**The Cord Factor: Structure, Biosynthesis and Application in Drug Research – Achilles Heel of** *Mycobacterium tuberculosis***?**

Ayssar A. Elamin, Matthias Stehr and Mahavir Singh *Department of Gene Regulation and Differentiation, Helmholtz Centre for Infection Research, Braunschweig Germany* 

## **1. Introduction**

186 Understanding Tuberculosis – New Approaches to Fighting Against Drug Resistance

Zhang, Y. The magic bullets and tuberculosis drug targets. Annual Review of Pharmacology

Recently, it was reported that the tuberculosis mortality in 2009 has dropped to 35% since 1990 (WHO, 2010). Nevertheless, the disease caused by the facultative intracellular bacterial pathogen *Mycobacterium tuberculosis* still remains the leading cause of death from a single bacterial species (Coker, 2004; Russell et al., 2010). The emergence of Multi-Drug Resistant (MDR) and Extreme Drug Resistant (XDR) strains of *M. tuberculosis* leads to prolonged treatment which drastically increases the therapy costs.

*M. tuberculosis* shows a remarkable property of existing in different states of invasion (infection), colonization and persistence (Casadevall & Pirofski, 2000). It also has outstanding mechanisms to escape from elimination and has a high degree of intrinsic resistance to most antibiotics, chemotherapeutic agents and immune eradication (Brennan & Nikaido, 1995; Coker, 2004). The major obstacle for host defence mechanisms and therapeutic intervention is the unusual robust cell wall which is unique among prokaryotes, and is a major determinant of virulence of the bacterium. The cell wall is critical for longterm persistence of *M. tuberculosis* in the hostile environment of the host's cells and for progression of tuberculosis (Barry et al., 1998). Approximately one-half of the cell wall mass is comprised of mycolic acids (Brennan & Nikaido, 1995). In the cell envelope, mycolic acids are esterified to the terminal pentaarabinofuranosyl unit of arabinogalactan, which is a peptidoglycan-linked polysaccharide. The outer envelope consists of trehalose 6,6' dimycolate (TDM; cord factor) and TMM (trehalose 6,6'-monomycolate, the biosynthetic precursor of TDM), where the mycolic acids of TDM interact with the mycolyl-residues from the layer beneath (Brennan & Nikaido, 1995). The mycolic acid-containing layers have width of ~10 nm and limit the penetration of hydrophilic substances, whereas the inner saccharide layer inhibits the penetration of lipophilic substances. The high abundance of mycolic acids in the outer cell envelope is the main barrier for water soluble antibiotics (Brennan, 2003; Coker, 2004).

The purpose of this review is to highlight the importance of the cord factor as one of the most unique determinant for *Mycobacterium tuberculosis* virulence. This article will especially focus on the steps of the cord factor biosynthesis, i.e., the transfer of mycolic acid from a

The Cord Factor: Structure, Biosynthesis and Application in

fever syndromes and cachexia (Silva & Faccioli, 1988).

**3. Drug targets in the biosynthesis of the cord factor** 

promising targets for anti-TB drug development.

al., 2009).

development.

**2.2 TDM as vaccine adjuvant** 

Drug Research - Achilles Heel of *Mycobacterium tuberculosis*? 189

receptor could not been identified. In 2009 Ishikawa et al. could demonstrate that macrophage inducible C-type lectin (Mincle) is an essential receptor for TDM (Ishikawa et

In the last years TDM has been used intensively as immunomodulatory and vaccine adjuvant (Behling et al., 1993b; Noll, 1956). TDM can reproduce several pathophysiologic properties of *M. tuberculosis* infection including granuloma formation and induction of proinflammatory cytokines, such as IL-1β, IL-6, and TNF in macrophages (Matsunaga & Moody, 2009). TNF (cachexin) has several toxic effects on host physiology, including sepsis,

Doses as little as 1–5 µg are granulomagenic in the lungs of mice (Bekierkunst et al., 1969). Moreover TDM increases the production of antibodies (Behling et al., 1993b; Perez et al., 1994; Perez et al., 2000) and it up-regulates the expression of MHCII on macrophages (Ryll et al., 2001). TDM also induces *in vivo* production of IL-12 (Oswald et al., 1997). Injected as mineral oil solution (Silva & Faccioli, 1988) TDM forms monolayers, where the mycolic acids are exposed. In the monolayer form TDM is highly toxic (lethal dose in mice: LD50 ~30 μg) and kills macrophages in minutes (Hunter et al., 2006b). In aqueous suspension TDM forms micelles, where the mycolic acid groups are completely covered and TDM is non-toxic (lethal dose in mice: LD50 >50,000 μg). Micellar TDM prevents phagosome/lysosome fusion and thus promotes the survival of mycobacteria in the macrophage. Nevertheless, until now there is no experimental evidence for the existence or formation of TDM micelles or monolayers *in vivo*. Due to its strong immunstimulatory effect, several studies have used TDM as a potential adjuvant in different vaccination models. In 1976 Saito et al. were the first who described the cord factor as good adjuvant in mice and rats but with only low adjuvant effect in guinea pigs (Saito et al., 1976). Lima et al. could show that microspheres, containing TDM with a Hsp65-encoding DNA plasmid, were able to protect vaccinated mice against virulent *M. tuberculosis* (Lima et al., 2003) and against *Leishmania major* infection (Coelho et al., 2006). The major problem using TDM as adjuvant is the relatively high toxicity of the mycolic acids and the accompanying contaminants during the preparation of TDM. A synthetic analog of the cord factor, trehalose-6,6-dibehenate (TDB), was shown to be an effective and safe alternative (Davidsen et al., 2005). TDB is less toxic compared with TDM and easier to produce, making it a potent candidate in the field of vaccine

The cord factor (trehalose 6,6´-di-mycolate) is composed of a sugar and a mycolic acid component. In the following section we present the trehalose and mycolic acid biosynthesis steps and the target enzymes in their biochemical context. Especially enzymes of mycolic acid biosynthesis, such as methyl transferase (PcaA) (Glickman et al., 2000), ß-ketoacyl-acyl carrier protein synthase (KasAB and FabH) (Bhatt et al., 2007), acyl-AMP ligase (Fad32) (Portevin et al., 2005) and polyketide synthase (Psk13) (Portevin et al., 2004), are regarded as

TMM to another TMM to form TDM by Ag85 complex enzymes. The Ag85 complex is one of the promising targets for novel antimycobacterial drugs and vaccines. We present our recently developed high throughput screening (HTS) assays suitable for the identification of potential inhibitors against Ag85.

## **2. Discovery of the cord factor (Trehalose 6,6´-di-mycolate; TDM)**

In 1884, Robert Koch described *M. tuberculosis* bacilli grown in culture as rope-like structures (Koch, 1884). More than half a century later, in 1947 Middlebrook found that the ability to form cords under specific conditions is an "essential accompaniment of virulence" (Middlebrook et al., 1947). In 1950 Bloch extracted the substance responsible for cord formation from virulent organisms and identified it as a "toxic substance" (Bloch, 1950).

The removal of the substance with petroleum ether resulted in somehow avirulent organisms but did not affect the growth of the bacilli. This suggested that the substance was located at the surface and since it was obtained only from "cordforming" organisms it was called "cord factor" (Bloch, 1950; Middlebrook et al., 1947). Six years later the cord factor was finally identified as trehalose 6,6´-di-mycolate (TDM) by Noll (Behling et al., 1993a; Noll, 1956). TDM is the most abundant glycolipid produced by virulent *M. tuberculosis*  (Hunter et al., 2006a). TDM molecules consist of trehalose (TDM glycan-head). Trehalose is abundant in mycobacteria as a free component (Elbein & Mitchell, 1973; Elbein et al., 2003). In the cord factor trehalose is esteried to two mycolic acid residues and the residues length is variable from species to species (Fig. 1). Mycobacterial mycolic acids contain generally 20– 80 carbons (Spargo et al., 1991).

Fig. 1. Structure of trehalose 6,6´-di-mycolate (TDM, cord factor).

The cord factor is comprised of trehalose which esterifed to two mycolic acid residues through their 6- and 6'-hydroxyl groups.

## **2.1 Effects of the cord factor on the immune system**

TDM inhibits the process of phagosome-lysosome fusion and is thus a key compound for the survival of the bacillus inside the host´s phagosomes (Indrigo et al., 2002). TDM induces a broad range of cytokine secretion in the host´s immune system, especially production of IL-1β, IL-6, and TNF in macrophages (Matsunaga & Moody, 2009). In the recent years it has been shown that TDM is a key driver of secondary and cavitary disease type of tuberculosis (Hunter et al., 2006b). Despite the various severe effects on the host immune system the host receptor could not been identified. In 2009 Ishikawa et al. could demonstrate that macrophage inducible C-type lectin (Mincle) is an essential receptor for TDM (Ishikawa et al., 2009).

#### **2.2 TDM as vaccine adjuvant**

188 Understanding Tuberculosis – New Approaches to Fighting Against Drug Resistance

TMM to another TMM to form TDM by Ag85 complex enzymes. The Ag85 complex is one of the promising targets for novel antimycobacterial drugs and vaccines. We present our recently developed high throughput screening (HTS) assays suitable for the identification of

In 1884, Robert Koch described *M. tuberculosis* bacilli grown in culture as rope-like structures (Koch, 1884). More than half a century later, in 1947 Middlebrook found that the ability to form cords under specific conditions is an "essential accompaniment of virulence" (Middlebrook et al., 1947). In 1950 Bloch extracted the substance responsible for cord formation from virulent organisms and identified it as a "toxic substance" (Bloch, 1950).

The removal of the substance with petroleum ether resulted in somehow avirulent organisms but did not affect the growth of the bacilli. This suggested that the substance was located at the surface and since it was obtained only from "cordforming" organisms it was called "cord factor" (Bloch, 1950; Middlebrook et al., 1947). Six years later the cord factor was finally identified as trehalose 6,6´-di-mycolate (TDM) by Noll (Behling et al., 1993a; Noll, 1956). TDM is the most abundant glycolipid produced by virulent *M. tuberculosis*  (Hunter et al., 2006a). TDM molecules consist of trehalose (TDM glycan-head). Trehalose is abundant in mycobacteria as a free component (Elbein & Mitchell, 1973; Elbein et al., 2003). In the cord factor trehalose is esteried to two mycolic acid residues and the residues length is variable from species to species (Fig. 1). Mycobacterial mycolic acids contain generally 20–

The cord factor is comprised of trehalose which esterifed to two mycolic acid residues

TDM inhibits the process of phagosome-lysosome fusion and is thus a key compound for the survival of the bacillus inside the host´s phagosomes (Indrigo et al., 2002). TDM induces a broad range of cytokine secretion in the host´s immune system, especially production of IL-1β, IL-6, and TNF in macrophages (Matsunaga & Moody, 2009). In the recent years it has been shown that TDM is a key driver of secondary and cavitary disease type of tuberculosis (Hunter et al., 2006b). Despite the various severe effects on the host immune system the host

**2. Discovery of the cord factor (Trehalose 6,6´-di-mycolate; TDM)** 

Fig. 1. Structure of trehalose 6,6´-di-mycolate (TDM, cord factor).

**2.1 Effects of the cord factor on the immune system** 

potential inhibitors against Ag85.

80 carbons (Spargo et al., 1991).

through their 6- and 6'-hydroxyl groups.

