**5.2. TLR mediated LD formation in** *M. bovis* **and** *M. leprae*

*Mycobacterium bovis* Bacillus Calmette-Guérin (BCG) and *M. leprae* are recognised by the Tolllike receptors (TLR) TLR6 and TLR2 [101,102]. Mycobacterium bovis Bacillus Calmette-Guérin induced lipid body formation is TLR2 mediated [103]. The mycobacterial surface molecule lipoarabinomannan (LAM) induces the formation of foamy macrophages by binding to TLR2 [104] (Figure 3).

*M. leprae* association to macrophages is mediated by binding of the bacteria to TLR2 and TLR6. Heterodimerization of TLR2 and TLR6 leads to downstream signalling and subsequent LD formation [102,105]. Macrophage association is not dependent on binding to TLR2 or TLR6. Neither a TLR2-/- or TLR6-/- knockout macrophage shows reduced binding to *M. leprae*. This suggests that both TLR2 and TLR6 can bind *M. leprae* alone, or/and the presence of other receptors, binding to *M. leprae*. The TLR2-/- or TLR6-/- knockout macrophages do also not completely abolish LD formation, but show only reduced LD formation [102]. This suggests the presence of additional signalling pathways for LD formation. In SCs TLR6, but not TLR2, is essential for *M. leprae*-induced LD biogenesis in [101]. In LL lesions, accumulated with LD enriched macropages the genes for ADRP and CD36 are up-regulated [30,92,102]. This suggests also an involvement of CD36 in LD formation of *M. leprae* (Figure 4) [99].

that that a set of oxygenated mycolic acids specifically produced by highly virulent myco‐ bacteria species (*M. tuberculosis*, *M. avium*) were responsible for the formation of foamy

Several enzymes of the mycobacterial lipid-biosynthesis are regarded as targets for new antitubercular compounds. The research focused on enzymes, involved in the biosynthesis of lipid compounds of the mycobacterial cell wall [107]. Especially the biosynthesis of the highly toxic cord factor is an attractive target. The cord factor is synthesized by the antigen 85 complex [108,109]. It was recently shown that one member of the complex, antigen 85A is involved in the formation of intracellular lipid bodies [71]. Antigen 85 is an important virulence factor. It has been shown that *M. tuberculosis* requires the expression of Ag85A for growth in macro‐ phages [110]. *M. tuberculosis* strain lacking Ag85C shows an decrease of 40% in the amount of cell wall linked mycolic acids [111,112]. The treatment by a trehalose analogue, 6-azido-6 deoxy-α,α'-trehalose (ADT) inhibits the activity of all members of Ag85 complex *in vitro* [108, 113]. Also ethambutol targets the synthesis of arabinogalactan, isoniazid and ethionamide

The most potent inhibitor for mycolic acid biosynthesis is isoniazid (INH). INH is a prodrug which is converted to the isonicotinoyl radical by KatG. INH forms a covalent adduct with NAD. This INH-NAD adduct inhibits FAS-II enoyl-ACP reductase InhA, which in conse‐ quence leads to inhibition of mycolic acid biosynthesis, and ultimately to cell death [114-117].

The inhibitors of fatty acid biosynthesis are summarized in Figure 2 and Table 2.

**Synthesis step Enzyme Compound / class References**

FAS-II KasA/KasB TLM (Thiolactomycin) [120-122] FAS-II KasA/KasB Platensimycin [123]

KasA/KasB Cerulenin (2R,3S-epoxy-4-oxo-7,10-trans,transdodecanoic acid amide

InhA INH (Isoniazid) [124] InhA ETH (Ethionamide) [125] InhA TRC (Triclosan) [126] InhA alkyl diphenyl ethers (Triclosan derivatives) [127] InhA 2-(o-Tolyloxy)-5-hexylphenol (PT70) [120]

MmaA4 TAC (Thiacetazone) [128]

TAC (Thiacetazone) [128]

[118] [119]

Lipid Inclusions in Mycobacterial Infections http://dx.doi.org/10.5772/54526 45

macrophages [19].

FAS-I and FAS-II

**6. Clinical implications**

inhibit biosynthesis of mycolic acids [107].

Cyclopro-panation CMASs (cmaA2, mmaA2 or pcaA)

**Table 2.** Inhibitors of fatty acid biosynthesis

**Figure 3.** Induction of lipid droplet biogenesis in macrophages by *Mycobacterium tuberculosis*. 1) Recognition of bac‐ teria by Toll-like receptors (TLR) trigger phagocytosis and subsequent formation of lipid droplets. 2) The infected mac‐ rophage produces reactive oxygen species (ROS), which oxidize LDL. 3) The binding of OxLDL to type 1 scavenger receptors CD36 and LOX1 induces increased surface expression of both receptors and increases uptake of host´s oxi‐ dized fatty acids. 4) Mycobacterium-laden phagosomes internalize lipid droplets. 5) Within the lipid droplets the bac‐ teria form lipid bodies and finally enter the dormant state. ApoB-100, apolipoprotein B-100.

**Figure 4.** Basic mechanisms of lipid droplet induction in *M. leprae* infected macrophages. *M. leprae* attaches to TLR2 and TLR6. Heterodimerization of TLR2 and TLR6 induces downstream signalling and subsequent accumulation by LD formation. [102,105]. In SCs TLR6, but not TLR2, is essential for *M. leprae*-induced LD biogenesis [101]. Cholesterol from the LDs accumulates at the site of mycobacterial entry and promotes mycobacterial uptake. Cholesterol also re‐ cruits TACO from the plasma membrane to the phagosome [61]. TACO prevents phagosome-lysosome fusion and pro‐ motes intracellular survival [62,63]. Hypothetical uptake of oxidized lipids by scavenger receptors in *M. leprae*: Reactive oxygen species might oxidize low-density lipoprotein (LDL) to oxLDL, which is thought to be subsequently bound and taken up by scavenger receptors CD36 and LOX1. CHO, cholesterol. Unknown mechanisms for LD induc‐ tion are indicated with a question mark.

#### **5.3. Mycolic acids induce the formation of foamy macrophages**

Mycolic acids and oxygenated mycolic acids are strong inducers of monocyte-derived macrophages differentiation into foamy macrophages [19,106]. Peyron et al. demonstrated that that a set of oxygenated mycolic acids specifically produced by highly virulent myco‐ bacteria species (*M. tuberculosis*, *M. avium*) were responsible for the formation of foamy macrophages [19].
