**4. Understanding the pathophysiology in light of protein synthesis**

Mycobacterium can maintain itself in the fluctuating environment inside the host and the diversity of its survival mechanism makes it one of the most successful pathogens in the world. The ability of the bacteria to enter a stealth mode and exhibit occult form in combination with its starvation strategies suggests that there exists a complex series of events that is modulated by differential gene expression and protein synthesis. When mycobacterium switches its lifestyle from luxury to mere survival the DNA replication goes to a standby mode (Wayne, 1994) and the protein synthesis process undergoes drastic changes (Hu et al., 1998). Though the knowledge in this research area is limited, understanding the bacterium in the light of protein synthesis and protein turnover opens new avenues to understand this 'culprit in disguise' better.

#### **4.1 Intricacies of macromolecular orchestration**

The protein synthesis process is a hotspot for action of antibiotic drugs, which interact directly with mycobacterial ribosome and sabotage 'protein synthesis', the most vital

of not only tuberculosis, but also candidiasis and melioidosis and many other pathogens (Kumar, 2009). The rationing mechanism for supply of the precursor amino acid and nucleic acids required for growth can be halted and paused by inhibitors of metabolic pathways and thus making it tough for the bug to proliferate and persist. The search for elixir of life continues with the effort to evolve a drug that has bactericidal activity both on the active and persistent form and a robust delivery mechanism that has ability to penetrate diverse

The best form of dormancy has been studied in gram positive bacteria forming spores. As far as mycobacterium is concerned the existence of spore is debatable. A recent study by a group led jointly by Leif Kirsebom and Santanu Dasgupta in Uppsala University put forward the evidence for existence of spores in mycobacterium (Ghosh et al., 2009). The study revealed existence of endospores in very late stationary phase cultures of *Mycobacterium marinum*, a common model for acute mycobacterial infections. Utilizing transmission electron microscopy the distinct outer coat and cortex of the spores could be identified. Furthermore, heat tolerance, malachite green staining, and the presence of dipicolinic acid was also shown. They were able to isolate rRNA from these sporulating cells and identified it as *M. marinum* rRNA. Extensive bioinformatics analysis identified possible homologues of spore forming genes in mycobacterial genome. Thus they suggested that mycobacteria can form spores under stress and presented the hypothesis that sporulation might be one of the adaptations causing dormancy. When genes encoding GFP or mCherry were introduced on plasmids or integrated in to mycobacterium genome fluorescent spore

However, a rebuttal to this work by several US labs challenging the concept of endospore formation in mycobacteria was published (Traag et al., 2010) which was responded in to a subsequent rebuttal (Singh et al., 2010). The controversies continue with more supporting evidences yet to be deciphered about a phenomenon not so common to many other species.

Mycobacterium can maintain itself in the fluctuating environment inside the host and the diversity of its survival mechanism makes it one of the most successful pathogens in the world. The ability of the bacteria to enter a stealth mode and exhibit occult form in combination with its starvation strategies suggests that there exists a complex series of events that is modulated by differential gene expression and protein synthesis. When mycobacterium switches its lifestyle from luxury to mere survival the DNA replication goes to a standby mode (Wayne, 1994) and the protein synthesis process undergoes drastic changes (Hu et al., 1998). Though the knowledge in this research area is limited, understanding the bacterium in the light of protein synthesis and protein turnover opens

The protein synthesis process is a hotspot for action of antibiotic drugs, which interact directly with mycobacterial ribosome and sabotage 'protein synthesis', the most vital

**4. Understanding the pathophysiology in light of protein synthesis** 

metabolic niche to eradicate these killer bugs.

**3.1 Sporulation speculations and beyond** 

were seen confirming their mycobacterial origin.

new avenues to understand this 'culprit in disguise' better.

**4.1 Intricacies of macromolecular orchestration** 

process of the cells. The mechanism of translation and the function of the ribosome in mycobacteria can thus be visited collectively in the light of action of the drugs which include aminoglycosides, macrolides, tetracycline and peptide antibiotics etc. (Inderlied, 1991). At the same time the study of the resistance mechanism in mycobacteria against these drugs, often involving certain mutations on rRNA or r-proteins, would shed light on the process.