In the last years TDM has been used intensively as immunomodulatory and vaccine adjuvant (Behling et al., 1993b; Noll, 1956). TDM can reproduce several pathophysiologic properties of *M. tuberculosis* infection including granuloma formation and induction of proinflammatory cytokines, such as IL-1β, IL-6, and TNF in macrophages (Matsunaga & Moody, 2009). TNF (cachexin) has several toxic effects on host physiology, including sepsis, fever syndromes and cachexia (Silva & Faccioli, 1988).

Doses as little as 1–5 µg are granulomagenic in the lungs of mice (Bekierkunst et al., 1969). Moreover TDM increases the production of antibodies (Behling et al., 1993b; Perez et al., 1994; Perez et al., 2000) and it up-regulates the expression of MHCII on macrophages (Ryll et al., 2001). TDM also induces *in vivo* production of IL-12 (Oswald et al., 1997). Injected as mineral oil solution (Silva & Faccioli, 1988) TDM forms monolayers, where the mycolic acids are exposed. In the monolayer form TDM is highly toxic (lethal dose in mice: LD50 ~30 μg) and kills macrophages in minutes (Hunter et al., 2006b). In aqueous suspension TDM forms micelles, where the mycolic acid groups are completely covered and TDM is non-toxic (lethal dose in mice: LD50 >50,000 μg). Micellar TDM prevents phagosome/lysosome fusion and thus promotes the survival of mycobacteria in the macrophage. Nevertheless, until now there is no experimental evidence for the existence or formation of TDM micelles or monolayers *in vivo*. Due to its strong immunstimulatory effect, several studies have used TDM as a potential adjuvant in different vaccination models. In 1976 Saito et al. were the first who described the cord factor as good adjuvant in mice and rats but with only low adjuvant effect in guinea pigs (Saito et al., 1976). Lima et al. could show that microspheres, containing TDM with a Hsp65-encoding DNA plasmid, were able to protect vaccinated mice against virulent *M. tuberculosis* (Lima et al., 2003) and against *Leishmania major* infection (Coelho et al., 2006). The major problem using TDM as adjuvant is the relatively high toxicity of the mycolic acids and the accompanying contaminants during the preparation of TDM. A synthetic analog of the cord factor, trehalose-6,6-dibehenate (TDB), was shown to be an effective and safe alternative (Davidsen et al., 2005). TDB is less toxic compared with TDM and easier to produce, making it a potent candidate in the field of vaccine development.

## **3. Drug targets in the biosynthesis of the cord factor**

The cord factor (trehalose 6,6´-di-mycolate) is composed of a sugar and a mycolic acid component. In the following section we present the trehalose and mycolic acid biosynthesis steps and the target enzymes in their biochemical context. Especially enzymes of mycolic acid biosynthesis, such as methyl transferase (PcaA) (Glickman et al., 2000), ß-ketoacyl-acyl carrier protein synthase (KasAB and FabH) (Bhatt et al., 2007), acyl-AMP ligase (Fad32) (Portevin et al., 2005) and polyketide synthase (Psk13) (Portevin et al., 2004), are regarded as promising targets for anti-TB drug development.

The Cord Factor: Structure, Biosynthesis and Application in

(extended by two carbons) and shuffled into the FAS II cycle.

**3.3 Mycolic acid biosynthesis** 

al., 2002; Takayama et al., 2005).

Drug Research - Achilles Heel of *Mycobacterium tuberculosis*? 191

Mycolic acids are -hydroxy fatty acids with a long -alkyl side chain. They are homologous series of fatty acids differing by a two-carbon unit (Asselineau & Lederer, 1950). The mycolic acids are composed of an branch at the alpha position in respect to the carboxylic group and a meromycolate branch. The "short" branch contains species dependent 20-26 saturated carbon atoms. The "long" meromycolate branch has 50-60 carbon atoms and its chemical composition is highly variable, containing cyclopropyl or unsaturated bonds ( mycolates), methoxy (methoxymycolates) and keto (ketomycolates) groups (Alahari et al., 2007). The confusing denotation "α-mycolates" refers not to their position in the molecule but to their position on thin layer chromatography. The "-mycolates" (cis, cisdicyclopropyl fatty acids) are the most abundant mycolic acids in *M. tuberculosis* (~57%), followed by methoxymycolates (32%) and ketomycolates (11%). The methoxy- and ketomycolates can have either the cis or trans configuration on the proximal cyclopropane ring. In summary there are five main classes of mycolic acids in *M. tuberculosis* (Schroeder et

In *M. tuberculosis* mycolic acids are essentially provided via conventional fatty acid biosynthesis. Mycobacteria contain both type I and type II FAS fatty acid biosynthesis systems. Fatty acid biosynthesis is initiated by the multifunctional FAS I enzyme (*Rv2524c*), catalyzing the de novo synthesis of long-chain acyl-CoAs (C16:0 and C18:0) from acetyl-CoA and using malonyl-CoA as an extender unit. The domains of the FAS-I multienzymecomplex are organized in the following order: acyltransferase, enoyl reductase, dehydratase, malonyl/palmitoyl transferase, acyl carrier protein (ACP), -keto reductase, -ketoacyl synthase (Fernandes & Kolattukudy, 1996). In *M. tuberculosis* the C16:0- and C18:0-S-ACP adducts, are converted either to the CoA derivatives or further elongated by FAS I to produce C26:0 (Kikuchi et al., 1992). In mycobacteria the de novo fatty acid biosynthesis is exclusively carried out by FAS-I, whereas the FAS-II system performs only the elongation of the fatty acids, generated by FAS-I. The FAS I and FAS II systems are connected by a key condensing enzyme, the -ketoacyl ACP synthase III or FabH, which catalyzes a decarboxylative condensation of malonyl-ACP with the acyl-CoA (C16:0–C20:0) products of the FAS-I system (Fig. 3). The resulting 3-ketoacyl-ACP product is reduced to an acyl-ACP

The ACP cycles the growing acyl chain between four enzymes MabA (ketoacyl reductase), -hydroxyacy dehydrase, InhA (enoyl reductase) and KasA/B (-ketoacyl synthase). *M. tuberculosis* contains two -ketoacyl synthases, KasA and KasB, which share 67% identity. KasA seems to be essential for growth, while KasB is not essential but produces longer carbon chains (Bhatt et al., 2005; Slayden & Barry, 2002; Swanson et al., 2009). The deletion of *KasB* in *M. tuberculosis* leads to mycolic acids that are 2-6 carbons shorter in length and a defect in trans-cycloproponation of oxygenated mycolic acids. Phenotypically leads a deletion of *KasB* to a loss of acid-fastness (Bhatt et al., 2007). 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 consequence leads to inhibition of mycolic acid biosynthesis, and ultimately to cell death (Mdluli et al., 1998; Takayama et al., 1972; 1975; Wilming & Johnsson, 1999). In *M. tuberculosis*, the C26:0

## **3.1 Biosynthesis of trehalose**

Mycobacteria possess three pathways for trehalose synthesis (Kaur et al., 2009). Trehalose can by synthesized from glucose-6-phosphate catalyzed by trehalose-6-phosphate synthase (OtsA, *Rv3490*) (Pan et al., 2002) and trehalose-6-phosphate phosphatase (OtsB2, *Rv3372*) (Pan et al., 2002). The second pathway generates trehalose from glycogen involving the maltooligosyltrehalose synthase (TreY, *Rv1653c*) and the maltooligosyltrehalose trehalohydrolase (TreZ, *Rv1562c*). In the third pathway maltose is converted to trehalose by the trehalose synthase (TreS, *Rv0126*). While all the three pathways are functional and essential for the proliferation *of M. smegmatis* (Woodruff et al., 2004), the OtsAB pathway is predominant and strictly essential in *M. tuberculosis* (Fig. 2). In the genome sequence of *M. tuberculosis* exist two o*tsB* homologues, but only OtsB2 (*Rv3372*) has a functional role in the pathway. OtsB2 has been suggested as an attractive target for novel drugs due to absence of trehalose in mammalian cell (Murphy et al., 2005).

#### **3.2 Trehalose transporters**

The LpqY-SugA-SugB-SugCATP-binding cassette transporter is highly specific for uptake of the disaccharide trehalose. Since trehalose is not present in mammals, it is unlikely that this system is used for sugar acquisition from the host. Trehalose release is known to occur as a byproduct of the biosynthesis of the mycolic acid cell envelope by *M. tuberculosis* antigen 85 complex. The mycolyltransferases of the antigen 85 complex transfer the lipid moiety of the glycolipid trehalose monomycolate (TMM) to arabinogalactan or another molecule of TMM, yielding trehalose dimycolate. These reactions lead to a constant release of trehalose from the cell. The LpqY-SugA-SugB-SugC ATP-binding cassette has been suggested as transporter system (Fig. 2), recycling the released trehalose. Perturbations in trehalose recycling strongly impaired virulence of *M. tuberculosis*. (Kalscheuer et al., 2010). These sugar transporters are thought to play an important role in bacterial pathogenesis and have been suggested as target for tuberculosis chemotherapy (Kalscheuer et al., 2010).

Fig. 2. Trehalose biosynthesis in *Mycobacterium tuberculosis*. 1, OtsAB pathway. 2, TreY-TreZ pathway. 3, TreS-pathway. 4, Trehalose import by an ATP-binding cassette transporter system.

#### **3.3 Mycolic acid biosynthesis**

190 Understanding Tuberculosis – New Approaches to Fighting Against Drug Resistance

Mycobacteria possess three pathways for trehalose synthesis (Kaur et al., 2009). Trehalose can by synthesized from glucose-6-phosphate catalyzed by trehalose-6-phosphate synthase (OtsA, *Rv3490*) (Pan et al., 2002) and trehalose-6-phosphate phosphatase (OtsB2, *Rv3372*) (Pan et al., 2002). The second pathway generates trehalose from glycogen involving the maltooligosyltrehalose synthase (TreY, *Rv1653c*) and the maltooligosyltrehalose trehalohydrolase (TreZ, *Rv1562c*). In the third pathway maltose is converted to trehalose by the trehalose synthase (TreS, *Rv0126*). While all the three pathways are functional and essential for the proliferation *of M. smegmatis* (Woodruff et al., 2004), the OtsAB pathway is predominant and strictly essential in *M. tuberculosis* (Fig. 2). In the genome sequence of *M. tuberculosis* exist two o*tsB* homologues, but only OtsB2 (*Rv3372*) has a functional role in the pathway. OtsB2 has been suggested as an attractive target for novel drugs due to absence of

The LpqY-SugA-SugB-SugCATP-binding cassette transporter is highly specific for uptake of the disaccharide trehalose. Since trehalose is not present in mammals, it is unlikely that this system is used for sugar acquisition from the host. Trehalose release is known to occur as a byproduct of the biosynthesis of the mycolic acid cell envelope by *M. tuberculosis* antigen 85 complex. The mycolyltransferases of the antigen 85 complex transfer the lipid moiety of the glycolipid trehalose monomycolate (TMM) to arabinogalactan or another molecule of TMM, yielding trehalose dimycolate. These reactions lead to a constant release of trehalose from the cell. The LpqY-SugA-SugB-SugC ATP-binding cassette has been suggested as transporter system (Fig. 2), recycling the released trehalose. Perturbations in trehalose recycling strongly impaired virulence of *M. tuberculosis*. (Kalscheuer et al., 2010). These sugar transporters are thought to play an important role in bacterial pathogenesis and have been suggested as target for

Fig. 2. Trehalose biosynthesis in *Mycobacterium tuberculosis*. 1, OtsAB pathway. 2, TreY-TreZ pathway. 3, TreS-pathway. 4, Trehalose import by an ATP-binding cassette transporter

**3.1 Biosynthesis of trehalose** 

**3.2 Trehalose transporters** 

system.

trehalose in mammalian cell (Murphy et al., 2005).

tuberculosis chemotherapy (Kalscheuer et al., 2010).

