**3.** *M. tuberculosis* **induces foamy macrophages in the host**

*M. tuberculosis* infects primarily alveolar macrophages, which reside within alveoli. The infected macrophage leaves the alveoli and migrates then towards the next lung draining lymph node. *M. tuberculosis* inhibits the generation of the phagolysosome and the bacteria begin to multiply within the macrophage [50]. The host's immune response seems to be unable to clear the bacillus from the infected macrophages. Infected macrophages secrete TNF-α and chemokines, which recruit systemic monocytes. The macrophages start to enlarge and accumulate TAG in lipid droplets. These lipid-filled foamy macrophages (FM) are surrounded by an outer layer of lymphocytes. Within the foamy macrophages the bacteria resist in phagosomes, packed with lipid droplets.

**Figure 1.** Development and structure of the human tuberculosis granuloma. 1, Uptake of *M. tuberculosis* by alveolar macrophages. 2, Migration of the infected macrophage towards the next lung draining lymph node. 3, Recruitment of systemic monocytes. 4, Granuloma formation. L, Lymphocytes at the periphery of the granuloma outside the fibrous outer layer. F, Fibrous capsule. Contains fibroblasts, collagen and other extracellular matrix proteins. M, Macrophage region with foamy macrophages. C, Caseum. Contains debris and lipids from necrotic macrophages. Orange, Lipid

Lipid Inclusions in Mycobacterial Infections http://dx.doi.org/10.5772/54526 35

**3.1. Lipid body formation in** *M. tuberculosis* **is critically dependent on lipid droplets from**

Host lipids from lipid droplets are used by the pathogen as substantial nutrient source. Middlebrook already demonstrated in the late 1940s that mycobacterial growth *in vitro* was enhanced by supplementation with oleic acid [54]. Over the last years several groups have reported that *M. tuberculosis* within foamy macrophages produces lipid bodies, suggesting that they are able to accumulate host cell lipids [19,55]. Mycobacterial growth inside adipocytes is strictly dependent upon TAG provided by lipid droplets in host cells [55], and it has been

The utilization of host lipids in vivo does not only promote survival but may also increases virulence and modulate the immune response to infection. Growth of *M. tuberculosis* on fatty acids such as such propionate or valerate during infection leads to increased production of the surface-exposed lipid virulence factors, phthiocerol dimycocerosate (PDIM) and sulfolipid-1

Cholesterol utilization was also identified to be required for mycobacterial persistence [57]. In 2008 Pandey and Sassetti found that *M. tuberculosis* can grow using cholesterol as a primary carbon source and that the mce4 transporter is required for cholesterol uptake. *M. tuberculo‐ sis* contains four homologous mce operons, mce1–mce4, which are thought to encode lipid

Especially *M. leprae* infected macrophages show an increased accumulation of cholesterol and cholesterol [10,30]. But in contrast to *M. tuberculosis* the *M. leprae* genome encodes only one

shown that *M. tuberculosis* incorporates intact host TAG into bacterial TAG [46].

droplets of the macrophage. Yellow dots, Lipid bodies.

**the host**

(SL-1) [56].

transporters [57,58].

Over the last years it has become evident that survival and persistence of *M. tuberculosis* is critically dependent on lipid body formation, and induction of foamy macrophages appears to be a key event in both sustaining persistent bacteria and and release of infectious bacilli [15].

*M. tuberculosis*-infected phagosomes engulf cellular lipid droplets and finally the bacteria are completely enclosed by cellular lipid droplets. Only enclosed by lipid droplets the bacteria form lipid bodies and cell replication comes to a halt and finally the bacteria enter the state of dormancy and induced drug resistance [19,28]. In the nonreplicative state *M. tuberculosis* induces several bacterial genes involved in lipid metabolism such as diacylglycerol acyltrans‐ ferase (tgs1), such as diacylglycerol acyltransferase (*tgs1*), lipase (*lipY*), and isocitrate lyase (*icl*) are upregulated [19,46]. In conclusion lipid body formation seems to be absolutely necessary for transition of *M. tuberculosis* to the dormant state. This goes along with the important observation that sputum from tuberculosis patients contains lipid body-laden bacilli [17].

The final granuloma consists of a core of infected, lipid-laden macrophages, which are surrounded by an outer layer of additional differentiated macrophages. The outer shell consists of T lymphocytes, B lymphocytes, dendritic cells, neutrophils, fibroblasts and an extracellular matrix [29,51-53].