In a study where several independent streptomycin-resistant mutants of *M. tuberculosis*  H37Rv were isolated, it was shown that while in the wild-type streptomycin inhibited the incorporation of 14C-amino acids into proteins, very little or no inhibition was observed in either high-level or low-level streptomycin-resistant strains (Shaila et al., 1973). This result on one hand points towards the activity of the drug against mycobacteria and on the other hand brings forward the potential problems arising from the development of resistance phenotypes. The Bottger-group has made very important contribution in the field in identifying potential drug candidates against mycobacteria (Hobbie et al., 2005; Hobbie et al., 2006a; Hobbie et al., 2006b; Hobbie et al., 2007). In addition, they have also identified potential hotspots for mutations leading to resistance against these drugs. Thus, these studies all together call for a continuous quest for identification of new and potential drug candidates for treatment of mycobacterial infections.

Protein synthesis involves a coordinated action of the ribosome, mRNA, tRNAs and translation factors; thus it needs an intelligent setup and precise orchestration between different components. The process, also known as translation, is divided into four distinct steps; namely initiation, elongation, termination and ribosome-recycling. The process initiates by association of large ribosomal subunit with an mRNA programmed small subunit bound with initiator tRNA and initiation factors. Once a 70S initiation complex is successfully formed, the initiation factors dissociate and the process proceeds to elongation. In the first step of elongation the elongation factor–Tu (EF-Tu) brings tRNA loaded with respective amino acids to the decoding center (DC) of the ribosome. This site includes highly conserved regions of 16S rRNA arranged in a highly ordered structure. Correct codon anticodon pairing between the tRNA and mRNA through initial selection and tRNA accommodation leads the process towards the most significant step of protein synthesis, i.e. peptide bond formation. A primarily rRNA (23S rRNA) constituted region of the large subunit, called the Peptidyl-transferase site, catalyses this step. After formation of the peptide bond the tRNA carrying the peptide chain moves one codon along the mRNA, a process called 'tRNA translocation', conducted by elongation factor-G (EF-G). Then the elongation steps repeat and the cycle continues until a stop codon on mRNA reaches the decoding center. The stop codons signal release factors to bind on the mRNA and release the peptide from the tRNA. The process ends by splitting of the ribosomal subunits, which recycle for the next round of protein synthesis. The detailed mechanism of these steps although studied mainly with components from gram-negative bacteria *E. coli*, can be extrapolated to gram-positive mycobacteria due to high degree of sequence conservation in the translation components in all eubacteria. It should be mentioned that although the protein synthesis machinery is highly conserved in general, the fine differences that exist in this system between the pathogenic bacteria and the host organisms provide ample opportunity for targeting the bacterial translation system in a rather specific manner.

Although in recent years, we have acquired a lot of knowledge on the mechanism of bacterial protein synthesis from high resolution crystal structure and fine-tuned biochemical

*Mycobacterium tuberculosis*: Dormancy, Persistence and Survival in the Light of Protein Synthesis 227

(Moazed & Noller, 1987). These studies suggested that antibiotics could act by interacting with highly conserved hotspots of the bacterial rRNA and thereby interfering with their natural functions. There are also evidences that antibiotics inhibit enzymes essential for creating modifications on rRNA vital for its function. Identification of the sites for resistant mutation often sheds light on the antibiotic action. Resistance to streptomycin which acts by causing misreading in the genetic code followed by inhibition of translation initiation and dubious proofreading (Moazed & Noller, 1987) is attributed to mutations in the aminoglycoside modifying enzyme (Benveniste & Davies, 1973) as well as in *rpsL* gene coding for r-protein S12 (Funatsu & Wittmann, 1972; Allen & Noller, 1989). Specific to mycobacteria, it has been shown that mutations in the *rpsL* gene that replaces Lys 43 or Lys 88 by arginine are associated with streptomycin resistance (Honore & Cole, 1994). There have been also evidences of mutations in 16S rRNA clustered in two regions in the 16SrRNA