Mycolic acids are -hydroxy fatty acids with a long -alkyl side chain. They are homologous series of fatty acids differing by a two-carbon unit (Asselineau & Lederer, 1950). The mycolic acids are composed of an branch at the alpha position in respect to the carboxylic group and a meromycolate branch. The "short" branch contains species dependent 20-26 saturated carbon atoms. The "long" meromycolate branch has 50-60 carbon atoms and its chemical composition is highly variable, containing cyclopropyl or unsaturated bonds ( mycolates), methoxy (methoxymycolates) and keto (ketomycolates) groups (Alahari et al., 2007). The confusing denotation "α-mycolates" refers not to their position in the molecule but to their position on thin layer chromatography. The "-mycolates" (cis, cisdicyclopropyl fatty acids) are the most abundant mycolic acids in *M. tuberculosis* (~57%), followed by methoxymycolates (32%) and ketomycolates (11%). The methoxy- and ketomycolates can have either the cis or trans configuration on the proximal cyclopropane ring. In summary there are five main classes of mycolic acids in *M. tuberculosis* (Schroeder et al., 2002; Takayama et al., 2005).

In *M. tuberculosis* mycolic acids are essentially provided via conventional fatty acid biosynthesis. Mycobacteria contain both type I and type II FAS fatty acid biosynthesis systems. Fatty acid biosynthesis is initiated by the multifunctional FAS I enzyme (*Rv2524c*), catalyzing the de novo synthesis of long-chain acyl-CoAs (C16:0 and C18:0) from acetyl-CoA and using malonyl-CoA as an extender unit. The domains of the FAS-I multienzymecomplex are organized in the following order: acyltransferase, enoyl reductase, dehydratase, malonyl/palmitoyl transferase, acyl carrier protein (ACP), -keto reductase, -ketoacyl synthase (Fernandes & Kolattukudy, 1996). In *M. tuberculosis* the C16:0- and C18:0-S-ACP adducts, are converted either to the CoA derivatives or further elongated by FAS I to produce C26:0 (Kikuchi et al., 1992). In mycobacteria the de novo fatty acid biosynthesis is exclusively carried out by FAS-I, whereas the FAS-II system performs only the elongation of the fatty acids, generated by FAS-I. The FAS I and FAS II systems are connected by a key condensing enzyme, the -ketoacyl ACP synthase III or FabH, which catalyzes a decarboxylative condensation of malonyl-ACP with the acyl-CoA (C16:0–C20:0) products of the FAS-I system (Fig. 3). The resulting 3-ketoacyl-ACP product is reduced to an acyl-ACP (extended by two carbons) and shuffled into the FAS II cycle.

The ACP cycles the growing acyl chain between four enzymes MabA (ketoacyl reductase), -hydroxyacy dehydrase, InhA (enoyl reductase) and KasA/B (-ketoacyl synthase). *M. tuberculosis* contains two -ketoacyl synthases, KasA and KasB, which share 67% identity. KasA seems to be essential for growth, while KasB is not essential but produces longer carbon chains (Bhatt et al., 2005; Slayden & Barry, 2002; Swanson et al., 2009). The deletion of *KasB* in *M. tuberculosis* leads to mycolic acids that are 2-6 carbons shorter in length and a defect in trans-cycloproponation of oxygenated mycolic acids. Phenotypically leads a deletion of *KasB* to a loss of acid-fastness (Bhatt et al., 2007). 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 consequence leads to inhibition of mycolic acid biosynthesis, and ultimately to cell death (Mdluli et al., 1998; Takayama et al., 1972; 1975; Wilming & Johnsson, 1999). In *M. tuberculosis*, the C26:0

The Cord Factor: Structure, Biosynthesis and Application in

(Takayama et al., 2005).

Drug Research - Achilles Heel of *Mycobacterium tuberculosis*? 193

mycolyl-β-D-mannopyranosyl -1-phosphoheptaprenol (Myc-PL) (Besra et al., 1994). Myc-PL migrates to the inner surface of the cell membrane and docks next to an ABC transporter, with its hydrophobic heptaprenol tail. The mycolyl group is transferred to trehalose 6-phosphate by a proposed membrane-associated mycolyltransferase II to form TMMphosphate, and the phosphate group is removed by the membrane-associated trehalose 6-phosphate phosphatase, yielding TMM. TMM is transported outside the cell by the ABC transporter (Fig. 4). There should be virtually no accumulation of TMM in the cytoplasm

Fig. 4. The proposed process of incorporation of newly synthesized mycolic acids into major cell wall components. The process starts inside the cell. Newly synthesized mycolic acids are

The antigen 85 complex is composed of Ag85A (FbpA), Ag85B (FbpB), and Ag85C (FbpC) as the predominant secreted proteins in *M. tuberculosis*. The corresponding genes are *fbpA* (*Rv3804c*), *fbpB* (*Rv1886c*), and *fbpC* (*Rv0129c*) (Belisle et al., 1997; Wiker & Harboe, 1992). The 85 complex proteins share 68–80% sequence identity (Belisle et al., 1997; Ronning et al., 2004). The mycolyltransferases of the antigen 85 complex are located outside the cell membrane and transfer the lipid moiety of the glycolipid trehalose monomycolate (TMM) to another molecule of TMM yielding trehalose dimycolate or to arabinogalactan to form cell

transferred to man-P-heptaprenol to produce 6-O-mycolyl-β-D-mannopyranosyl-1 phosphoheptaprenol (Myc-PL) and after that to trehalose 6-phosphate to yield TMM-P by the proposed membrane-associated mycolyltransferase II (reaction 1). TMM is produced by dephosphorylation of TMM-P by the membrane-bound TMM-P phosphatase (reaction 2). The transportation of TMM to the outside is catalyzed by a proposed ABC transporter cassette (TMM transporter) (reaction 3). Outside the cell the Ag85 complex catalyzes the transfer of mycolate to another TMM and arabinogalactan to yield TDM (reaction 4) or

arabinogalactan-mycolate (reaction 5) (Takayama et al., 2005).

wall arabinogalactan-mycolate (Fig. 4) (Sanki et al., 2009a).

**3.5 TDM biosynthesis by Ag85** 

fatty acids synthesized by FAS I will become the substrate of a dedicated acyl-CoA carboxylase (ACCase) to generate the -carboxy C26:0 fatty acid used as one of the substrate by the Pks 13 in the biosynthesis of mycolic acids (Gavalda et al., 2009). The last steps of the biosynthesis of mycolic acids are catalyzed by proteins encoded by the *fadD32*-*pks13*-*accD4* cluster. Pks13 ultimately condenses the two loaded fatty acyl chains to produce -alkyl -ketoacids, the precursors of mycolic acids (Gavalda et al., 2009). FadD32 has been shown essential for growth (Carroll et al., 2011). Double bonds at specific sites on mycolic acid precursors are modified by the action of cyclopropane mycolic acid synthases (CMASs) such as MmaA1-A4, PcaA and CmaA2, which are S-adenosyl-methionine-dependent methyl transferases (Alahari et al., 2007). The antitubercular drug, thiacetazone (TAC) and its chemical analogues acts on CMASs, inhibiting mycolic acid cyclopropanation (Alahari et al., 2007; Alahari et al., 2009).

Fig. 3. Biosynthesis of mycolic acids for cord factor synthesis. Enzymes are in bold letters. Selected inhibitors are depicted in red bold letters. TLM, thiolactomycin. CER, cerulenin. ETH, ethionamide. INH, isoniazid. TRC, triclosan. TAC, thiacetazone. DEP, diethyl phosphate. ADT, 6-azido-6-deoxy-α,α′-trehalose (See text for details).

#### **3.4 TMM biosynthesis**

TDM is thought to be synthesized exclusively outside the cell and its precursor TMM is transported outside the cell. In addition TMM has to be exported from the cytoplasm, to prevent the degradation of TMM inside the cell by the ubiquitously present Ag85/Fbp (Kilburn et al., 1982; Sathyamoorthy & Takayama, 1987). The mycolyl group is first transferred from mycolyl-S-Pks13 (mycolyl-S-PPB) to D-mannopyranosyl-1 phosphoheptaprenol by a proposed cytoplasmic mycolyltransferase I to yield Myc-PL 6-O-

fatty acids synthesized by FAS I will become the substrate of a dedicated acyl-CoA carboxylase (ACCase) to generate the -carboxy C26:0 fatty acid used as one of the substrate by the Pks 13 in the biosynthesis of mycolic acids (Gavalda et al., 2009). The last steps of the biosynthesis of mycolic acids are catalyzed by proteins encoded by the *fadD32*-*pks13*-*accD4* cluster. Pks13 ultimately condenses the two loaded fatty acyl chains to produce -alkyl -ketoacids, the precursors of mycolic acids (Gavalda et al., 2009). FadD32 has been shown essential for growth (Carroll et al., 2011). Double bonds at specific sites on mycolic acid precursors are modified by the action of cyclopropane mycolic acid synthases (CMASs) such as MmaA1-A4, PcaA and CmaA2, which are S-adenosyl-methionine-dependent methyl transferases (Alahari et al., 2007). The antitubercular drug, thiacetazone (TAC) and its chemical analogues acts on CMASs, inhibiting mycolic acid cyclopropanation (Alahari et al.,

Fig. 3. Biosynthesis of mycolic acids for cord factor synthesis. Enzymes are in bold letters. Selected inhibitors are depicted in red bold letters. TLM, thiolactomycin. CER, cerulenin. ETH, ethionamide. INH, isoniazid. TRC, triclosan. TAC, thiacetazone. DEP, diethyl

TDM is thought to be synthesized exclusively outside the cell and its precursor TMM is transported outside the cell. In addition TMM has to be exported from the cytoplasm, to prevent the degradation of TMM inside the cell by the ubiquitously present Ag85/Fbp (Kilburn et al., 1982; Sathyamoorthy & Takayama, 1987). The mycolyl group is first transferred from mycolyl-S-Pks13 (mycolyl-S-PPB) to D-mannopyranosyl-1 phosphoheptaprenol by a proposed cytoplasmic mycolyltransferase I to yield Myc-PL 6-O-

phosphate. ADT, 6-azido-6-deoxy-α,α′-trehalose (See text for details).

2007; Alahari et al., 2009).

**3.4 TMM biosynthesis** 

mycolyl-β-D-mannopyranosyl -1-phosphoheptaprenol (Myc-PL) (Besra et al., 1994). Myc-PL migrates to the inner surface of the cell membrane and docks next to an ABC transporter, with its hydrophobic heptaprenol tail. The mycolyl group is transferred to trehalose 6-phosphate by a proposed membrane-associated mycolyltransferase II to form TMMphosphate, and the phosphate group is removed by the membrane-associated trehalose 6-phosphate phosphatase, yielding TMM. TMM is transported outside the cell by the ABC transporter (Fig. 4). There should be virtually no accumulation of TMM in the cytoplasm (Takayama et al., 2005).

