Mycobacterium tuberculosiscell envelope lipids and the host immune response

Mycobacterium tuberculosis infects about one-third of the world's population, causing ≈ 3 million deaths annually (Dye et al., 1999). The organism is a slow-growing bacillus that is transmitted by the respiratory route. Although it is capable of causing disease in most organs, pulmonary involvement is most common. Soon after infection, the bacilli are phagocytosed by alveolar macrophages and survive within early phagosomes. Innate immune responses directed by macrophages predominate early in infection. Subsequent recruitment of dendritic cells leads to cell-mediated responses involving CD4+ and CD8+ T cells and eventual granuloma formation (for a review, see Fenton and Vermeulen, 1996; Flynn and Chan, 2001). The vast majority of immunocompetent individuals are able to contain, but not eliminate, the pathogen in pulmonary granulomas, leading to latent tuberculosis infection (Manabe and Bishai, 2000). In a small minority of cases, through unclear mechanisms, persistent bacilli can reactivate to form disease many years to decades after initial infection. An interesting feature of mycobacteria is the extraordinarily high lipid content of the cell envelope, constituting up to 40% of their dry weight (Anderson, 1940). In addition to conferring unique tinctorial properties, which aid in the diagnosis of infected clinical specimens, the mycobacterial cell envelope (see Fig. 1) is important in directing host–pathogen interactions (Besra and Chatterjee, 1994; Brennan and Nikaido, 1995). Schematic representation of the cell envelope of M. tuberculosis (Besra and Chatterjee, 1994; Brennan and Nikaido, 1995; Iseman, 2000). The components include the (A) plasma membrane, (B) peptidoglycans, (C) arabinogalactan, (D) mannose-capped lipoarabinomannan, (E) plasma membrane- and cell envelope-associated proteins, (F) mycolic acids and (G) glycolipid surface molecules associated with the mycolic acids. Certain cell envelope-associated lipids, such as phthiocerol dimycocerosate (PDIM), have received significant attention recently (Camacho et al., 1999; Cox et al., 1999), because they are considered to be important virulence factors. Recent advances in mycobacterial genetic techniques and the availability of the M. tuberculosis genome (Cole et al., 1998) have provided the opportunity to test directly the virulence of PDIM-deficient mutants. For example, mutation of mmpL7 (Cox et al., 1999), the gene product of which may be involved in PDIM transport, and mutation of genes involved in PDIM synthesis, including msl7 and pks10 (Sirakova et al., 2003) and pks7 (Rousseau et al., 2003a), yield strains with attenuated growth in the murine model. Although experimental evidence supports the hypothesis that PDIM is important for virulence, the effects of this molecule on host immune responses have not been elucidated. Other virulence factors have been implicated in the immunopathogenesis of tuberculosis (TB) and may therefore represent potential targets for future therapeutic or preventive interventions. This review will focus on the immunomodulatory properties of the following cell envelope-associated lipids of M. tuberculosis: (i) lipoarabinomannan; (ii) sulpholipids; (iii) mycolic acid-containing glycolipids; and (iv) the 19 kDa lipoprotein. Lipoarabinomannan (LAM) is a phosphatidylinositol-anchored lipoglycan composed of a mannan core with oligoarabinosyl-containing side-chains with diverse biological activities (Hunter and Brennan, 1990; Chatterjee et al., 1991; Chatterjee and Khoo, 1998). LAM is a prominent component of the mycobacterial cell envelope, accounting for up to 5 mg g−1 bacterial weight (Hunter et al., 1986). LAM structure differs according to mycobacterial species, and three general classes of LAM have been described: (i) ManLAM, from the virulent strains Erdman and H37Rv and the avirulent strains H37Ra and BCG (Chatterjee et al., 1992a; Prinzis et al., 1993), which is characterized by extensive mannose capping of the arabinan termini; (ii) phospho-myo-inositol-capped LAM (PILAM), found in the rapidly growing mycobacteria M. smegmatis and M. fortuitum (Nigou et al., 2003); and (iii) AraLAM, which was described in the rapidly growing M. chelonae and lacks mannosylation in its arabinan termini (Guerardel et al., 2002). Two distinct pools of ManLAM have been described, namely parietal and cellular, which differ based on their phosphatidylinositol anchor and the extent of mannose capping (Gilleron et al., 2000), and in their immunological properties (Nigou et al., 1997). Although there is significant heterogeneity between LAM molecules with respect to glycosylation and acylation (Nigou et al., 2003), differences in biological activity between the major classes of LAM have been attributed primarily to the heavy