Macrolides are bacteriostatic in nature and inhibit the petidyl transferase function of the 50S ribosomal subunit by blocking the peptidyl exit-tunnel. The clinically acquired resistance against macrolides is described as the MLS phenotype (resistant to Macrolide, Lincomycin and Stretogramin B) which is thought to be collectively mediated by methylases coded by *erm* genes (Leclercq & Courvalin, 1991). These methylases brings about changes in the conserved loop of domain V of 23S rRNA (A2058 equivalent of *E.coli*) which is implicated to have definitive role in peptidyl transfer. Recent studies have shown paromomycin, an aminoglycoside, to be effective both *in vitro* and *in vivo* against MDR-TB (Kanyok et al., 1994). A study in *E.coli* has mapped the changes in rRNA that confer resistance by transfecting mutated rRNA operons in case of paromomycin(De Stasio et al., 1989). It would be interesting to see if similar mutations in mycobacteria would also confer resistance for

In case of tetracycline studies it was revealed that the drug does not inhibit the growth of *M. tuberculosis* but inhibits protein synthesis *in vitro* suggesting that intact cells are impermeable to the drug (Bottger, 1994). Ribosomal mutations that confer tetracycline resistance are difficult to

map because the mutations occur frequently in the system which pumps the drugs out.

**Protein synthesis: The portfolio for inhibition and innovating novel combat** 

amikacin, kanamycin, and capreomycin) and oxazolidinones (linezolid).

**Peptidyl transfer inhibitor:** Chloroamphenicol, Macrolides

 **Initiation:** Streptomycin / Rifampicin **Elongation and translocation:** Fusidic acid **Amino acyl tRNA inhibitors:** Tetracyclines **Proofreading inhibitors:** Aminoglycosides

**50S:** Chloroamphenicol, Oxazolidinones, Macrolides

 **EFG inhibitors:** Fusidic acid Inhibitors binding to ribosome

**30S:** Aminoglycosides and Tetracyclines

The contemporary treatment of tuberculosis includes aminoglycosides (streptomycin,

leading to resistance to streptomycin (Douglass & Steyn, 1993).

this antibiotic.

**strategies** 

experiments, very little information specific to mycobacteria have been available. Generally it is believed that bacterial growth rate is highly dependent on the rate of protein synthesis and varies according to the number of translationally active ribosomes in the bacterial cells (Dennis & Bremer, 1974). Thus, mycobacterium, a remarkably slow growing pathogenic bacterium, provides a very interesting system for studying the rate of protein synthesis in different stages of its life.

In 2008, a strong step was taken to study the basic components of mycobacterial protein synthesis. It is known that although very similar, mycobacterial ribosomes differ from the well characterized ribosomes from *E. coli* to some extent. Firstly, the mutations on rRNA lead to somewhat different phenotype in these two bacteria. Secondly, the ribosomal stalk, a visible protuberance on the large subunit of the ribosome composed of the multicopy protein L12, differs in its composition and structural conformation between the two. In *E. coli,* the ribosomal stalk is composed of two dimers of L12 protein in contrast to three dimers in *M. smegmatis.* Also in *E. coli,* the stalk changes its conformation from compact to extended form depending on its state of interaction with the translation factors. In comparison, the stalk in mycobacteria seems to be extended most of the time, the functional significance of such behaviour is not known. It should also be mentioned that mycobacterial species often harbour only one or two rRNA operons in contrast to seven rRNA operons in *E. coli.* Thus mycobacteria provide a very good system to test the effect of mutations in the significant positions of rRNA towards the viability of the bacteria (Hobbie et al., 2007; Long et al., 2009). Also, homogeneous populations of the ribosomes carrying mutations in the rRNA could be isolated and characterized relatively easily from the mutated mycobacterial strains.

The study, based on translation components from *M. smegmatis* enriched the knowledge about the translation machinery in mycobacteria. A combined effort by the research groups of Erik C. Böttger and Marina Rodnina led the study where the genes for various translation factors were mapped, cloned, and expressed in heterologous (*E. coli*) expression system. Further, these factors were used together with mycobacterial ribosomes in a complete *in vitro* translation system complemented with some components from *E. coli*. In fast kinetics assay, the rate of initiation and elongation was measured and compared with those obtained from purely *E. coli* origin. Under these conditions, the rates of fundamental reactions of initiation and elongation of protein synthesis were found remarkably similar in the two systems. Thus, these results suggested that under *in* vitro conditions the basic mechanisms of protein synthesis are highly conserved in these two widely separated species of bacteria (Bruell et al., 2008). At the same time, this study provides an alternative system to identify and test the action of the antibiotics. However, it fails to provide any insight about the rate of protein synthesis in slow growing phases of mycobacteria and calls for detailed investigation of the same under controlled *in vivo* conditions using methods such as radiolabelled amino acid incorporation or beta-galactosidase synthesis assay.