Fig. 4. The proposed process of incorporation of newly synthesized mycolic acids into major cell wall components. The process starts inside the cell. Newly synthesized mycolic acids are transferred to man-P-heptaprenol to produce 6-O-mycolyl-β-D-mannopyranosyl-1 phosphoheptaprenol (Myc-PL) and after that to trehalose 6-phosphate to yield TMM-P by the proposed membrane-associated mycolyltransferase II (reaction 1). TMM is produced by dephosphorylation of TMM-P by the membrane-bound TMM-P phosphatase (reaction 2). The transportation of TMM to the outside is catalyzed by a proposed ABC transporter cassette (TMM transporter) (reaction 3). Outside the cell the Ag85 complex catalyzes the transfer of mycolate to another TMM and arabinogalactan to yield TDM (reaction 4) or arabinogalactan-mycolate (reaction 5) (Takayama et al., 2005).

## **3.5 TDM biosynthesis by Ag85**

The antigen 85 complex is composed of Ag85A (FbpA), Ag85B (FbpB), and Ag85C (FbpC) as the predominant secreted proteins in *M. tuberculosis*. The corresponding genes are *fbpA* (*Rv3804c*), *fbpB* (*Rv1886c*), and *fbpC* (*Rv0129c*) (Belisle et al., 1997; Wiker & Harboe, 1992). The 85 complex proteins share 68–80% sequence identity (Belisle et al., 1997; Ronning et al., 2004). The mycolyltransferases of the antigen 85 complex are located outside the cell membrane and transfer the lipid moiety of the glycolipid trehalose monomycolate (TMM) to another molecule of TMM yielding trehalose dimycolate or to arabinogalactan to form cell wall arabinogalactan-mycolate (Fig. 4) (Sanki et al., 2009a).

The Cord Factor: Structure, Biosynthesis and Application in

**3.6 Ag85 as a putative drug target for tuberculosis treatment** 

development for TB (Ronning et al., 2000; 2004).

**mycolytransferase 85A** 

1991).

Drug Research - Achilles Heel of *Mycobacterium tuberculosis*? 195

The ongoing treatment battle of tuberculosis is worsened by the emergence of new strains of *M. tuberculosis* which are resistant to standard antibiotics. In the urgent need of new targets the biogenesis of fatty acids, mycolic acids and glycolipids stay as hotspots. There is hope that the crystal structure of antigen 85A, 85B and 85C shall help in rational drug

The treatment by a trehalose analogue, 6-azido-6-deoxy-α,α′-trehalose (ADT) inhibited the activity of all members of Ag85 complex *in vitro* and the growth of *Mycobacterium aurum,*  and it also increased the efficacy of various antibiotics, supporting the importance of TDM (Belisle et al., 1997; Mizuguchi et al., 1983). *M. tuberculosis* strain lacking Ag85C has a 40% decrease in the amount of cell wall linked mycolic acid, but with no change in the relative amounts of TMM and TDM (Jackson et al., 1999; Sanki et al., 2009a). Furthermore, an Ag85A knockout strain lost the ability to grow in macrophage-like cell-lines and poor media which highlights the role of Ag85A in virulence and survival of the organism (Armitige et al., 2000). In the last decades several antitubercular drugs have focused on targets in the mycobacterial cell wall (Johnson et al., 2006). Most commonly, ethambutol targets the synthesis of arabinogalactan. Isoniazid and ethionamide inhibit biosynthesis of mycolic acids (Johnson et al., 2006). Obviously, the crystal structure of antigen 85 complex is expected to accelerate the design of new drugs against Ag85 activity and cord factor

biosynthesis (Table 1) (Gobec et al., 2004; Sanki et al., 2009b; Wang et al., 2004).

**4. Drug development: Novel high-throughput screening assays for** 

Since the protein/substrate interactions and co-crystal structure of Ag85 are now known, the search for rapid assays for high-throughput screening (HTS) of large substance libraries has increased considerably. Most of the mycolyltransferase assays previously published are not suitable for HTS, due to their complexity or use of radioactive substances. The first one is a widely used radioassay which monitors enzymatic transfer of mycolic acids from a lipid-soluble TMM molecule to a radioactive water-soluble trehalose. Manipulation of the radioactive products in a two-phase reaction, extraction and thin layer chromatography allows visualization of the products (Kremer et al., 2002; Sathyamoorthy & Takayama, 1987). Another test published uses the substrate analogue p-nitrophenyl-6-O-octanoyl-H-Dglucopyranoside that functions as the acyl donor but it may not represent the natural enzymatic activity (Boucau et al., 2009). Also an excess of D-glucose is added to the reaction to function as an acyl acceptor and to promote turnover of the enzyme. Recently new assay

smaller Leu230 in Ag85C is highlighted by an arrow. Also shown: Arginine 233, which covers the second carbohydrate binding pocket in Ag85C is replaced by smaller polar amino acids Thr235 and Ser235 in Ag85A and B, respectively. The surface is colored by electrostatic potential: The red and blue coloring represent negative and positive electrostatic potential, respectively. For Ag85A, B and C the coordinates from 1SFR, 1F0P and 1DQZ were used, respectively. The position of the trehalose molecules in Ag85A and Ag85C were modeled using the coordinates from 1F0P. The figure was prepared using GRASP (Nicholls et al.,

There is also evidence, the Ag85 complex proteins bind to fibronectin and the fibronectinbinding property of the Ag85 complex is important for mycobacterium life cycle in the host and macrophages (Klegerman et al., 1994; Ronning et al., 2004). The crystal structures of the three 30–32 kDa proteins (Ag85A, B and C) have been determined (Anderson et al., 2001; Ronning et al., 2004). These proteins contain a carboxylesterase domain bearing the highlyconserved consensus sequence GXXSXXG. The interaction between Ag85 and fibronectin is mediated by the sequence homologous to residues 56–66 (FEEYYQSGLSV) of the recombinant *M. tuberculosis* Ag85C (Ronning et al., 2004). Up to date, the question remains open why *Mycobacterium tuberculosis* has three antigen 85 enzymes sharing the similar sequence and substrate specificity (Daffe, 2000; Ronning et al., 2004).

Ag85 complex members from *M. tuberculosis* belong to the α/β hydrolase superfamily and catalyze the hydrolysis of ester and amide bonds using a catalytic triad comprised of Ser126, Glu230 and His262 in Ag85A/B and Ser124, Glu228 and His260 in Ag85C. All three enzymes contain two carbohydrate binding sites. The active site carbohydrate binding pocket binds TMM to form a temporary mycolate ester with the catalytic serine. The second carbohydrate binding site binds the incoming trehalose monomycolate, which "swings over" to the active site to displace the mycolate from its serine ester (Anderson et al., 2001; Ronning et al., 2004). The second trehalose binding site is separated from the acyl binding pocket by a bulky phenylalanine in Ag85A/B or a smaller leucine in Ag85C. All residues that form the active site carbohydrate binding pocket are 100% conserved in the *M. tuberculosis* antigen 85 proteins, while the surface of the acyl binding pocket, which is supposed to bind the long mycolate chains of TMM, exhibits slight differences. The conserved Leu152 in Ag85A and B is replaced by the bulky Phe150 in Ag85C, which in consequence leads to changes of surface topology in the mycolate binding portion (Fig. 5). The differences may alter substrate specificity and thus Ag85A, B and C might prefer different mycolic acids (Ronning et al., 2000; 2004).

Fig. 5. Surface representation of Ag85A, B and C with two bound trehalose molecules. The trehalose molecules are depicted as ball-and-stick model. The position of the catalytic serine is indicated by a yellow asterisk. The carbohydrate binding pocket of all three proteins is 100% conserved (Arg43, Gln45 Ile53, Asn54, Trp264 in Ag85A and B) and Arg41, Gln43, Ile51, Asn52, Trp264 in Ag85C. Corresponding residues, which differ among the three proteins are shown with red labels. The separation of the second carbohydrate binding pocket from the acyl binding pocket by the Phe232 in Ag85A/B and the corresponding

There is also evidence, the Ag85 complex proteins bind to fibronectin and the fibronectinbinding property of the Ag85 complex is important for mycobacterium life cycle in the host and macrophages (Klegerman et al., 1994; Ronning et al., 2004). The crystal structures of the three 30–32 kDa proteins (Ag85A, B and C) have been determined (Anderson et al., 2001; Ronning et al., 2004). These proteins contain a carboxylesterase domain bearing the highlyconserved consensus sequence GXXSXXG. The interaction between Ag85 and fibronectin is mediated by the sequence homologous to residues 56–66 (FEEYYQSGLSV) of the recombinant *M. tuberculosis* Ag85C (Ronning et al., 2004). Up to date, the question remains open why *Mycobacterium tuberculosis* has three antigen 85 enzymes sharing the similar

Ag85 complex members from *M. tuberculosis* belong to the α/β hydrolase superfamily and catalyze the hydrolysis of ester and amide bonds using a catalytic triad comprised of Ser126, Glu230 and His262 in Ag85A/B and Ser124, Glu228 and His260 in Ag85C. All three enzymes contain two carbohydrate binding sites. The active site carbohydrate binding pocket binds TMM to form a temporary mycolate ester with the catalytic serine. The second carbohydrate binding site binds the incoming trehalose monomycolate, which "swings over" to the active site to displace the mycolate from its serine ester (Anderson et al., 2001; Ronning et al., 2004). The second trehalose binding site is separated from the acyl binding pocket by a bulky phenylalanine in Ag85A/B or a smaller leucine in Ag85C. All residues that form the active site carbohydrate binding pocket are 100% conserved in the *M. tuberculosis* antigen 85 proteins, while the surface of the acyl binding pocket, which is supposed to bind the long mycolate chains of TMM, exhibits slight differences. The conserved Leu152 in Ag85A and B is replaced by the bulky Phe150 in Ag85C, which in consequence leads to changes of surface topology in the mycolate binding portion (Fig. 5). The differences may alter substrate specificity and thus Ag85A, B and C might prefer

Fig. 5. Surface representation of Ag85A, B and C with two bound trehalose molecules. The trehalose molecules are depicted as ball-and-stick model. The position of the catalytic serine is indicated by a yellow asterisk. The carbohydrate binding pocket of all three proteins is 100% conserved (Arg43, Gln45 Ile53, Asn54, Trp264 in Ag85A and B) and Arg41, Gln43, Ile51, Asn52, Trp264 in Ag85C. Corresponding residues, which differ among the three proteins are shown with red labels. The separation of the second carbohydrate binding pocket from the acyl binding pocket by the Phe232 in Ag85A/B and the corresponding

sequence and substrate specificity (Daffe, 2000; Ronning et al., 2004).

different mycolic acids (Ronning et al., 2000; 2004).

smaller Leu230 in Ag85C is highlighted by an arrow. Also shown: Arginine 233, which covers the second carbohydrate binding pocket in Ag85C is replaced by smaller polar amino acids Thr235 and Ser235 in Ag85A and B, respectively. The surface is colored by electrostatic potential: The red and blue coloring represent negative and positive electrostatic potential, respectively. For Ag85A, B and C the coordinates from 1SFR, 1F0P and 1DQZ were used, respectively. The position of the trehalose molecules in Ag85A and Ag85C were modeled using the coordinates from 1F0P. The figure was prepared using GRASP (Nicholls et al., 1991).