The development and composition of a human tuberculosis granuloma is depicted in Figure 1.

typically non-replicating dormant state, primarily in hypoxic granulomas in the lung [1]. The otherwise drug-susceptible dormant bacteria develop drug resistance within the granulomas of the host. These nonreplicative drug-resistant bacteria within the host´s tissues are called

It has been observed that persisters store large amounts of intracellular triacylglycerol lipid bodies (LBs) [15,17,28,45,46]. *M. tuberculosis* uses TAG from the lipid bodies as energy and carbon source under conditions such as starvation [47], oxygen depletion [48], and pathogen reactivation [49]. The observation that sputum from tuberculosis patients contains lipid bodyladen bacilli, proves the importance of lipids for the survival of the bacterium in the host [17].

*M. tuberculosis* infects primarily alveolar macrophages, which reside within alveoli. The infected macrophage leaves the alveoli and migrates then towards the next lung draining lymph node. *M. tuberculosis* inhibits the generation of the phagolysosome and the bacteria begin to multiply within the macrophage [50]. The host's immune response seems to be unable to clear the bacillus from the infected macrophages. Infected macrophages secrete TNF-α and chemokines, which recruit systemic monocytes. The macrophages start to enlarge and accumulate TAG in lipid droplets. These lipid-filled foamy macrophages (FM) are surrounded by an outer layer of lymphocytes. Within the foamy macrophages the bacteria resist in

Over the last years it has become evident that survival and persistence of *M. tuberculosis* is critically dependent on lipid body formation, and induction of foamy macrophages appears to be a key event in both sustaining persistent bacteria and and release of infectious bacilli [15].

*M. tuberculosis*-infected phagosomes engulf cellular lipid droplets and finally the bacteria are completely enclosed by cellular lipid droplets. Only enclosed by lipid droplets the bacteria form lipid bodies and cell replication comes to a halt and finally the bacteria enter the state of dormancy and induced drug resistance [19,28]. In the nonreplicative state *M. tuberculosis* induces several bacterial genes involved in lipid metabolism such as diacylglycerol acyltrans‐ ferase (tgs1), such as diacylglycerol acyltransferase (*tgs1*), lipase (*lipY*), and isocitrate lyase (*icl*) are upregulated [19,46]. In conclusion lipid body formation seems to be absolutely necessary for transition of *M. tuberculosis* to the dormant state. This goes along with the important observation that sputum from tuberculosis patients contains lipid body-laden bacilli [17].

The final granuloma consists of a core of infected, lipid-laden macrophages, which are surrounded by an outer layer of additional differentiated macrophages. The outer shell consists of T lymphocytes, B lymphocytes, dendritic cells, neutrophils, fibroblasts and an

The development and composition of a human tuberculosis granuloma is depicted in Figure 1.

**3.** *M. tuberculosis* **induces foamy macrophages in the host**

phagosomes, packed with lipid droplets.

34 Tuberculosis - Current Issues in Diagnosis and Management

extracellular matrix [29,51-53].

persisters [2].

**Figure 1.** Development and structure of the human tuberculosis granuloma. 1, Uptake of *M. tuberculosis* by alveolar macrophages. 2, Migration of the infected macrophage towards the next lung draining lymph node. 3, Recruitment of systemic monocytes. 4, Granuloma formation. L, Lymphocytes at the periphery of the granuloma outside the fibrous outer layer. F, Fibrous capsule. Contains fibroblasts, collagen and other extracellular matrix proteins. M, Macrophage region with foamy macrophages. C, Caseum. Contains debris and lipids from necrotic macrophages. Orange, Lipid droplets of the macrophage. Yellow dots, Lipid bodies.

#### **3.1. Lipid body formation in** *M. tuberculosis* **is critically dependent on lipid droplets from the host**

Host lipids from lipid droplets are used by the pathogen as substantial nutrient source. Middlebrook already demonstrated in the late 1940s that mycobacterial growth *in vitro* was enhanced by supplementation with oleic acid [54]. Over the last years several groups have reported that *M. tuberculosis* within foamy macrophages produces lipid bodies, suggesting that they are able to accumulate host cell lipids [19,55]. Mycobacterial growth inside adipocytes is strictly dependent upon TAG provided by lipid droplets in host cells [55], and it has been shown that *M. tuberculosis* incorporates intact host TAG into bacterial TAG [46].