mannose capping of ManLAM or to differences in the phosphatidylinositol anchor (Barnes et al., 1992; Chatterjee et al., 1992b). The sulpholipids of M. tuberculosis are five structurally related sulphatides. Sulpholipid-I (SL-I), the principal sulphatide of the family, has been identified as a 2,3,6,6′-tetraacyl-α,α′-d-trehalose 2′-sulphate (Goren et al., 1976a). The sulpholipids are mycobacterial cell envelope molecules with pleiotrophic biological activities, including antitumour activity (Yarkoni et al., 1979). The extent of sulpholipid expression by various M. tuberculosis strains has been correlated with strain virulence in the guinea pig model (Goren, 1982). Recent work has identified essential genes for SL-I biosynthesis, including pks2 (Sirakova et al., 2001) and mmpL8 (Converse et al., 2003), thus providing the opportunity to study the virulence of these gene mutants in animal models. Interestingly, although neither mutant can produce SL-I, pks2 mutants demonstrate no reduction in replication, persistence or pathogenicity in mice or guinea pigs (Rousseau et al., 2003b), whereas mmpL8 mutants are attenuated for growth in the mouse model of TB (Converse et al., 2003), suggesting that SL-I per se is not required for bacillary growth in vivo. The difference in growth phenotypes between these mutants may be explained if the mmpL8 gene product acts as a transporter for other surface molecules in addition to SL-I that mediate host–pathogen interactions, or if the accumulation of a precursor molecule in the sulpholipid biosynthesis pathway in mmpL8 mutants interferes with in vivo growth-related pathways. Mycolic acids are high-molecular-weight α-alkyl, β-hydroxy fatty acids found in the genera Mycobacterium, Nocardia, Corynebacterium and Rhodococcus (Brennan and Nikaido, 1995). Covalently attached to arabinogalactan, mycolic acids, together with other lipids forming the outer leaflet, constitute a very hydrophobic barrier (Draper, 1998), responsible for resistance to certain drugs (Nikaido, 2001). Mycolic acid-containing glycolipids and, in particular, trehalose-6,6′-dimycolate (TDM), which is the most prominent and best-studied representative of this class of molecules, exert a number of immunomodifying effects. TDM, also known as cord factor, was first identified in virulent M. tuberculosis, which led to speculation concerning its role in virulence and in the process of serpentine cording, a phenomenon observed in virulent mycobacteria (Bloch, 1950). However, its role in cording was later challenged when TDM was also found in other non-cording mycobacterial species (Goren, 1972). TDM has a number of biological activities including high toxicity to mice (Behling et al., 1993), potent adjuvant activity by stimulating cell-mediated immunity with co-administration of protein antigen (Ribi et al., 1982), induction of non-specific resistance to infectious agents (Parant et al., 1977) and antitumour (Nolibe et al., 1986) and proangiogenic activities (Saita et al., 2000; Sakaguchi et al., 2000). The 19 kDa lipoprotein is a member of a family of prokaryotic lipoproteins that have been found extensively in both Gram-positive and Gram-negative bacteria, including Treponema pallidum, Mycoplasma species and Borrelia burgdorferi (Brightbill et al., 1999). In members of the M. tuberculosis complex, this lipoprotein is anchored in the cell wall by its lipid moiety (Young and Garbe, 1991). Although its function remains largely unknown, the 19 kDa lipoprotein has been implicated in a range of immunological responses. The immunological activity of the lipoprotein has been attributed to the NH2-terminal triacylated lipopeptide region, as removal of the fatty acyl moieties abolishes the immunoregulatory functions of the parent molecule (Brightbill et al., 1999). Mycobacterium tuberculosis cell envelope lipids have been shown to have effects on neutrophil and monocyte migration. AraLAM, but not ManLAM, induces the chemotaxis of monocyte-derived macrophages and peripheral blood monocytes (Bernardo et al., 1998). Other studies have shown that, compared with ManLAM, AraLAM is a more potent inducer of the chemoattractant cytokines JE (murine homologue of MCP-1), KC (murine homologue of GROα), MCP-3, pentraxin-3 and interleukin (IL)-8 (Roach et al., 1993; Zhang et al., 1995; Vouret-Craviari et al., 1997a,b). In a mouse model, intratracheal administration of AraLAM or BCG LAM induced significant neutrophil recruitment into the lungs, but intratracheally administered ManLAM failed to induce neutrophil migration (Strohmeier and Fenton, 1999). Therefore, mannose capping of LAM may represent a strategy by virulent M. tuberculosis to circumvent the activation of a pulmonary granulocytic response, which may be important in the early immune response to mycobacterial infection (Appelberg et al., 