#### **3.6 Ag85 as a putative drug target for tuberculosis treatment**

The ongoing treatment battle of tuberculosis is worsened by the emergence of new strains of *M. tuberculosis* which are resistant to standard antibiotics. In the urgent need of new targets the biogenesis of fatty acids, mycolic acids and glycolipids stay as hotspots. There is hope that the crystal structure of antigen 85A, 85B and 85C shall help in rational drug development for TB (Ronning et al., 2000; 2004).

The treatment by a trehalose analogue, 6-azido-6-deoxy-α,α′-trehalose (ADT) inhibited the activity of all members of Ag85 complex *in vitro* and the growth of *Mycobacterium aurum,*  and it also increased the efficacy of various antibiotics, supporting the importance of TDM (Belisle et al., 1997; Mizuguchi et al., 1983). *M. tuberculosis* strain lacking Ag85C has a 40% decrease in the amount of cell wall linked mycolic acid, but with no change in the relative amounts of TMM and TDM (Jackson et al., 1999; Sanki et al., 2009a). Furthermore, an Ag85A knockout strain lost the ability to grow in macrophage-like cell-lines and poor media which highlights the role of Ag85A in virulence and survival of the organism (Armitige et al., 2000). In the last decades several antitubercular drugs have focused on targets in the mycobacterial cell wall (Johnson et al., 2006). Most commonly, ethambutol targets the synthesis of arabinogalactan. Isoniazid and ethionamide inhibit biosynthesis of mycolic acids (Johnson et al., 2006). Obviously, the crystal structure of antigen 85 complex is expected to accelerate the design of new drugs against Ag85 activity and cord factor biosynthesis (Table 1) (Gobec et al., 2004; Sanki et al., 2009b; Wang et al., 2004).

## **4. Drug development: Novel high-throughput screening assays for mycolytransferase 85A**

Since the protein/substrate interactions and co-crystal structure of Ag85 are now known, the search for rapid assays for high-throughput screening (HTS) of large substance libraries has increased considerably. Most of the mycolyltransferase assays previously published are not suitable for HTS, due to their complexity or use of radioactive substances. The first one is a widely used radioassay which monitors enzymatic transfer of mycolic acids from a lipid-soluble TMM molecule to a radioactive water-soluble trehalose. Manipulation of the radioactive products in a two-phase reaction, extraction and thin layer chromatography allows visualization of the products (Kremer et al., 2002; Sathyamoorthy & Takayama, 1987). Another test published uses the substrate analogue p-nitrophenyl-6-O-octanoyl-H-Dglucopyranoside that functions as the acyl donor but it may not represent the natural enzymatic activity (Boucau et al., 2009). Also an excess of D-glucose is added to the reaction to function as an acyl acceptor and to promote turnover of the enzyme. Recently new assay

The Cord Factor: Structure, Biosynthesis and Application in

(Fig. 6).

(Elamin et al., 2009).

following equations:

Drug Research - Achilles Heel of *Mycobacterium tuberculosis*? 197

2005) and reflects natural activity, allowing to get the most accurate kinetic parameters. In the reaction Ag85A produces one molecule of trehalose as product per reaction cycle and by adding trehalase the trehalose converted to glucose, which can be easily measured

Fig. 6. Scheme for the new mycolyltransferase activity assay. Using trehalose that is produced as one of nal products of mycolyltransferase reaction by trehalase to produce glucose, which is oxidized to gluconic acid and hydrogen peroxidase by glucose oxidase. Hydrogen peroxide reacts with o-dianisidine in the presence of peroxidase to produce a colored product (oxidized o-dianisidine), which will be converted to a stable colored

Quantication of glucose is nally achieved by the glucose oxidase assay (Washko & Rice, 1961). The amount of glucose is proportional to the TMM concentration. The assay showed that the antigen 85A can be assayed in the presence of methanol or mixture of chloroform/methanol, which is usually used to extract and purify the glycolipids from mycobacterium cell wall fractions. The results from substrate/solvent experiments showed that the enzyme activity was reduced in the presence of organic solvents than in standard buffer reaction alone. This indicates and proves that this method is useful to quantify the

One molecule of trehalose produced from TMM processed by Ag85 complex, which by our method is converted to two molecules of glucose. We can calculate the original concentration of TMM and concentration of TDM and trehalose in the reaction by the

One has to keep in mind that the extracted total lipids contain free trehalose and glucose and will affect the final calculations. In this case and to calculate the TMM concentration in total lipids one should run different negative controls. The Z′ factor (Zhang et al., 1999) measurement of the current assay in different volumes indicates an excellent signal/noise

(S/N) ratio for the assay and its high potential for HTS applications (Table 2).

Concentration of [TMM] = concentration of [glucose] (1)

Concentration of [TDM] = concentration of [glucose]/2 (2)

Concentration of [trehalose] = concentration of [glucose]/2 (3)

product by sulfuric acid. The colored product is measured at 540 nm

TMM from the total lipid of mycobacterium cells.


for Ag85 was developed based on the use of mono and dihexanoyl trehalose substrates, followed by quantitation of the acyl-transfer to the unnatural trehalose by mass spectrometry (Backus et al., 2011).

Table 1. Inhibitors of cord factor biosynthesis

The mycobacterial glycolipids and TMM levels in the cell wall might give an indirect indication of the fitness of the cell inside the host cells specially in macrophages. Thus the quantitation of the mycobacterial TMM status after drug treatment may allow the estimation of drug effectiveness. Unfortunately, there is no method for measurement of the amount of glycolipids that is suitable for HTS. We designed an assay (Elamin et al., 2009) based on the use of natural substrate, and this mycolyltransferase assay offers a novel means to determine the TMM status of the mycobacterium cell wall and reects the natural activity of mycolyltransferase enzyme based on simple steps. The new assay uses the natural substrate TMM, which can be easily purified from mycobacteria (Fujita et al.,

for Ag85 was developed based on the use of mono and dihexanoyl trehalose substrates, followed by quantitation of the acyl-transfer to the unnatural trehalose by mass

7,10-trans,trans-dodecanoic

InhA INH (Isoniazid) (Slayden et al., 2000) InhA ETH (Ethionamide) (Kremer et al., 2003) InhA TRC (Triclosan) (McMurry et al.,

Unknown N-octanesulfonylacetamide (Parrish et al., 2001)

MmaA4 TAC (Thiacetazone) (Alahari et al., 2007)

Ag85C Methyl β-D-arabinofuranoside (Sanki et al., 2009b)

arabinofuranoside analogues

Ag85 complex Trehalose analogues (Wang et al., 2004)

Ag85 complex Phosphonate compounds (Gobec et al., 2004;

Ag85C DEP (Diethyl phosphate) (Ronning et al., 2000)

TAC (Thiacetazone) (Alahari et al., 2007)

(Schroeder et al., 2002) (Johansson et

Kremer et al., 2000; Luckner et al., 2010)

(Sullivan et al., 2006)

(Luckner et al., 2010)

(Sanki et al., 2008)

(Belisle et al., 1997; Mizuguchi et al.,

Gobec et al., 2007)

1983)

al., 2008)

1999)

**Synthesis step Enzyme Compound / class References** 

KasA/KasB Cerulenin (2R,3S-epoxy-4-oxo-

acid amide

**FAS-II** KasA/KasB TLM (Thiolactomycin) (Douglas et al., 2002;

**FAS-II** KasA/KasB Platensimycin (Brown et al., 2009)

InhA alkyl diphenyl ethers (Triclosan derivatives)

InhA 2-(o-Tolyloxy)-5-hexylphenol (PT70)

Ag85 complex ADT (6-azido-6-deoxy-α,α′ trehalose)

The mycobacterial glycolipids and TMM levels in the cell wall might give an indirect indication of the fitness of the cell inside the host cells specially in macrophages. Thus the quantitation of the mycobacterial TMM status after drug treatment may allow the estimation of drug effectiveness. Unfortunately, there is no method for measurement of the amount of glycolipids that is suitable for HTS. We designed an assay (Elamin et al., 2009) based on the use of natural substrate, and this mycolyltransferase assay offers a novel means to determine the TMM status of the mycobacterium cell wall and reects the natural activity of mycolyltransferase enzyme based on simple steps. The new assay uses the natural substrate TMM, which can be easily purified from mycobacteria (Fujita et al.,

Ag85C 5-S-alkyl-5-thio-

CMASs (cmaA2, mmaA2 or pcaA)

Table 1. Inhibitors of cord factor biosynthesis

spectrometry (Backus et al., 2011).

**FAS-I and FAS-II** 

**Mycolic acid biosynthesis** 

**Cyclopropanation** 

**Cord-factor biosynthesis**  2005) and reflects natural activity, allowing to get the most accurate kinetic parameters. In the reaction Ag85A produces one molecule of trehalose as product per reaction cycle and by adding trehalase the trehalose converted to glucose, which can be easily measured (Fig. 6).

Fig. 6. Scheme for the new mycolyltransferase activity assay. Using trehalose that is produced as one of nal products of mycolyltransferase reaction by trehalase to produce glucose, which is oxidized to gluconic acid and hydrogen peroxidase by glucose oxidase. Hydrogen peroxide reacts with o-dianisidine in the presence of peroxidase to produce a colored product (oxidized o-dianisidine), which will be converted to a stable colored product by sulfuric acid. The colored product is measured at 540 nm (Elamin et al., 2009).

Quantication of glucose is nally achieved by the glucose oxidase assay (Washko & Rice, 1961). The amount of glucose is proportional to the TMM concentration. The assay showed that the antigen 85A can be assayed in the presence of methanol or mixture of chloroform/methanol, which is usually used to extract and purify the glycolipids from mycobacterium cell wall fractions. The results from substrate/solvent experiments showed that the enzyme activity was reduced in the presence of organic solvents than in standard buffer reaction alone. This indicates and proves that this method is useful to quantify the TMM from the total lipid of mycobacterium cells.

One molecule of trehalose produced from TMM processed by Ag85 complex, which by our method is converted to two molecules of glucose. We can calculate the original concentration of TMM and concentration of TDM and trehalose in the reaction by the following equations:

$$\text{Concentration of [TMM]} = \text{concentration of [glucose]} \tag{1}$$

$$\text{Concentration of [TDM]} = \text{concentration of [glucose]} / 2 \tag{2}$$

Concentration of [trehalose] = concentration of [glucose]/2 (3)

One has to keep in mind that the extracted total lipids contain free trehalose and glucose and will affect the final calculations. In this case and to calculate the TMM concentration in total lipids one should run different negative controls. The Z′ factor (Zhang et al., 1999) measurement of the current assay in different volumes indicates an excellent signal/noise (S/N) ratio for the assay and its high potential for HTS applications (Table 2).

