not fuse with lysosomal granules. Mycobacteria are also internalized by neutrophils, which possess at least two types of granules, specific and azurophil granules, the latter being specialized lysosomes. Here, we investigated the ability of mycobacteria to inhibit the fusion of these granules with their phagosomes in human neutrophils. It was found that when pathogenic (Mycobacterium kansasii and Mycobacterium avium) or nonpathogenic (Mycobacterium smegmatis and Mycobacterium phlei) mycobacteria were internalized by neutrophils, they induced the inhibition of azurophil granule fusion with phagosomes even when they were serum opsonized. In contrast, secretion of specific granule content and production of O2−, both of which contribute to the neutrophil bactericidal response, were triggered. Hck is a Src family tyrosine kinase associated with azurophil granules. During internalization of zymosan, azurophil granules fused with phagosomes and Hck was activated and translocated to the phagosomal membrane, whereas in neutrophils engulfing mycobacteria, Hck did not translocate and remained unactivated. The activation of the tyrosine kinase Fgr was not affected. These results indicate that 1) pathogenic and nonpathogenic mycobacteria trigger similar bactericidal responses in neutrophils, 2) phagocytosis and fusion of azurophil granules can be uncoupled by mycobacteria, and 3) Hck could be one of the key elements of the azurophil secretory pathway that are altered during phagocytosis of mycobacteria.

Glycopeptidolipids (GPLs) are a class of species- or type-specific mycobacterial lipids and major constituents of the cell envelopes of many non-tuberculous mycobacteria. To determine the function of GPLs in the physiology of these bacteria, a mutant of Mycobacterium smegmatis in which the gene encoding a mycobacterial nonribosomal peptide synthetase has been inactivated by transposon mutagenesis was analysed. Labelling experiments indicated that half of the bacterial GPLs were located on the cell surface and represented 85% of the surface-exposed lipids of the parent strain whereas the mutant was defective in the production of the GPLs. Compared to the parent smooth morphotype strain, the GPL-deficient mutant strain exhibited a rough colony morphology, an increase of the cell hydrophobicity and formed huge aggregates. As a consequence, the mutant cells were no longer able to bind ruthenium red, as observed by transmission electron microscopy. The altered surface properties of the mutant cells also affected the phagocytosis of individual bacilli by human monocyte-derived macrophages since mutant cells were internalized more rapidly than cells from the parent strain. Nevertheless, no specific release of surface constituents into the culture broth of the mutant was observed, indicating that the cell surface is composed of substances other than GPLs and that these are essential for maintaining the architecture of the outermost layer of the cell envelope. Importantly, the absence of these major extractable lipids of M. smegmatis from the mutant strain has a profound effect on the uptake of the hydrophobic chenodeoxycholate by cells, indicating that GPLs are involved in the cell wall permeability barrier of M. smegmatis. Altogether, these data showed that, in addition to being distinctive markers of numerous mycobacterial species, GPLs play a role in the bacterial phenotype, surface properties and cell wall permeability.

Two subfamilies of the polar glycopeptidolipids (GPLs) located on the surface of Mycobacterium smegmatis, along with unknown phospholipids, were recently shown to participate in the nonopsonic phagocytosis of mycobacteria by human macrophages (Villeneuve, C., G. Etienne, V. Abadie, H. Montrozier, C. Bordier, F. Laval, M. Daffe, I. Maridonneau-Parini, and C. Astarie-Dequeker. 2003. Surface-exposed glycopeptidolipids of Mycobacterium smegmatis specifically inhibit the phagocytosis of mycobacteria by human macrophages. Identification of a novel family of glycopeptidolipids. J. Biol. Chem. 278: 51291–51300). As demonstrated herein, a phospholipid mixture that derived from the methanol-insoluble fraction inhibited the phagocytosis of M. smegmatis. Inhibition was essentially attributable to phosphatidylinositol mannosides (PIMs), namely PIM2 and PIM6, because the purified phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylinositol were inactive. This was further confirmed using purified PIM2 and PIM6 from M. bovis BCG that decreased by half the internalization of M. smegmatis. Both compounds also inhibited the uptake of M. tuberculosis and M. avium but had no effect on the internalization of zymosan used as a control particle of the phagocytic process. When coated on latex beads, PIM2 and polar GPL (GPL III) favored the particle entry through complement receptor 3. GPL III, but not PIM2, also directed particle entry through the mannose receptor. Therefore, surface-exposed mycobacterial PIM and polar GPL participate in the receptor-dependent internalization of mycobacteria in human macrophages.

In macrophages, lysosomes are suspected to have a heterogenous population of vesicles. This study was thus undertaken to identify and to characterize lysosomal compartments in human macrophages. Hck is a Src-family tyrosine kinase associated with secretory lysosomes in neutrophils and with cytoplasmic vesicles in macrophages that fuse with phagosomes. We identified these Hck-positive vesicles and compared them to CD63-positive, M6PR-negative vesicles known as classical lysosomes. Hck vesicles exhibited lysosomal features. Indeed, Hck-positive vesicles could be loaded with rhodamine-dextran, which has been shown to accumulate in lysosomal compartments. Hck was delivered to zymosan-containing phagosomes at a late stage of the maturation process, which occurs after the fusion with CD63-positive lysosomes. Finally, when mycobacteria were used to prevent phagolysosome biogenesis, Hck was not recruited to phagosomes. Moreover, Hck lysosomes were physically and functionally distinct from CD63-lysosomes. For instance, sucrose induced swelling of CD63-lysosomes without affecting Hck-positive ones. Only CD63-lysosomes fused with phagosomes in a microtubule-dependent manner. Entry of particles through the mannose receptor and Fcγ receptors drove the phagosome towards a fusion with CD63-lysosomes, whereas only Fcγ receptors induced the mobilisation of Hck-lysosomes. This study provides further evidence for the existence of sub-populations of lysosomes in macrophages: one stained by CD63 and another one characterized by the presence of Hck. Therefore, Hck represents a new tool to study the fusion dynamics of lysosomal compartments and their subversion by several intracellular pathogens.