1995; Saunders and Cheers, 1996) and the enhancement of mycobactericidal activity in the lungs (Fulton et al., 2002). Like AraLAM, TDM has chemotactic activity for many immune cells, including neutrophils, monocytes and macrophages (Ofek and Bekierkunst, 1976; Lima et al., 2001). Whereas intraperitoneal inoculation of delipidated M. tuberculosis in mice results in intense recruitment of polymorphonuclear cells into the peritoneal cavity, intraperitoneal injection of intact M. tuberculosis or of purified TDM results in greater recruitment of mononuclear cells (Lima et al., 2001). Mycobacterium tuberculosis cell envelope lipids appear to induce neutrophil activation. Both M. bovis BCG ManLAM and M. smegmatis phosphatidylinositol-capped LAM (PILAM) were able to activate the protein tyrosine kinase Hck in human neutrophils (Astarie-Dequeker et al., 2000). Interestingly, complete deacylation of ManLAM abolished its effect on Hck activity, demonstrating that acylation of LAM, but not mannosylation, is critical for Hck activation (Astarie-Dequeker et al., 2000). Like LAM, SL-I also activates neutrophils in vitro. At low SL-I can neutrophils to whereas SL-I directly neutrophils to et al., 1991). respect to the effects of M. tuberculosis cell envelope lipids appear to be more capping of LAM to be critical in directing responses. AraLAM from avirulent mycobacteria to be more ManLAM in responses from macrophages (for see Chatterjee and Khoo, and Fenton, 1999). AraLAM is a potent inducer of whereas ManLAM from the virulent M. tuberculosis Erdman strain induces or no in macrophages et al., 1999). The which to expression in murine was more induced in macrophages and murine macrophages by AraLAM compared with ManLAM and 1995). In addition to a inducer of ManLAM may directly thus the of M. ManLAM and expression in human mononuclear and induces monocyte expression of a tyrosine that may function to responses et al., 1998). ManLAM from M. tuberculosis and M. bovis BCG failed to induce activation of macrophages et al., 1999). Although the role of activation in host M. tuberculosis is not there is evidence that T cell activation is important et al., 2002). of ManLAM to induce may therefore be important strategy by which M. tuberculosis the host immune In addition to activation et al., 1993), ManLAM can induce growth et al., which may and T cell activation and to of responses and immunity M. tuberculosis et al., 1998). to macrophages the mannose LAM is through the pathway to it may to are to the cell they can be by T cells et al., 1995; et al., 1997). is evidence that LAM may be from this pathway as a virulence LAM has been observed in the M. tuberculosis et al., and LAM and other lipid have been to the host the and et al., 2000). LAM have been found in the after of mycobacteria by in cells et al., 2000). In to their effect on neutrophil sulpholipids have been implicated in resistance of M. tuberculosis to to be involved in the of (Goren et al., sulpholipids were shown to of the pathogen by activation (Goren et al., with sulphatide of human monocytes by or in to that of monocytes after with et al., This effect of SL-I on monocyte was when SL-I was after of monocytes by suggesting that SL-I directly with monocyte and not involved in However, a study challenged these by that sulphatide of monocytes with in activity of these monocytes after in et al., The in the results of these studies was attributed to differences in experimental including of SL-I, of in of monocytes and the of monocyte et al., these the effects of SL-I on function and their in TB TDM can immune responses in M. tuberculosis infection by of the cell et al., 1996; et al., ManLAM and SL-I, TDM has been shown to activate macrophages and induce non-specific resistance to of with TDM in and of et al., and (Yarkoni and Bekierkunst, by TDM the of a response through induction of and et al., and by of cells et al., 1999). However, the effects of TDM on M. there is no evidence to that these effects in of bacilli by the of M. tuberculosis of the pathogen in mouse bacterial in macrophages was of the with purified TDM et al., 2002). et that in reduction in of and which were with purified TDM has been implicated in the of because it between in et al., 1991). these a role for TDM in activation of macrophages and induction of a response after M. tuberculosis infection, but also in enhancement of mycobacterial within infected as a of early lipoproteins have been shown to be molecules of the immune The M. tuberculosis 19 kDa lipoprotein was identified as a major inducer of by human macrophages and of and of (Brightbill et al., 1999). activation are through a human and the fatty acyl moieties of the lipoprotein (Brightbill et al., 1999). the other infection of human monocyte-derived macrophages with M. smegmatis the 19 kDa lipoprotein in of