The Cord Factor: Structure, Biosynthesis and Application in

1994), pp. 12735-12739, ISSN 0027-8424

7596-7606, ISSN 0021-9193

ISSN 0027-8424

91-97, ISSN 1472-9792

2009), pp. e6306, ISSN 1932-6203

Drug Research - Achilles Heel of *Mycobacterium tuberculosis*? 199

Behling, C.A.;Bennett, B.;Takayama, K., & Hunter, R.L. (1993a) Development of a trehalose

Bekierkunst, A.;Levij, I.S.;Yarkoni, E.;Vilkas, E.;Adam, A., & Lederer, E. (1969) Granuloma

*bacteriology*, Vol.100, No.1, (October 1969), pp. 95-102, ISSN 0021-9193 Belisle, J.T.;Vissa, V.D.;Sievert, T.;Takayama, K.;Brennan, P.J., & Besra, G.S. (1997) Role of

*York, N.Y.)*, Vol.276, No.5317, (May 1997), pp. 1420-1422, ISSN 0036-8075 Besra, G.S.;Sievert, T.;Lee, R.E.;Slayden, R.A.;Brennan, P.J., & Takayama, K. (1994)

Bhatt, A.;Kremer, L.;Dai, A.Z.;Sacchettini, J.C., & Jacobs, W.R., Jr. (2005) Conditional

Bhatt, A.;Fujiwara, N.;Bhatt, K.;Gurcha, S.S.;Kremer, L.;Chen, B.;Chan, J.;Porcelli,

Bloch, H. (1950) Studies on the virulence of tubercle bacilli; isolation and biological

*medicine*, Vol.91, No.2, (February 1950), pp. 197-218, pl, ISSN 0022-1007 Boucau, J.;Sanki, A.K.;Voss, B.J.;Sucheck, S.J., & Ronning, D.R. (2009) A coupled assay

*biochemistry*, Vol.385, No.1, (February 2009), pp. 120-127, ISSN 1096-0309 Brennan, P.J., & Nikaido, H. (1995) The envelope of mycobacteria. *Annual review of* 

Brennan, P.J. (2003) Structure, function, and biogenesis of the cell wall of *Mycobacterium* 

Brown, A.K.;Taylor, R.C.;Bhatt, A.;Futterer, K., & Besra, G.S. (2009) Platensimycin activity

Carroll, P.;Faray-Kele, M.C., & Parish, T. (2011) Identifying Vulnerable Pathways in

*biochemistry*, Vol.64, (July 1995), pp. 29-63, ISSN 0066-4154

No.4, (July-August 1993b), pp. 256-266, ISSN 0091-7370

6,6'-dimycolate model which explains cord formation by *Mycobacterium tuberculosis*. *Infection and immunity*, Vol.61, No.6, (June 1993a), pp. 2296-2303, ISSN 0019-9567 Behling, C.A.;Perez, R.L.;Kidd, M.R.;Staton, G.W., Jr., & Hunter, R.L. (1993b) Induction of

pulmonary granulomas, macrophage procoagulant activity, and tumor necrosis factor-alpha by trehalose glycolipids. *Annals of clinical and laboratory science*, Vol.23,

formation induced in mice by chemically defined mycobacterial fractions. *Journal of* 

the major antigen of *Mycobacterium tuberculosis* in cell wall biogenesis. *Science (New* 

Identification of the apparent carrier in mycolic acid synthesis. *Proceedings of the National Academy of Sciences of the United States of America*, Vol.91, No.26, (December

depletion of KasA, a key enzyme of mycolic acid biosynthesis, leads to mycobacterial cell lysis. *Journal of bacteriology*, Vol.187, No.22, (November 2005), pp.

S.A.;Kobayashi, K.;Besra, G.S., & Jacobs, W.R., Jr. (2007) Deletion of *kasB* in *Mycobacterium tuberculosis* causes loss of acid-fastness and subclinical latent tuberculosis in immunocompetent mice. *Proceedings of the National Academy of Sciences of the United States of America*, Vol.104, No.12, (March 2007), pp. 5157-5162,

properties of a constituent of virulent organisms. *The Journal of experimental* 

measuring *Mycobacterium tuberculosis* antigen 85C enzymatic activity. *Analytical* 

*tuberculosis*. *Tuberculosis (Edinburgh, Scotland)*, Vol.83, No.1-3, (February 2003), pp.

against mycobacterial beta-ketoacyl-ACP synthases. *PLoS One*, Vol.4, No.7, (July

*Mycobacterium tuberculosis* by Using a Knockdown Approach. *Applied and* 


Table 2. The calculated Z′ factor at different volumes from 96-well plate format assays.

#### **5. Concluding remarks**

Large gaps remain in our understanding of mycobacterium pathogenesis and persistence including the critical questions how bacteria survive in host cells and escape from the therapy. Future work on mycobacterial cell wall biosynthesis especially glycolipids and related pathways is expected to reveal *in vivo* drug-resistance mechanism. Perhaps more notably, the described new and low-cost colorimetric method based on use of TMM as natural substrate could brings flexibility and convenience in HT-screening of substance libraries and help in the development of novel drugs against tuberculosis.

#### **6. References**


**Reaction volume 200 µl 300 µl 350 µl**  Z′ factor 0.67 ±0.021 0.72 ± 0.014 0.73 ±0.012

Large gaps remain in our understanding of mycobacterium pathogenesis and persistence including the critical questions how bacteria survive in host cells and escape from the therapy. Future work on mycobacterial cell wall biosynthesis especially glycolipids and related pathways is expected to reveal *in vivo* drug-resistance mechanism. Perhaps more notably, the described new and low-cost colorimetric method based on use of TMM as natural substrate could brings flexibility and convenience in HT-screening of substance

Alahari, A.;Trivelli, X.;Guerardel, Y.;Dover, L.G.;Besra, G.S.;Sacchettini, J.C.;Reynolds,

Alahari, A.;Alibaud, L.;Trivelli, X.;Gupta, R.;Lamichhane, G.;Reynolds, R.C.;Bishai,

*microbiology*, Vol.71, No.5, (March 2009), pp. 1263-1277, ISSN 1365-2958 Anderson, D.H.;Harth, G.;Horwitz, M.A., & Eisenberg, D. (2001) An interfacial mechanism

Armitige, L.Y.;Jagannath, C.;Wanger, A.R., & Norris, S.J. (2000) Disruption of the genes

Asselineau, J., & Lederer, E. (1950) Structure of the mycolic acids of Mycobacteria. *Nature*,

Backus, K.M.;Boshoff, H.I.;Barry, C.S.;Boutureira, O.;Patel, M.K.;D'Hooge, F.;Lee, S.S.;Via,

Barry, C.E., 3rd;Lee, R.E.;Mdluli, K.;Sampson, A.E.;Schroeder, B.G.;Slayden, R.A., & Yuan,

Vol.166, No.4227, (November 1950), pp. 782-783, ISSN 0028-0836

R.C.;Coxon, G.D., & Kremer, L. (2007) Thiacetazone, an antitubercular drug that inhibits cyclopropanation of cell wall mycolic acids in mycobacteria. *PLoS One*,

W.R.;Guerardel, Y., & Kremer, L. (2009) Mycolic acid methyltransferase, MmaA4, is necessary for thiacetazone susceptibility in *Mycobacterium tuberculosis*. *Molecular* 

and a class of inhibitors inferred from two crystal structures of the *Mycobacterium tuberculosis* 30 kDa major secretory protein (Antigen 85B), a mycolyl transferase. *Journal of molecular biology*, Vol.307, No.2, (March 2001), pp. 671-681, ISSN 0022-

encoding antigen 85A and antigen 85B of *Mycobacterium tuberculosis H37Rv*: effect on growth in culture and in macrophages. *Infection and immunity*, Vol.68, No.2,

L.E.;Tahlan, K.;Barry, C.E., 3rd, & Davis, B.G. (2011) Uptake of unnatural trehalose analogs as a reporter for *Mycobacterium tuberculosis*. *Nature chemical biology*, Vol.7,

Y. (1998) Mycolic acids: structure, biosynthesis and physiological functions. *Progress in lipid research*, Vol.37, No.2-3, (July-August 1998), pp. 143-179, ISSN 0163-

Table 2. The calculated Z′ factor at different volumes from 96-well plate format assays.

libraries and help in the development of novel drugs against tuberculosis.

Vol.2, No.12, (December 2007), pp. e1343, ISSN 1932-6203

(February 2000), pp. 767-778, ISSN 0019-9567

No.4, (April 2011), pp. 228-235, ISSN 1552-4469

**5. Concluding remarks** 

**6. References** 

2836

7827


The Cord Factor: Structure, Biosynthesis and Application in

No.13, (July 2004), pp. 3559-3562, ISSN 0960-894X

0021-9258

0002-9440

9538

1573-1587, ISSN 0950-382X

2006), pp. 97-111, ISSN 1467-3037

Drug Research - Achilles Heel of *Mycobacterium tuberculosis*? 201

Glickman, M.S.;Cox, J.S., & Jacobs, W.R., Jr. (2000) A novel mycolic acid cyclopropane

Gobec, S.;Plantan, I.;Mravljak, J.;Svajger, U.;Wilson, R.A.;Besra, G.S.;Soares, S.L.;Appelberg,

*medicinal chemistry*, Vol.42, No.1, (January 2007), pp. 54-63, ISSN 0223-5234 Hunter, R.L.;Olsen, M.;Jagannath, C., & Actor, J.K. (2006a) Trehalose 6,6'-dimycolate and

Hunter, R.L.;Olsen, M.R.;Jagannath, C., & Actor, J.K. (2006b) Multiple roles of cord factor in

crosstalk for the biosynthesis of mycolic acids in *Mycobacterium tuberculosis*. *The Journal of biological chemistry*, Vol.284, No.29, (July 2009), pp. 19255-19264, ISSN

synthetase is required for cording, persistence, and virulence of *Mycobacterium tuberculosis*. *Molecular cell*, Vol.5, No.4, (April 2000), pp. 717-727, ISSN 1097-2765 Gobec, S.;Plantan, I.;Mravljak, J.;Wilson, R.A.;Besra, G.S., & Kikelj, D. (2004) Phosphonate

inhibitors of antigen 85C, a crucial enzyme involved in the biosynthesis of the *Mycobacterium tuberculosis* cell wall. *Bioorganic & medicinal chemistry letters*, Vol.14,

R., & Kikelj, D. (2007) Design, synthesis, biochemical evaluation and antimycobacterial action of phosphonate inhibitors of antigen 85C, a crucial enzyme involved in biosynthesis of the mycobacterial cell wall. *European journal of* 

lipid in the pathogenesis of caseating granulomas of tuberculosis in mice. *The American journal of pathology*, Vol.168, No.4, (April 2006a), pp. 1249-1261, ISSN

the pathogenesis of primary, secondary, and cavitary tuberculosis, including a revised description of the pathology of secondary disease. *Annals of clinical and laboratory science*, Vol.36, No.4, (Autumn 2006b), pp. 371-386, ISSN 0091-7370 Indrigo, J.;Hunter, R.L., Jr., & Actor, J.K. (2002) Influence of trehalose 6,6'-dimycolate (TDM)

during mycobacterial infection of bone marrow macrophages. *Microbiology (Reading, England)*, Vol.148, No.Pt 7, (July 2002), pp. 1991-1998, ISSN 1350-0872 Ishikawa, E.;Ishikawa, T.;Morita, Y.S.;Toyonaga, K.;Yamada, H.;Takeuchi, O.;Kinoshita,

T.;Akira, S.;Yoshikai, Y., & Yamasaki, S. (2009) Direct recognition of the mycobacterial glycolipid, trehalose dimycolate, by C-type lectin Mincle. *The Journal of experimental medicine*, Vol.206, No.13, (December 2009), pp. 2879-2888, ISSN 1540-

D.;Gicquel, B., & Daffe, M. (1999) Inactivation of the antigen 85C gene profoundly affects the mycolate content and alters the permeability of the *Mycobacterium tuberculosis* cell envelope. *Molecular microbiology*, Vol.31, No.5, (March 1999), pp.