#### **4.2 Combat targets and troubleshooting**

There are basically three main hotspots that can confer resistance to inhibitors against protein synthesis. The first is by mutations in the rRNA, the second being by mutations in the ribosomal proteins, and finally by post-transcriptional modification, especially methylation of the ribosomal RNA. Concerted efforts have revealed in the recent past that antibiotics might interfere with chemical probe binding to specific nucleotides in the rRNA

experiments, very little information specific to mycobacteria have been available. Generally it is believed that bacterial growth rate is highly dependent on the rate of protein synthesis and varies according to the number of translationally active ribosomes in the bacterial cells (Dennis & Bremer, 1974). Thus, mycobacterium, a remarkably slow growing pathogenic bacterium, provides a very interesting system for studying the rate of protein synthesis in

In 2008, a strong step was taken to study the basic components of mycobacterial protein synthesis. It is known that although very similar, mycobacterial ribosomes differ from the well characterized ribosomes from *E. coli* to some extent. Firstly, the mutations on rRNA lead to somewhat different phenotype in these two bacteria. Secondly, the ribosomal stalk, a visible protuberance on the large subunit of the ribosome composed of the multicopy protein L12, differs in its composition and structural conformation between the two. In *E. coli,* the ribosomal stalk is composed of two dimers of L12 protein in contrast to three dimers in *M. smegmatis.* Also in *E. coli,* the stalk changes its conformation from compact to extended form depending on its state of interaction with the translation factors. In comparison, the stalk in mycobacteria seems to be extended most of the time, the functional significance of such behaviour is not known. It should also be mentioned that mycobacterial species often harbour only one or two rRNA operons in contrast to seven rRNA operons in *E. coli.* Thus mycobacteria provide a very good system to test the effect of mutations in the significant positions of rRNA towards the viability of the bacteria (Hobbie et al., 2007; Long et al., 2009). Also, homogeneous populations of the ribosomes carrying mutations in the rRNA could be

isolated and characterized relatively easily from the mutated mycobacterial strains.

labelled amino acid incorporation or beta-galactosidase synthesis assay.

**4.2 Combat targets and troubleshooting** 

The study, based on translation components from *M. smegmatis* enriched the knowledge about the translation machinery in mycobacteria. A combined effort by the research groups of Erik C. Böttger and Marina Rodnina led the study where the genes for various translation factors were mapped, cloned, and expressed in heterologous (*E. coli*) expression system. Further, these factors were used together with mycobacterial ribosomes in a complete *in vitro* translation system complemented with some components from *E. coli*. In fast kinetics assay, the rate of initiation and elongation was measured and compared with those obtained from purely *E. coli* origin. Under these conditions, the rates of fundamental reactions of initiation and elongation of protein synthesis were found remarkably similar in the two systems. Thus, these results suggested that under *in* vitro conditions the basic mechanisms of protein synthesis are highly conserved in these two widely separated species of bacteria (Bruell et al., 2008). At the same time, this study provides an alternative system to identify and test the action of the antibiotics. However, it fails to provide any insight about the rate of protein synthesis in slow growing phases of mycobacteria and calls for detailed investigation of the same under controlled *in vivo* conditions using methods such as radio-

There are basically three main hotspots that can confer resistance to inhibitors against protein synthesis. The first is by mutations in the rRNA, the second being by mutations in the ribosomal proteins, and finally by post-transcriptional modification, especially methylation of the ribosomal RNA. Concerted efforts have revealed in the recent past that antibiotics might interfere with chemical probe binding to specific nucleotides in the rRNA

different stages of its life.

