**2. Glyoxylate pathway: The saviour of the anarchist**

Tuberculosis is characterized by periods in which the disease may be non-obvious or even clinically inapparent but even in these regimens mycobacteria persist with the potential to reactivate the disease opportunistically. Persistence may be defined as a stage where the metabolic downshift to anaerobiosis brings about a nutritionally suspended condition. In the granulomas the bacterium does not replicate and becomes inert yet surviving in occult forms to get activated in immune-compromised situations. In persistent phase the *ala carte* is changed from glucose to delicious lipids, glycolysis is decreased and the glyoxylate shunt is upregulated allowing anaplerotic maintenance of the tricarboxylic acid (TCA) cycle (McKinney et al., 2000). The glyoxylate shunt converts isocitrate to succinate and glyoxylate, catalyzed by the enzyme isocitrate lyase (ICL), followed by the addition of Acetyl-CoA to

*Mycobacterium tuberculosis*: Dormancy, Persistence and Survival in the Light of Protein Synthesis 221

governed by subtle ionic interactions (Kumar & Bhakuni, 2011). Antibodies to MS have been discovered in 90% of patients during incipient subclinical tuberculosis (Singh et al., 2003; Singh et al., 2005). Bishai (Gyanu Lamichhane, 2003) reported the random insertion of transposons into the MtbMS gene (*glcB*, annotated Rv1837c) resulted in a non-viability under normal growth conditions; in contrast, Sassetti (Sassetti & Rubin, 2003) determined that MtbMS is not required for tuberculosis infection in mice using a similar transposon insertion method. However, attempts to knockout Mtb *glcB* have been unsuccessful (J. D. McKinney, personal communication). This consolidates the role of MS in metabolic up

In enzymology, an isocitrate lyase (EC 4.1.3.1) is an enzyme that catalyzes the reversible aldol cleavage of threo-DS(+)-isocitric acid to succinic and glyoxylic acids (Rua et al., 1989)

Isocitrate Succinate + Glyoxylate

Hence, this enzyme has one substrate, isocitrate, and two products, succinate and glyoxylate. This enzyme belongs to the family of lyases, specifically the oxo-acid-lyases, which cleave carbon-carbon bonds. The systematic name of this enzyme class is isocitrate glyoxylate-lyase (succinate-forming). Isocitrate lyase is the first enzyme unique to the metabolic pathway known as the glyoxylate cycle which is required for the assimilation of fatty acids and acetate (Kelly et al., 2002). Recent reports have alluded to an additional role for this enzyme in *M. tuberculosis* metabolism, specifically for growth on propionate. A product of betaoxidation of odd-chain fatty acids is propionyl-CoA. Clearance of propionyl-CoA and the

keeping of the bacterium when hostile environment prevails.

Acetyl-CoA + H2O + Glyoxylate (S)-Malate + CoA Fig. 2. Reaction scheme of glyoxylate shunt.

**2.2 The rescue saga** 

glyoxylate to form malate by malate synthase (MS) (Sharma et al., 2000). The glyoxylate shunt allows the bacteria to avoid the carbon dioxide generating steps of the Krebs cycle, enabling them to shunt carbons from fats to carbohydrate synthesis (Wayne, 1994). The drugs used today in combination therapy for treating tuberculosis were discovered 40 years ago and none of them has been effective against these robust persisters (Reddy et al., 2009). The global mandate today is focused to reduce the treatment time line which is six to nine months at present. Targeting pathways that get triggered during persistence phase can yield potential leads. As humans do not have functional glyoxylate pathway the enzymes of the pathway are promising drug target (Kumar & Bhakuni, 2008). The relative abundance of genes pertaining to fatty acid degradation (more than 150) in mycobacterial genome highlights the importance of survival on lipids derived from host *in vivo* (Cole, 1999).

### **2.1 The arsenal of survival**

Enzymes of the glyoxylate shunt operate by diverting carbon from beta-oxidation of fatty acids into the glyoxylate pathway to sustain a nutrient starved intracellular infection they have been implicated for their roles in both virulence and persistence in candida (Lorenz & Fink, 2001) and salmonella (Allen et al., 2000) too. One protein present at significantly higher level in the phagocytized population was identified by microsequencing as isocitrate lyase, a key enzyme of the glyoxylate cycle (Manabe et al., 1999). Furthermore, cDNA selection technique to identify genes upregulated upon phagocytosis revealed Isocitrate lyase as one of the 11 genes identified and was the only metabolic gene in the set (Graham & Clark-Curtiss, 1999).Expression of ICL is upregulated under certain challenged growth conditions (Honer Zu Bentrup et al., 1999) and during infection of macrophages by Mycobacterium spp. It has also been demonstrated that ICL is important for survival of *M. tuberculosis* in the lungs of mice during the persistent phase of infection (2–16 weeks), but is not essential during the acute phase (0–2 weeks) of infection (McKinney et al., 2000). Recent report suggests that suppressing the apoptosis of host macrophage may be one of the important mechanisms for their increased intracellular survival (LI Jun-ming, 2008). Common to all ICL is a signature sequence 'KKCGH'. It has the nucleophilic cysteine residue that sits on a flexible loop which undergoes large conformational change after binding of substrate resulting in complete closing of the active site from the bulk solvent (Smith et al., 2004). Interestingly it is quite important to note that although the genome of *M. tuberculosis* encodes orthologues of two of the three enzymes of the methylcitrate cycle, methylcitrate synthase and methylcitrate dehydratase, it does not appear to contain a distinct 2-methyl isocitrate lyase (MCL). ICL from *M. tuberculosis* can clearly function as a MCL thus metabolizing both Acetyl and Propionyl CoA generated by β-oxidation of even and odd chain fatty acids facilitating the lipid lunch (Gould et al., 2006).

The other enzyme downstream in the pathway is Malate Synthase G (MtbMS) that drives the reaction ahead. A single malate synthase gene called *glcB* (Rv1837c) has been identified in *M. tuberculosis* encoding MtbMS (Smith et al., 2004). MtbMS is a very important housekeeping enzyme involved in persistence of the bacteria. Its intracellular/extracellular localization acting as adhesion and virulence factor together with persistence is quite enigmatic. A recent study further showed the existence of active dimeric form of the enzyme that may add to multiplicity of function that the enzyme exhibits (Kumar & Bhakuni, 2010). It was further revealed in a recent study that the enzyme in mycobacterium differ substantially as compared to *Escherichia coli* by having differences in molecular assembly governed by subtle ionic interactions (Kumar & Bhakuni, 2011). Antibodies to MS have been discovered in 90% of patients during incipient subclinical tuberculosis (Singh et al., 2003; Singh et al., 2005). Bishai (Gyanu Lamichhane, 2003) reported the random insertion of transposons into the MtbMS gene (*glcB*, annotated Rv1837c) resulted in a non-viability under normal growth conditions; in contrast, Sassetti (Sassetti & Rubin, 2003) determined that MtbMS is not required for tuberculosis infection in mice using a similar transposon insertion method. However, attempts to knockout Mtb *glcB* have been unsuccessful (J. D. McKinney, personal communication). This consolidates the role of MS in metabolic up keeping of the bacterium when hostile environment prevails.