Grininger, M. (2008) Inhibition of the fungal fatty acid synthase type I multienzyme complex. *Proceedings of the National Academy of Sciences of the United States of* 

Drug resistance in *Mycobacterium tuberculosis*. *Curr Issues Mol Biol*, Vol.8, No.2, (July

Jackson, M.;Raynaud, C.;Laneelle, M.A.;Guilhot, C.;Laurent-Winter, C.;Ensergueix,

Johansson, P.;Wiltschi, B.;Kumari, P.;Kessler, B.;Vonrhein, C.;Vonck, J.;Oesterhelt, D., &

*America*, Vol.105, No.35, (September 2008), pp. 12803-12808, ISSN 1091-6490 Johnson, R.;Streicher, E.M.;Louw, G.E.;Warren, R.M.;van Helden, P.D., & Victor, T.C. (2006)

*environmental microbiology*, Vol.77, No.14, (July 2011), pp. 5040-5043, ISSN 1098- 5336


Casadevall, A., & Pirofski, L.A. (2000) Host-pathogen interactions: basic concepts of

Coelho, E.A.;Tavares, C.A.;Lima Kde, M.;Silva, C.L.;Rodrigues, J.M., Jr., & Fernandes, A.P.

*Parasitology research*, Vol.98, No.6, (May 2006), pp. 568-575, ISSN 0932-0113 Coker, R.J. (2004) Review: multidrug-resistant tuberculosis: public health challenges.

Daffe, M. (2000) The mycobacterial antigens 85 complex - from structure to function and

Davidsen, J.;Rosenkrands, I.;Christensen, D.;Vangala, A.;Kirby, D.;Perrie, Y.;Agger, E.M., &

Douglas, J.D.;Senior, S.J.;Morehouse, C.;Phetsukiri, B.;Campbell, I.B.;Besra, G.S., & Minnikin,

Elamin, A.A.;Stehr, M.;Oehlmann, W., & Singh, M. (2009) The mycolyltransferase 85A, a

Elbein, A.D., & Mitchell, M. (1973) Levels of glycogen and trehalose in *Mycobacterium* 

Fernandes, N.D., & Kolattukudy, P.E. (1996) Cloning, sequencing and characterization of a

Fujita, Y.;Naka, T.;Doi, T., & Yano, I. (2005) Direct molecular mass determination of

Gavalda, S.;Leger, M.;van der Rest, B.;Stella, A.;Bardou, F.;Montrozier, H.;Chalut, C.;Burlet-

*Gene*, Vol.170, No.1, (April 1996), pp. 95-99, ISSN 0378-1119

*bacteriology*, Vol.113, No.2, (February 1973), pp. 863-873, ISSN 0021-9193 Elbein, A.D.;Pan, Y.T.;Pastuszak, I., & Carroll, D. (2003) New insights on trehalose: a

No.12, (December 2000), pp. 6511-6518, ISSN 0019-9567

5336

40, ISSN 1360-2276

pp. 22-31, ISSN 0006-3002

ISSN 0959-6658

1443-1452, ISSN 1350-0872

2002), pp. 3101-3109, ISSN 1350-0872

0966-842X

*environmental microbiology*, Vol.77, No.14, (July 2011), pp. 5040-5043, ISSN 1098-

microbial commensalism, colonization, infection, and disease. *Infect Immun*, Vol.68,

(2006) *Mycobacterium* hsp65 DNA entrapped into TDM-loaded PLGA microspheres induces protection in mice against *Leishmania* (*Leishmania*) *major* infection.

*Tropical medicine & international health : TM & IH*, Vol.9, No.1, (January 2004), pp. 25-

beyond. *Trends in microbiology*, Vol.8, No.10, (October 2000), pp. 438-440, ISSN

Andersen, P. (2005) Characterization of cationic liposomes based on dimethyldioctadecylammonium and synthetic cord factor from *M. tuberculosis* (trehalose 6,6'-dibehenate)-a novel adjuvant inducing both strong CMI and antibody responses. *Biochimica et biophysica acta*, Vol.1718, No.1-2, (December 2005),

D.E. (2002) Analogues of thiolactomycin: potential drugs with enhanced antimycobacterial activity. *Microbiology (Reading, England)*, Vol.148, No.Pt 10, (October

putative drug target of *Mycobacterium tuberculosis*: development of a novel assay and quantification of glycolipid-status of the mycobacterial cell wall. *Journal of microbiological methods*, Vol.79, No.3, (December 2009), pp. 358-363, ISSN 1872-8359

*smegmatis* and the purification and properties of the glycogen synthetase. *Journal of* 

multifunctional molecule. *Glycobiology*, Vol.13, No.4, (April 2003), pp. 17R-27R,

fatty acid synthase-encoding gene from *Mycobacterium tuberculosis* var. *bovis* BCG.

trehalose monomycolate from 11 species of mycobacteria by MALDI-TOF mass spectrometry. *Microbiology (Reading, England)*, Vol.151, No.Pt 5, (May 2005), pp.

Schiltz, O.;Marrakchi, H.;Daffe, M., & Quemard, A. (2009) The Pks13/FadD32

crosstalk for the biosynthesis of mycolic acids in *Mycobacterium tuberculosis*. *The Journal of biological chemistry*, Vol.284, No.29, (July 2009), pp. 19255-19264, ISSN 0021-9258


The Cord Factor: Structure, Biosynthesis and Application in

pp. 1607-1610, ISSN 0036-8075

425-431, ISSN 0385-5600

318,

ISSN 0019-9567

6100, ISSN 0014-2956

Drug Research - Achilles Heel of *Mycobacterium tuberculosis*? 203

Matsunaga, I., & Moody, D.B. (2009) Mincle is a long sought receptor for mycobacterial cord factor. *J Exp Med*, Vol.206, No.13, (December 2009), pp. 2865-2868, ISSN 1540-9538 McMurry, L.M.;McDermott, P.F., & Levy, S.B. (1999) Genetic evidence that InhA of

Middlebrook, G.;Dubos, R.J., & Pierce, C. (1947) Virulence and Morphological

Mizuguchi, Y.;Udou, T., & Yamada, T. (1983) Mechanism of antibiotic resistance in

Murphy, H.N.;Stewart, G.R.;Mischenko, V.V.;Apt, A.S.;Harris, R.;McAlister, M.S.;Driscoll,

*chemistry*, Vol.280, No.15, (April 2005), pp. 14524-14529, ISSN 0021-9258 Nicholls, A.;Sharp, K.A., & Honig, B. (1991) Protein folding and association: insights from

Noll, H., H. Bloch, J. Asselineau, and E. Lederer (1956) The chemical structure of the cord

Oswald, I.P.;Dozois, C.M.;Petit, J.F., & Lemaire, G. (1997) Interleukin-12 synthesis is a

Pan, Y.T.;Carroll, J.D., & Elbein, A.D. (2002) Trehalose-phosphate synthase of *Mycobacterium* 

Parrish, N.M.;Houston, T.;Jones, P.B.;Townsend, C., & Dick, J.D. (2001) In vitro activity of a

Perez, R.L.;Roman, J.;Staton, G.W., Jr., & Hunter, R.L. (1994) Extravascular coagulation and

Perez, R.L.;Roman, J.;Roser, S.;Little, C.;Olsen, M.;Indrigo, J.;Hunter, R.L., & Actor, J.K.

Vol.149, No.2 Pt 1, (February 1994), pp. 510-518, ISSN 1073-449X

Vol.86, No.2, (July 1947), pp. 175-184, ISSN 0022-1007

No.4, (December 1991), pp. 281-296, ISSN 0887-3585

(April 2001), pp. 1143-1150, ISSN 0066-4804

*chemotherapy*, Vol.43, No.3, (March 1999), pp. 711-713, ISSN 0066-4804 Mdluli, K.;Slayden, R.A.;Zhu, Y.;Ramaswamy, S.;Pan, X.;Mead, D.;Crane, D.D.;Musser, J.M.,

*Mycobacterium smegmatis* is a target for triclosan. *Antimicrobial agents and* 

& Barry, C.E., 3rd (1998) Inhibition of a *Mycobacterium tuberculosis* beta-ketoacyl ACP synthase by isoniazid. *Science (New York, N.Y.)*, Vol.280, No.5369, (June 1998),

Characteristics of Mammalian Tubercle Bacilli. *The Journal of experimental medicine*,

*Mycobacterium intracellulare*. *Microbiology and immunology*, Vol.27, No.5, 1983), pp.

P.C.;Young, D.B., & Robertson, B.D. (2005) The OtsAB pathway is essential for trehalose biosynthesis in *Mycobacterium tuberculosis*. *The Journal of biological* 

the interfacial and thermodynamic properties of hydrocarbons. *Proteins*, Vol.11,

factor of *Mycobacterium tuberculosis*. *Biochim. Biophys. Acta*, Vol.20, 1956), pp. 299-

required step in trehalose dimycolate-induced activation of mouse peritoneal macrophages. *Infection and immunity*, Vol.65, No.4, (April 1997), pp. 1364-1369,

*tuberculosis*. Cloning, expression and properties of the recombinant enzyme. *European journal of biochemistry / FEBS*, Vol.269, No.24, (December 2002), pp. 6091-

novel antimycobacterial compound, N-octanesulfonylacetamide, and its effects on lipid and mycolic acid synthesis. *Antimicrobial agents and chemotherapy*, Vol.45, No.4,

fibrinolysis in murine lung inflammation induced by the mycobacterial cord factor trehalose-6,6'-dimycolate. *American journal of respiratory and critical care medicine*,

(2000) Cytokine message and protein expression during lung granuloma formation and resolution induced by the mycobacterial cord factor trehalose-6,6'-dimycolate.


Kalscheuer, R.;Weinrick, B.;Veeraraghavan, U.;Besra, G.S., & Jacobs, W.R., Jr. (2010)

Kaur, D.;Guerin, M.E.;Skovierova, H.;Brennan, P.J., & Jackson, M. (2009) Chapter 2:

Kikuchi, S.;Rainwater, D.L., & Kolattukudy, P.E. (1992) Purification and characterization of

Kilburn, J.O.;Takayama, K., & Armstrong, E.L. (1982) Synthesis of trehalose dimycolate

Klegerman, M.E.;Oner, F.;Morris, P.;Son, K., & Groves, M.J. (1994) Isolation of a fibronectin-

Koch, R. (1884) Die aetiologie der tuberkulose. *Mittheilungen aus dem Kaiserlichen* 

Kremer, L.;Douglas, J.D.;Baulard, A.R.;Morehouse, C.;Guy, M.R.;Alland, D.;Dover,

Kremer, L.;Maughan, W.N.;Wilson, R.A.;Dover, L.G., & Besra, G.S. (2002) The *M. tuberculosis*

Kremer, L.;Dover, L.G.;Morbidoni, H.R.;Vilcheze, C.;Maughan, W.N.;Baulard, A.;Tu,

Lima, K.M.;Santos, S.A.;Lima, V.M.;Coelho-Castelo, A.A.;Rodrigues, J.M., Jr., & Silva, C.L.