and which may to T cell activation et al., 2001). with although the rapidly growing species M. and M. which not the 19 kDa induce in mice with M. tuberculosis et al., et al., this immunity was when mice were with a M. smegmatis strain the 19 kDa lipoprotein et al., 2001). In addition to their effects on bacterial lipoproteins have been shown to induce of cells through human et al., 1999). the 19 kDa lipoprotein has been identified as a molecule M. of monocytes and macrophages et al., 2000). Although evidence that is associated with mycobacterial et al., 2000), other studies have shown that induction of alveolar by avirulent mycobacterial species, results in bacillary et al., 2000; et al., The between the and properties of the 19 kDa lipoprotein has been attributed to the of infection in the et al., 2000). In the of has a in which to and activation and 1998). cells are critical in host as they are capable of leading to of immune responses. ManLAM has been shown to have effects on dendritic cell et found that ManLAM from M. bovis BCG was able to by human dendritic cells with of the mannose or the phosphatidylinositol anchor this mannan as as a for the mannose by dendritic cells, the that of the mannose by ManLAM to dendritic cells that may to the persistence of virulent mycobacteria within cells (Nigou et al., 2001). a dendritic was found to mediate and of M. bovis BCG by dendritic cells et al., of ManLAM to or of dendritic cells and by dendritic cells in response to et al., 2003), a role for ManLAM in mycobacterial of host immune responses. Interestingly, et found that the parietal form of ManLAM from M. bovis BCG from human dendritic cells, and parietal were more potent of from dendritic cells compared with In the the immunological activities of ManLAM were after deacylation and were not observed with which lacks the suggesting that this anchor may essential role in the immunomodulatory properties of the (Nigou et al., 1997). In to the effects of ManLAM on dendritic cell the effects of the 19 kDa lipoprotein on these cells appear to be more M. tuberculosis 19 kDa lipoprotein and other have been shown to dendritic cell as by expression of class molecules, and and expression and activity et al., 2001). dendritic cells T cell activity in as by T cell and et al., 2001). the other et found that or 19 kDa lipoprotein of M. tuberculosis expression of molecules and of a with these it was recently that, through activation of the 19 kDa lipoprotein in human macrophages by in antigen and by CD4+ T cells et al., The by which the 19 kDa lipoprotein expression is of of and activation of but to induction of class et al., that the 19 kDa lipoprotein may activation of and immune functions early in infection later of infection, this molecule may expression and antigen a thus the of M. tuberculosis and by T The M. tuberculosis to immune and infection et al., et al., In addition to its effects on neutrophil and LAM also induces T cell migration. from M. human alveolar macrophages can induce T cell but this response can be by the addition of et al., Interestingly, although purified ManLAM from both virulent H37Rv and attenuated H37Ra can induce T cell from macrophages infected with the virulent strain this chemotactic response, suggesting that virulent strains a for LAM et al., Both AraLAM and M. tuberculosis Erdman strain ManLAM are chemotactic for CD4+ and CD8+ peripheral blood T cells in whereas M. bovis BCG ManLAM lacks these chemotactic properties et al., to its effects on ManLAM can T cell and activation in vitro. M. tuberculosis ManLAM was found to of human CD4+ T cell for and of peripheral human T cells after with mycobacterial antigen et al., This effect on T cell to be related to the of antigen by cells, as the phenomenon was observed when the T cells were with intact and not when they were with the et al., Interestingly, although ManLAM from M. the of T cells, this effect was not in response to with M. tuberculosis ManLAM, suggesting that differences within the ManLAM family may for differences in immunological M. ManLAM has been found to T cell activation in as by expression of for including and in T cells with and et al., ManLAM, TDM induces of T cells from and in and In TDM has been found to non-specific resistance in mice to infection by a of T cells that in lungs et al., 1997). of TDM into mice results in a of the T cell population, by of CD4+ cells after and of cells after 3 et al., CD8+ T cells to M. tuberculosis 19 kDa lipoprotein were found to be in a small study of with pulmonary and in most with response to et al., An composed of of the 19 kDa lipoprotein was found to be by CD8+ T cells from both individuals and with but not by et al., 1998). dendritic cells with this induced from the T cells of and T cells for this were shown to monocytes that been infected with the