(Moazed & Noller, 1987). These studies suggested that antibiotics could act by interacting with highly conserved hotspots of the bacterial rRNA and thereby interfering with their natural functions. There are also evidences that antibiotics inhibit enzymes essential for creating modifications on rRNA vital for its function. Identification of the sites for resistant mutation often sheds light on the antibiotic action. Resistance to streptomycin which acts by causing misreading in the genetic code followed by inhibition of translation initiation and dubious proofreading (Moazed & Noller, 1987) is attributed to mutations in the aminoglycoside modifying enzyme (Benveniste & Davies, 1973) as well as in *rpsL* gene coding for r-protein S12 (Funatsu & Wittmann, 1972; Allen & Noller, 1989). Specific to mycobacteria, it has been shown that mutations in the *rpsL* gene that replaces Lys 43 or Lys 88 by arginine are associated with streptomycin resistance (Honore & Cole, 1994). There have been also evidences of mutations in 16S rRNA clustered in two regions in the 16SrRNA leading to resistance to streptomycin (Douglass & Steyn, 1993).

Macrolides are bacteriostatic in nature and inhibit the petidyl transferase function of the 50S ribosomal subunit by blocking the peptidyl exit-tunnel. The clinically acquired resistance against macrolides is described as the MLS phenotype (resistant to Macrolide, Lincomycin and Stretogramin B) which is thought to be collectively mediated by methylases coded by *erm* genes (Leclercq & Courvalin, 1991). These methylases brings about changes in the conserved loop of domain V of 23S rRNA (A2058 equivalent of *E.coli*) which is implicated to have definitive role in peptidyl transfer. Recent studies have shown paromomycin, an aminoglycoside, to be effective both *in vitro* and *in vivo* against MDR-TB (Kanyok et al., 1994). A study in *E.coli* has mapped the changes in rRNA that confer resistance by transfecting mutated rRNA operons in case of paromomycin(De Stasio et al., 1989). It would be interesting to see if similar mutations in mycobacteria would also confer resistance for this antibiotic.

In case of tetracycline studies it was revealed that the drug does not inhibit the growth of *M. tuberculosis* but inhibits protein synthesis *in vitro* suggesting that intact cells are impermeable to the drug (Bottger, 1994). Ribosomal mutations that confer tetracycline resistance are difficult to map because the mutations occur frequently in the system which pumps the drugs out.

#### **Protein synthesis: The portfolio for inhibition and innovating novel combat strategies**

The contemporary treatment of tuberculosis includes aminoglycosides (streptomycin, amikacin, kanamycin, and capreomycin) and oxazolidinones (linezolid).


**50S:** Chloroamphenicol, Oxazolidinones, Macrolides

**30S:** Aminoglycosides and Tetracyclines

*Mycobacterium tuberculosis*: Dormancy, Persistence and Survival in the Light of Protein Synthesis 229

(Roberts, 1994). A detailed account of the process in slow growing mycobacteria is still not available, which would yield valuable insight about the antibiotic in treatment of

The complex milieu, in which the mycobacterium survives, necessitates dynamic adjustments and fine-tuning in protein synthesis and turn-over. During the transfer from active to dormant stage a change in the nature of the proteins expressed is bound to occur. It remains an open question whether protein synthesis continues in a slow rate in the dormant stage or stops completely following the change of the growth rate in mycobacteria. It is very important to understand how protein synthesis machinery work in the dormant or persistent stage since under favourable conditions this dormant form can again get triggered and reactivated leading to a full blown infection in the immune compromised host; thus posing a big threat. One recent report suggested that the protein synthesis is almost shut down in dormant mycobacterium and the conditions can be reversed by providing stimuli like oxygen or heat shock to anaerobic stationary cultures (Hu et al., 1998). It also illustrates the fact that the mycobacteria are quite responsive to stimuli and can trigger protein synthesis quite rapidly even in the dormant state. Hu *et al*. showed a 98% decrease in protein synthesis using 35S-met pulse labeling experiments with microaerophilic cultures of mycobacterium tuberculosis. However, it is still not understood fully whether there is complete shutdown of protein synthesis or a minimal protein synthesis continues in this state to facilitate survival. Another recent report by Murphy *et.al*, suggests that during dormancy *M. tuberculosis* performs limited protein synthesis and the pathogen spends its available resources in maintaining cell wall, membrane potential, genome integrity as well as resisting host defence systems (Murphy & Brown, 2007). The transcriptiome analysis further revealed five times more downregulated genes in persisters than upregulated ones (1,408 versus 282) under dormant condition suggesting that the growth and energy metabolism pathways are significantly downregulated (Keren et al., 2011). Even the synthesis of the ribosomal proteins tends to get down regulated in the state of dormancy as revealed in several different models (Wayne & Hayes, 1996; Betts et al., 2002; Keren et al., 2011). Although these studies are insufficient to resolve whether proteins synthesis comes to a complete halt or not in the dormant stage, these also open up a new question regarding the availability of the vital proteins in this stage. In other words, these studies indicate