The utilization of host lipids in vivo does not only promote survival but may also increases virulence and modulate the immune response to infection. Growth of *M. tuberculosis* on fatty acids such as such propionate or valerate during infection leads to increased production of the surface-exposed lipid virulence factors, phthiocerol dimycocerosate (PDIM) and sulfolipid-1 (SL-1) [56].

Cholesterol utilization was also identified to be required for mycobacterial persistence [57]. In 2008 Pandey and Sassetti found that *M. tuberculosis* can grow using cholesterol as a primary carbon source and that the mce4 transporter is required for cholesterol uptake. *M. tuberculo‐ sis* contains four homologous mce operons, mce1–mce4, which are thought to encode lipid transporters [57,58].

Especially *M. leprae* infected macrophages show an increased accumulation of cholesterol and cholesterol [10,30]. But in contrast to *M. tuberculosis* the *M. leprae* genome encodes only one operon for cholesterol uptake (mce1). All *M. leprae* five *mce* genes were overexpressed during intracellular growth in mouse and human biopsies [59,60]. This observation suggests, that the intracellular bacilli population induces cholersterol uptake of the infected cell and subse‐ quently uses the stored cholesterol as carbon and energy source.

following two-carbon fragments are added sequentially to yield fatty acids of the desired length. *M. tuberculosis* uses both type I and type II FAS systems for fatty acid elongation. The multifunctional FAS I enzyme (*Rv2524c*) catalyzes the de novo synthesis of C16- and C18-S-ACP. These fatty acids are converted to the CoA derivative and used primarily for the synthesis of membrane phospholipids. By continuous elongation of these fatty acids FAS I produces specifically the C20- and C26-S-ACP products, and these fatty acids are released as the CoA derivatives. The C20 fatty acid is transferred to the FAS II system for the synthesis of the verylong-chain mero segment of α-, methoxy-, and ketomycolic acids [64]. The transfer from the FAS I to the FAS II system occurs by a key condensing enzyme, the ketoacyl ACP synthase III (FabH). FabH catalyzes the decarboxylative condensation of malonyl-ACP with the acyl-CoA products of the FAS I system (Figure 2). Two distinct cyclopropane synthases, MmaA2 and

Lipid Inclusions in Mycobacterial Infections http://dx.doi.org/10.5772/54526 37

**Figure 2.** Fatty acid biosynthesis in *Mycobacterium tuberculosis*. The FAS-II elongation module uses the substrates R-CO-S-ACP and malonyl-S-ACP derived from malonyl-S-CoA, generated by FabD. FabH condenses both substrates R, long-chain alkyl group. Enzymes involved in these reactions are as follows: FabG1, a β-ketoacyl-ACP reductase catalyz‐ es the reduction of beta-ketoacyl-ACP substrates to beta-hydroxyacyl-ACP. β-hydroxyacyl-ACP dehydrase. 2-trans-eno‐ yl-ACP reductase (InhA). The β-ketoacyl-ACP synthase (KasA/KasB) catalyzes the addition of of two carbons from malonyl-ACP to R-CO-S-ACP (See text for details). R, long-chain alkyl group. ACP, acyl carrier protein. Enzymes are in bold letters. Selected inhibitors are depicted in red bold letters. TLM, thiolactomycin. CER, cerulenin. ETH, ethionamide.

Esterification of fatty acids with glycerol-3-phosphate occurs via sequential acylation of the sn-1,2 and 3 positions of glycerol-3-phosphate, and removal of the phosphate group before the last acylation step. The terminal reaction is the esterification of diacylglycerol (DAG) with acyl-CoA by an diacylglycerol acyltransferase [40]. Animals and plants use diacylglycerol acyl‐transferases (DGAT) for the terminal esterification. DGATs catalyze ex‐ clusively the esterification of acyl-CoA with diacylglycerol. Bacteria do not contain

INH, isoniazid. TRC, triclosan. TAC, thiacetazone.

PcaA introduce cyclopropane rings into the the growing acyl chain [64-66].

Cholesterol is also essential for uptake of *M. tuberculosis* and *M. leprae* in macrophages. Cholesterol accumulates at the site of mycobacterial entry in macrophages and promotes mycobacterial uptake. Cholesterol mediates the recruitment of TACO from the plasma membrane to the phagosome [61]. TACO, also termed as CORO1A, is a coat protein that prevents phagosome-lysosome fusion and thus degradation of mycobacteria in phagolyso‐ somes (Figure 4) [61,62]. This mechanism for the formation of TACO-coated phagosomes promotes intracellular survival [62,63].