H37Ra strain of M. tuberculosis et al., 1998). the 19 kDa lipoprotein was shown to of antigen by macrophages and to CD8+ T cells et al., The important role of CD8+ T cells in immunity M. tuberculosis was in with mice CD8+ T mice to infection with M. tuberculosis et al., Subsequent studies that mice also have to tuberculosis et al., 1999; et al., 2000), other evidence that M. may be to et al., 1999). potential by which mycobacterial may be to CD8+ T cells was recently by et that of M. macrophages of mycobacterial which are by cells and can be through and to CD8+ T when as a protein in rapidly growing the 19 kDa lipoprotein was found to be from the mycobacterial within the first after and was able to for by class T cells by a et al., 2001). immune responses to M. tuberculosis infection T granuloma formation in to the their and for their by cell-mediated immunity et al., The of cord factors to induce pulmonary in mice after mycobacterial infection was many years TDM has been shown to both and in mice by as a non-specific and T antigen et al., 2001). In the the that CD4+ cells, with the cytokines and and the were more in of mice with TDM compared with mice after TDM et al., 2001). cytokines are to be components in the process of granuloma formation and injection of TDM in induces weight prominent and pulmonary and and from whereas injection of sulpholipid has of these effects et al., 2000). injection of purified TDM, but not SL-I, from M. tuberculosis in mice results in and pulmonary by et al., 1997). effects were attributed to induction of as they were by administration of et al., 1997). In the has been in the cell the of were identified in as class molecules responsible for lipid to T cells and, human was identified as the involved in mycolic et al., of and of lipid in the pathway et al., 1998). The interactions of mycolic with the of et al., 2001) and to the T cell have been described recently et al., 2000). immune responses to LAM, SL-I, TDM and the 19 kDa lipoprotein have been described in after experimental administration of these and in the of M. tuberculosis infection. immunomodulatory effects for these have been was to mediate which may be important for of bacilli by mononuclear et al., 1998). In and of administered LAM in mice et al., 2000). The of TDM to induce responses was many years et al., The injection of TDM antigen administration in the of mice in to the antigen compared with that from antigen injection into the et al., with TDM the of the response of mice to et al., Recent studies have shown that injection of TDM into mice or of macrophages in to TDM results in the induction of a of including and et al., 2000; Lima et al., which may a to In addition to TDM can induce its including its moiety and mycolic components et al., lipoproteins are potent of et al., as adjuvant in a lipoprotein was found to and the immune response to in a from a to a immune response et al., Whereas the human infection is et al., 1993), of mice with various within the 19 kDa lipoprotein that these of a et al., Although these lipid induce the role of immunity to these within the host immune response to M. tuberculosis infection to mycobacterial lipid have been as potential in human In based on have and and is for in TB more of for TB is the of this Mycobacterium tuberculosis is a in because of its to and survive within the tuberculosis infection a between pathogen virulence factors and the host immune response, leading to but not of persistent bacilli (Manabe and Bishai, 2000). host immune such as with infection with human the in of the in bacillary and tuberculosis The cell envelope of M. tuberculosis has components that a role in directing these host–pathogen This review the immunomodulatory properties of the cell envelope-associated molecules lipoarabinomannan, mycolic acid-containing glycolipids and the 19 kDa lipoprotein. The pleiotrophic and immunological effects of these molecules it to the by which to the immunopathogenesis of have been in the the immunological effects of potential for of these may be that they are a function of the experimental to study the other they may a strategy by which M. tuberculosis is able to survive within the host for of The immunological effect by these cell envelope-associated lipids may be to and activate in for the pathogen to into these cells, to the of these cells and of the The of genetic to mycobacteria and the availability of the M. tuberculosis genome (Cole et al., 1998) have provided the opportunity to in the genes required for biosynthesis of these cell envelope-associated molecules and to test directly the role of molecule in the immunopathogenesis of M. these strains attenuated in they may as potential of the involved in the of these molecules may targets for drugs to this This work was by a from the the of and and a from the of