Continuous translation is not the only crucial process that defines the abundance of a protein or enzyme in the cell; instead it is often regulated by the parameters controlling its propensity to degradation and stabilization. Thus, regulation of protein amounts in a nonfavourable and fluctuating environment can be more dependent on turnover of proteins rather than the synthesis (Neher et al., 2003). Our knowledge regarding protein turnover in dormant phases in mycobacteria is still in infancy. However research in the last decade has thrown some light on it by attempting to examine the protein turnover at global level. Two seminal papers (Rao & Li, 2009; Rao et al., 2009) have helped in understanding the molecular basis of persistence in mycobacterium. Rao et.al showed that the protein turnover was much more effective to maintain relative protein abundance in the dormant phases of mycobacteria. These studies also help explain how the bacteria manage to exist as a submissive pathogen in the host for a prolonged period. The molecular detail behind the

mycobacteria.

**4.3 The Vendetta in light of protein turnover** 

towards a longer turn-over of the proteins in this stage.

Fig. 3. Translation scheme showing various steps that can be targets of therapeutic intervention. (Adopted from Mandava CS, Ribosomal Stalk Protein L12 Structure Function and Application, thesis submitted to Uppsala University, Sweden 2011)

Nevertheless two important resistant determinants have been found in *E. coli TetO* and *TetM*  genes. TetM acts by protecting the ribosome from antibiotic attack and the resistance to tetracycline encoded by these genes are located on plasmids or transposable elements (Roberts, 1994). A detailed account of the process in slow growing mycobacteria is still not available, which would yield valuable insight about the antibiotic in treatment of mycobacteria.

### **4.3 The Vendetta in light of protein turnover**

228 Understanding Tuberculosis – Deciphering the Secret Life of the Bacilli

**Initiation** 

**GTP GDP Pi** 

**Polypeptide** 

**Termination** 

**Elongation** 

**Ternary complex** 

**Translocation** 

**Recycling** 

**GTP**

Fig. 3. Translation scheme showing various steps that can be targets of therapeutic

and Application, thesis submitted to Uppsala University, Sweden 2011)

intervention. (Adopted from Mandava CS, Ribosomal Stalk Protein L12 Structure Function

Nevertheless two important resistant determinants have been found in *E. coli TetO* and *TetM*  genes. TetM acts by protecting the ribosome from antibiotic attack and the resistance to tetracycline encoded by these genes are located on plasmids or transposable elements

**30S Preinitiation complex**

The complex milieu, in which the mycobacterium survives, necessitates dynamic adjustments and fine-tuning in protein synthesis and turn-over. During the transfer from active to dormant stage a change in the nature of the proteins expressed is bound to occur. It remains an open question whether protein synthesis continues in a slow rate in the dormant stage or stops completely following the change of the growth rate in mycobacteria. It is very important to understand how protein synthesis machinery work in the dormant or persistent stage since under favourable conditions this dormant form can again get triggered and reactivated leading to a full blown infection in the immune compromised host; thus posing a big threat. One recent report suggested that the protein synthesis is almost shut down in dormant mycobacterium and the conditions can be reversed by providing stimuli like oxygen or heat shock to anaerobic stationary cultures (Hu et al., 1998). It also illustrates the fact that the mycobacteria are quite responsive to stimuli and can trigger protein synthesis quite rapidly even in the dormant state. Hu *et al*. showed a 98% decrease in protein synthesis using 35S-met pulse labeling experiments with microaerophilic cultures of mycobacterium tuberculosis. However, it is still not understood fully whether there is complete shutdown of protein synthesis or a minimal protein synthesis continues in this state to facilitate survival. Another recent report by Murphy *et.al*, suggests that during dormancy *M. tuberculosis* performs limited protein synthesis and the pathogen spends its available resources in maintaining cell wall, membrane potential, genome integrity as well as resisting host defence systems (Murphy & Brown, 2007). The transcriptiome analysis further revealed five times more downregulated genes in persisters than upregulated ones (1,408 versus 282) under dormant condition suggesting that the growth and energy metabolism pathways are significantly downregulated (Keren et al., 2011). Even the synthesis of the ribosomal proteins tends to get down regulated in the state of dormancy as revealed in several different models (Wayne & Hayes, 1996; Betts et al., 2002; Keren et al., 2011). Although these studies are insufficient to resolve whether proteins synthesis comes to a complete halt or not in the dormant stage, these also open up a new question regarding the availability of the vital proteins in this stage. In other words, these studies indicate towards a longer turn-over of the proteins in this stage.

