The mycobacterial cell wall 13

Mycobacteria are characterized by a stable, rigid and hydrophobic cell wall which protects against lysosomal enzymes, reactive oxygen and nitrogen metabolites and other defense mechanisms of the host. The cell wall is composed of a complex mixture of various compounds and is unique for mycobacteria (Brennan and Nikaido, 1995). The primary cell wall structure is composed of a plasma-membrane stabilized by a murein backbone.

Attached to this is a arabinogalactan layer, esterified with mycolic acids. Free lipids and a capsule composed of phosphatidylinositol-mannosides (PIMs) are attached to the outer face of the cell wall. The plasma membrane is mainly composed of phosphatidylethanolamin, phosphatidylinositol and diphosphatidylglycerol (Cardiolipid, CL). Lipoarabinomannan (LAM) is a major constituent of the mycobacterial cell wall. LAM is composed of a PIM core, anchored to the plasma membrane and a polymer composed of arabinose residues to which mannose residues are attached.

1.7.2 Immune responses to mycobacterial infection

Mycobacteria have developed extensive mechanisms to avoid degradation by the microbicidal arsenal of macrophages and to interfere with lymphocyte activation.

Mycobacteria mainly persist intracellularly in phagosomes of macrophages. Phagocytosis of bacteria usually leads to their degradation in the endosomal pathway. Endosomes mature, are acidified and fuse with lysosomes which contain a variety of decomposition enzymes like proteases and lipases as well as reactive oxygen and nitrogen metabolites (Kornfeld and Mellman, 1989). However, mycobacteria, although taken up into the phagosome, prevent their degradation by the host. They inhibit the acidification of the phagosome and the fusion of the phagosome with endosomes and lysosomes which disconnects them from the endosomal/lysosomal maturation and degradation pathway (Hasan et al., 1997; Sturgill-Koszycki et al., 1994). Moreover, they compete with the host for intracellular iron (Russell et al., 1996; Schaible et al., 1998). Components of the mycobacterial cell wall are responsible for mycobacterial survival within phagosomes of macrophages through the modulation of macrophage functions and lymphocyte stimulation (Chan et al., 1991a; Vergne and Daffe, 1998). Mycobacterial LAM inhibits T cell stimulation and Protein Kinase C activity, interferes with IFNγ mediated macrophage activation and neutralizes oxygen radicals (Kaplan et al.,

1987). Additionally, inhibition of antigen-specific monocyte activation can be inhibited by PIMs.

However, there are several ways the immune system competes with mycobacterial infection.

IFNγ activated macrophages exhibit a range of anti-mycobacterial devises. Activated macrophages downregulate the amount of transferrin-receptor and ferritin leading to decreased availability of iron within the phagosome (Schaible and Kaufmann, 2004).

Furthermore, INFγ induces the production of reactive oxygen and nitrogen metabolites by the macrophage empowering them to attack and destroy intracellular bacteria (Fazal, 1997).

Mycobacterial protein antigens are presented by MHC class II molecules to CD4⁺ T cells.

These T lymphocytes are essential for establishing an immune response to mycobacteria, since mice lacking CD4⁺ T cells are highly susceptible to mycobacterial infection (Caruso et al., 1999). The main function of these T cells in defence against mycobacteria is the production of IFNγ. Despite CD4⁺ T cells, other lymphocytes are important for anti-mycobacterial immune responses. Cross presentation mediates activation of CD8⁺ T cells which lyse infected target cells with the residing mycobacteria via perforin / granzme B and also secrete IFNγ (Flynn and Chan, 2001; Schaible et al., 2003). Antibody production against mycobacterial lipids and proteins was detected in human tuberculosis patients. Although antibodies against mycobacterial LAM, PIM, trehalose dimycolate and cardiolipin could be detected, these do not play a role in immune defence against mycobacteria (Fournie et al., 1991; Julian et al., 1997; Pan et al., 1999; Santiago et al., 1989). Furthermore, lipids shed by mycobacteria during infection (Fischer et al., 2001) can be presented by CD1 molecules and are recognized by lipid-antigen specific T cells. These lipid specific T cells could have an important role in immune responses against bacterial pathogens (Moody et al., 2000)

1.8 CD1 lipid antigens and CD1 reactive T cells

CD1 molecules, except CD1a contain a signalling sequence that direct their trafficking to late endosomes and lysosomes (Porcelli, 1995). Mycobacterial lipids have access to these compartments as well. It was shown that lipoarabinomannan (LAM) is taken up via the mannose receptor and could be detected in late endosomes and MIIC compartments together with CD1b. This implies that CD1 molecules could meet different (mycobacterial) lipid antigens in specialized compartments, bind them and present them on the cell surface to T cells (Prigozy et al., 1997). This is supported by the presence of a wide range of degradative enzymes, such as glycosidases and lipases in the acidified endosomal/lysosomal vesicles. These enzymes could be involved in the processing of lipid antigens, derived from the mycobacterial phagosome (Reimann and Kaufmann, 1997).

The identification of the first antigen presented by CD1 molecules to T cells demonstrated that CD1 presented antigens are lipids (Beckman et al., 1994). So far, it has been shown that both hydrophobic peptides and lipids/glycolipids can be presented by CD1 molecules (Porcelli and Modlin, 1999). The foreign antigens presented by CD1 molecules that have been characterized include a range of diverse lipids found in the cell walls of mycobacteria.

Mycolic acid, glucose-monomycolate (GMM) and lipoarabinomannan (LAM) are presented by CD1b molecules (Beckman et al., 1994; Moody et al., 1997; Sieling et al., 1995).

Mycobacterial mannosyl-β1-phosphoisoprenoid, a glycophospholipid with only a single short lipid tail, is recognized by a CD1c-restricted T cell line (Moody et al., 2000). CD1a-restricted T cells recognize a lipopeptide (didehydroxymycobactin) from Mycobacterium tuberculosis, defining a new biochemical class of antigens for CD1-restricted T cells (Moody et al., 2004).

T cells that are group I CD1 restricted display a CD4^-CD8^- double negative (DN) or CD8α/β⁺ or CD4⁺ phenotype. DN T cells predominantly express an α/β-TCR but can also carray a γ/δ-TCR (Shamshiev et al., 2000; Spada et al., 2000). The common structural feature of all lipid antigens presented by CD1a, b and c is a hydrophobic moiety composed of 2 acyl chains which bind to the 2 hydrophobic pockets of the CD1 molecule, and a polar head group displayed on the surface of the CD1 molecule and recognized by the TCR. Removal of acyl chains leads to loss of CD1 binding and removal or modification of the polar head groups diminishes T cell recognition and activation (Moody et al., 1997).

Microbial lipids or lipopeptide antigens presented by CD1 have been confirmed only for CD1a, b, and c. There have been conflicting reports whether murine CD1d-restricted T cell (NKT cells) recognize glycosylphosphatidylinositol (GPI)-anchored glycoproteins from Plasmodium or Trypanosoma spp. in vitro or affect the IgG response to GPI-linked proteins in vivo (Duthie et al., 2002; Procopio et al., 2002; Romero et al., 2001; Schofield et al., 1999).

Mycobacterial phosphatidyinositol-dimannosid (PIM2) was shown to elicit a NKT cell response. However, this response was CD1d independent (Gilleron et al., 2001). Until recently, the only CD1d-presented antigen that elicits a NKT cell response was α-Galactosyl-ceramide (αGalCer) (Kawano et al., 1997). It strongly induces actvation of NKT cells and rapid production of cytokines. However, this sphingolipids is derived from the marine sponge Agelas mauritianus and is of unclear physiological relevance.