Luckner, S.R.;Liu, N.;am Ende, C.W.;Tonge, P.J., & Kisker, C. (2010) A slow, tight binding

*Microbios*, Vol.80, No.324, 1994), pp. 173-180, ISSN 0026-2633

Vol.275, No.22, (June 2000), pp. 16857-16864, ISSN 0021-9258

Vol.278, No.23, (June 2003), pp. 20547-20554, ISSN 0021-9258

*Gesundheitsamte*, Vol.2, 1884), pp. 1-88,

No.4, (April 2002), pp. 233-237.,

685, ISSN 0969-7128

14337, ISSN 1083-351X

21766, ISSN 1091-6490

2164

ISSN 0003-9861

ISSN 0006-291X

Trehalose-recycling ABC transporter LpqY-SugA-SugB-SugC is essential for virulence of *Mycobacterium tuberculosis*. *Proceedings of the National Academy of Sciences of the United States of America*, Vol.107, No.50, (December 2010), pp. 21761-

Biogenesis of the cell wall and other glycoconjugates of *Mycobacterium tuberculosis*. *Advances in applied microbiology*, Vol.69, (September 2009), pp. 23-78, ISSN 0065-

an unusually large fatty acid synthase from *Mycobacterium tuberculosis* var. *bovis* BCG. *Archives of biochemistry and biophysics*, Vol.295, No.2, (June 1992), pp. 318-326,

(cord factor) by a cell-free system of *Mycobacterium smegmatis*. *Biochemical and biophysical research communications*, Vol.108, No.1, (September 1982), pp. 132-139,

binding tryptic peptide from the antigen 85A protein of *Mycobacterium bovis* BCG.

L.G.;Lakey, J.H.;Jacobs, W.R., Jr.;Brennan, P.J.;Minnikin, D.E., & Besra, G.S. (2000) Thiolactomycin and related analogues as novel anti-mycobacterial agents targeting KasA and KasB condensing enzymes in *Mycobacterium tuberculosis*. *J Biol Chem*,

antigen 85 complex and mycolyltransferase activity. *Lett Appl Microbiol.*, Vol.34,

S.C.;Honore, N.;Deretic, V.;Sacchettini, J.C.;Locht, C.;Jacobs, W.R., Jr., & Besra, G.S. (2003) Inhibition of InhA activity, but not KasA activity, induces formation of a KasA-containing complex in mycobacteria. *The Journal of biological chemistry*,

(2003) Single dose of a vaccine based on DNA encoding mycobacterial hsp65 protein plus TDM-loaded PLGA microspheres protects mice against a virulent strain of *Mycobacterium tuberculosis*. *Gene therapy*, Vol.10, No.8, (April 2003), pp. 678-

inhibitor of InhA, the enoyl-acyl carrier protein reductase from *Mycobacterium tuberculosis*. *The Journal of biological chemistry*, Vol.285, No.19, (May 2010), pp. 14330-


The Cord Factor: Structure, Biosynthesis and Application in

1472-9792

1554-8937

ISSN 1878-4186

Drug Research - Achilles Heel of *Mycobacterium tuberculosis*? 205

Sathyamoorthy, N., & Takayama, K. (1987) Purification and characterization of a novel

Schroeder, E.K.;de Souza, N.;Santos, D.S.;Blanchard, J.S., & Basso, L.A. (2002) Drugs that

Slayden, R.A.;Lee, R.E., & Barry, C.E., 3rd (2000) Isoniazid affects multiple components of

*microbiology*, Vol.38, No.3, (November 2000), pp. 514-525, ISSN 0950-382X Slayden, R.A., & Barry, C.E., 3rd (2002) The role of KasA and KasB in the biosynthesis of

Spargo, B.J.;Crowe, L.M.;Ioneda, T.;Beaman, B.L., & Crowe, J.H. (1991) Cord factor

Sullivan, T.J.;Truglio, J.J.;Boyne, M.E.;Novichenok, P.;Zhang, X.;Stratton, C.F.;Li, H.J.;Kaur,

Swanson, S.;Gokulan, K., & Sacchettini, J.C. (2009) *KasA*, another brick in the mycobacterial

Takayama, K.;Wang, L., & David, H.L. (1972) Effect of isoniazid on the in vivo mycolic acid

*agents and chemotherapy*, Vol.2, No.1, (July 1972), pp. 29-35, ISSN 0066-4804 Takayama, K.;Schnoes, H.K.;Armstrong, E.L., & Boyle, R.W. (1975) Site of inhibitory action

Takayama, K.;Wang, C., & Besra, G.S. (2005) Pathway to synthesis and processing of

Wang, J.;Elchert, B.;Hui, Y.;Takemoto, J.Y.;Bensaci, M.;Wennergren, J.;Chang, H.;Rai, R., &

Washko, M.E., & Rice, E.W. (1961) Determination of glucose by an improved enzymatic procedure. *Clinical chemistry*, Vol.7, (October 1961), pp. 542-545, ISSN 0009-9147

*lipid research*, Vol.16, No.4, (July 1975), pp. 308-317, ISSN 0022-2275

No.1, (January 2005), pp. 81-101, ISSN 0893-8512

No.24, (December 2004), pp. 6397-6413, ISSN 0968-0896

Vol.88, No.3, (February 1991), pp. 737-740, ISSN 0027-8424

*Biotechnol*, Vol.3, No.3, (September 2002), pp. 197-225, ISSN 1389-2010 Silva, C.L., & Faccioli, L.H. (1988) Tumor necrosis factor (cachectin) mediates induction of

(December 1988), pp. 3067-3071, ISSN 0019-9567

mycolic acid exchange enzyme from *Mycobacterium smegmatis*. *The Journal of biological chemistry*, Vol.262, No.28, (October 1987), pp. 13417-13423, ISSN 0021-9258

inhibit mycolic acid biosynthesis in *Mycobacterium tuberculosis*. *Curr Pharm* 

cachexia by cord factor from mycobacteria. *Infection and immunity*, Vol.56, No.12,

the type II fatty acid synthase system of *Mycobacterium tuberculosis*. *Molecular* 

meromycolic acids and isoniazid resistance in *Mycobacterium tuberculosis*. *Tuberculosis (Edinburgh, Scotland)*, Vol.82, No.4, (August 2002), pp. 149-160, ISSN

(alpha,alpha-trehalose 6,6'-dimycolate) inhibits fusion between phospholipid vesicles. *Proceedings of the National Academy of Sciences of the United States of America*,

T.;Amin, A.;Johnson, F.;Slayden, R.A.;Kisker, C., & Tonge, P.J. (2006) High affinity InhA inhibitors with activity against drug-resistant strains of *Mycobacterium tuberculosis*. *ACS chemical biology*, Vol.1, No.1, (February 2006), pp. 43-53, ISSN

cell wall. *Structure (London, England : 1993)*, Vol.17, No.7, (July 2009), pp. 914-915,

synthesis, cell growth, and viability of *Mycobacterium tuberculosis*. *Antimicrobial* 

of isoniazid in the synthesis of mycolic acids in *Mycobacterium tuberculosis*. *Journal of* 

mycolic acids in *Mycobacterium tuberculosis*. *Clinical microbiology reviews*, Vol.18,

Chang, C.W. (2004) Synthesis of trehalose-based compounds and their inhibitory activities against *Mycobacterium smegmatis*. *Bioorganic & medicinal chemistry*, Vol.12,

*Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research*, Vol.20, No.9, (September 2000), pp. 795-804, ISSN 1079-9907


Portevin, D.;De Sousa-D'Auria, C.;Houssin, C.;Grimaldi, C.;Chami, M.;Daffe, M., & Guilhot,

Portevin, D.;de Sousa-D'Auria, C.;Montrozier, H.;Houssin, C.;Stella, A.;Laneelle,

Ronning, D.R.;Vissa, V.;Besra, G.S.;Belisle, J.T., & Sacchettini, J.C. (2004) *Mycobacterium* 

Russell, D.G.;Barry, C.E., 3rd, & Flynn, J.L. (2010) Tuberculosis: what we don't know can,

Ryll, R.;Kumazawa, Y., & Yano, I. (2001) Immunological properties of trehalose dimycolate

Saito, R.;Tanaka, A.;Sugiyama, K.;Azuma, I., & Yamamura, Y. (1976) Adjuvant effect of cord

Sanki, A.K.;Boucau, J.;Srivastava, P.;Adams, S.S.;Ronning, D.R., & Sucheck, S.J. (2008)

Sanki, A.K.;Boucau, J.;Ronning, D.R., & Sucheck, S.J. (2009a) Antigen 85C-mediated acyl-

*bioSystems*, Vol.5, No.9, (September 2009b), pp. 945-956, ISSN 1742-2051

*and immunology*, Vol.45, No.12, 2001), pp. 801-811, 0385-5600

ISSN 1079-9907

314-319, ISSN 0027-8424

2000), pp. 141-146, ISSN 1072-8368

856, ISSN 1095-9203

776-781, ISSN 0019-9567

2008), pp. 5672-5682, ISSN 1464-3391

(August 2004), pp. 36771-36777, ISSN 0021-9258

*Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research*, Vol.20, No.9, (September 2000), pp. 795-804,

C. (2004) A polyketide synthase catalyzes the last condensation step of mycolic acid biosynthesis in mycobacteria and related organisms. *Proceedings of the National Academy of Sciences of the United States of America*, Vol.101, No.1, (January 2004), pp.

M.A.;Bardou, F.;Guilhot, C., & Daffe, M. (2005) The acyl-AMP ligase FadD32 and AccD4-containing acyl-CoA carboxylase are required for the synthesis of mycolic acids and essential for mycobacterial growth: identification of the carboxylation product and determination of the acyl-CoA carboxylase components. *The Journal of biological chemistry*, Vol.280, No.10, (March 2005), pp. 8862-8874, ISSN 0021-9258 Ronning, D.R.;Klabunde, T.;Besra, G.S.;Vissa, V.D.;Belisle, J.T., & Sacchettini, J.C. (2000)

Crystal structure of the secreted form of antigen 85C reveals potential targets for mycobacterial drugs and vaccines. *Nature structural biology*, Vol.7, No.2, (February

*tuberculosis* antigen 85A and 85C structures confirm binding orientation and conserved substrate specificity. *The Journal of biological chemistry*, Vol.279, No.35,

and does, hurt us. *Science (New York, N.Y.)*, Vol.328, No.5980, (May 2010), pp. 852-

(cord factor) and other mycolic acid-containing glycolipids--a review. *Microbiology* 

factor, a mycobacterial lipid. *Infection and immunity*, Vol.13, No.3, (March 1976), pp.

Synthesis of methyl 5-S-alkyl-5-thio-D-arabinofuranosides and evaluation of their antimycobacterial activity. *Bioorganic & medicinal chemistry*, Vol.16, No.10, (May

transfer between synthetic acyl donors and fragments of the arabinan. *Glycoconjugate journal*, Vol.26, No.5, (July 2009a), pp. 589-896, ISSN 1573-4986 Sanki, A.K.;Boucau, J.;Umesiri, F.E.;Ronning, D.R., & Sucheck, S.J. (2009b) Design, synthesis

and biological evaluation of sugar-derived esters, alpha-ketoesters and alphaketoamides as inhibitors for *Mycobacterium tuberculosis* antigen 85C. *Molecular* 


**Part 2** 

**New Drugs to Face Resistance** 