Continuous translation is not the only crucial process that defines the abundance of a protein or enzyme in the cell; instead it is often regulated by the parameters controlling its propensity to degradation and stabilization. Thus, regulation of protein amounts in a nonfavourable and fluctuating environment can be more dependent on turnover of proteins rather than the synthesis (Neher et al., 2003). Our knowledge regarding protein turnover in dormant phases in mycobacteria is still in infancy. However research in the last decade has thrown some light on it by attempting to examine the protein turnover at global level. Two seminal papers (Rao & Li, 2009; Rao et al., 2009) have helped in understanding the molecular basis of persistence in mycobacterium. Rao et.al showed that the protein turnover was much more effective to maintain relative protein abundance in the dormant phases of mycobacteria. These studies also help explain how the bacteria manage to exist as a submissive pathogen in the host for a prolonged period. The molecular detail behind the

*Mycobacterium tuberculosis*: Dormancy, Persistence and Survival in the Light of Protein Synthesis 231

and block the initiation of translation (Waksman & Schatz, 1943). As early as in 1948 reports of streptomycin resistance hit the scene where streptomycin seems no more effective at all (1948). It was in 1950's that the field had many new drugs hitting the market and the situation looked quite under control. The most recent drug to hit the space is still 35 years old. Modern therapy relies on a combination of potent bactericidal agents, such as isoniazid, rifampicin and pyrazinamide, in a treatment with six month duration. Isoniazid and Rifampicin are the two most effective drugs in the treatment bouquet that is administered throughout the treatment (Zhang, 2005). These are complemented with Ethambutol and Pyrazinamide in the early phase of treatment. Since there has been no new drugs developed in the last four decades and with excessive use of antibiotics there has emerged a new family of multidrug and extremely drug resistance strains leading to what we call MDR and XDR-TB. MDR refers to a resistance against the frontline drugs i.e. Isoniazid and Rifampicin whereas XDR is a complex form which is coupled to MDR and resistant against second line of potent antibiotics like Amikacin, Kanamycin and Capreomycin etc. (Organization, 2010). The perennial problem of non-compliance led the WHO to come up with universal treatment adherence programs, through a process currently known as the directly observed

The complex milieu of *Mycobacterium tuberculosis* and its intelligent survival instinct by virtue of adaptive diversification in tuberculosis disease have been a major obstacle that hinders the development of shorter treatment regimens to eradicate the disease. Currently the TB Alliance, a global initiative in newer TB drug development is trying to come up with affordable, shorter, safer and effective alternatives to target tuberculosis. At present they have 20 promising projects in the pipeline which includes three crucial drug candidates and a novel treatment regimen under phase three clinical trials (REMOX TB) that would considerably shorten the treatment span by at least two months without compromising efficacy. Another blue- eyed boy is a potential compound TMC207 that promises to be active on both drug sensitive and drug resistance form is under phase two trials. A novel cocktail NC001 is also under clinical trial which would shorten treatment regimen and alternatively

On the vaccination front BCG still occupies the central position being one of the most widely used vaccine against TB to be administered globally without any serious side effects. Its low cost of production ability to confer lifelong protection without booster dose in single immunization, availability of oral alternatives makes it popular in infant immunization programs. BCG has been effective to prevent meningeal TB in children, but it does not confer immunity to pulmonary TB in adults (Orme, 2001b). An interesting investigation in India during the 1960's by WHO in order to establish the efficacy of the BCG vaccine on two separate groups comprising 375,000 people in the province of Madras where one group was vaccinated against TB and the other group was not finally concluded that: "The efficacy of the TB vaccine is 0%" (www.whale.to/a/tb\_q.html). Since then in last decade most of the European countries has removed it from their immunization programme. The result is further substantiated by the argument that contracting TB doesn't provide any immunity against a second infection; and if a natural infection doesn't provide protection then a vaccination certainly won't provide protection either. As of now no promising vaccine has come in to the scene so far for tuberculosis in spite of various attempts in coming up with a DNA vaccine (Young et al., 1988; Lowrie et al., 1999; Orme, 2001b). The vaccine development has again come to picture after lacunae of several decades where the

provide affordable treatment without compromise (Alliance, 2010 ).

treatment short-course (DOTS).

prolonged protein-turnover mechanism in mycobacteria is not available yet. We speculate that under the dormant and stress condition, the protein degradation systems also slow down significantly as the protein synthesis system, thereby maintaining a good-enough concentration of the vital proteins in the cell and ensure survival of the bacteria.

Using the two-dimensional electrophoresis-based proteomics approach, a protein has been identified in *Escherichia coli* (Almiron et al., 1992), which expresses preferentially under starvation conditions. This protein is homologous to a family of proteins called Dps (DNA binding Protein from Starved cells) that are known to protect DNA under various kinds of environmental stresses. Although Dps kind of proteins have, so far, not been yet reported in mycobacteria, it is highly possible that similar system exists. Using comparative modelling it was possible to demonstrate that Dps from *Mycobacterium smegmatis* could form a dodecamer structure similar to the Dps from *Escherichia coli* (Gupta et al., 2002). The intriguing properties related to protein stability, DNA binding property and protection was further revisited to consolidate the structure function attribute of this unique protein (Ceci et al., 2005). These studies put emphasis on the need of global proteome analysis not only for actively growing cells but also for cells under dormant conditions. The proteomics can also be useful to identify the proteins related to starvation response of the pathogen.

Applying a systems biology approach the contemporary knowledge reveals approximately 4000 genes in mycobacterium genome and proteomic studies on the persistence reveals only very few which are differentially expressed during dormancy. A big proportion of these correspond to stress response proteins and metabolic enzymes (Cho et al., 2006). Unfortunately, there seems to be no exact correlation between the transcriptiome and the proteome data, which would otherwise be very useful (Mattow et al., 2006). The research group led by KVS Rao at ICGEB, New Delhi came up with an innovative strategy to comprehend the host key players that are involved in the onset and persistence of mycobacterium. They employed exhaustive genome-wide small interfering RNA (Si RNA) and identified 275 key molecules that are intricately associated in a network. This has led to a better understanding of host intracellular adaptation as result of bacterial diversification and host-pathogen interactions (Kumar et al., 2010). A bird's eye view from this aspect is bound to yield innovative insights in the pathophysiology of host pathogen interactions.

#### **5. Future perspective and therapeutic interventions**

There has been a great interest in looking for better alternatives and treatment modules throughout the globe to address the issue of this deadly pathogen. The focus has further intensified because of its proven role in immunodeficiency syndromes such as AIDS. The Global TB Alliance estimates that the disease will be a financial burden with expenditure ranging from \$ 1-3 trillion in coming decades for poor countries (www.tballiance.org). Developing countries are at the hotspots where 94% of TB cases and 98% of death takes place. The present treatment employs the strategies being used since decades with longer treatment regimen. The standard treatment regimen of six to nine month was based on clinical practices dating almost half a century back. The puzzle is that the exact molecular mechanism still needs to be deciphered with evidence that Isoniazid one of the key cocktail medicine only acts on actively growing form (Fox et al., 1999) and not against the anaerobic occult form (Thadepalli et al., 1979) makes situation draconian. The first real rescue for tuberculosis came in the form of Streptomycin in 1943 that was found to bind to 16S rRNA

prolonged protein-turnover mechanism in mycobacteria is not available yet. We speculate that under the dormant and stress condition, the protein degradation systems also slow down significantly as the protein synthesis system, thereby maintaining a good-enough

Using the two-dimensional electrophoresis-based proteomics approach, a protein has been identified in *Escherichia coli* (Almiron et al., 1992), which expresses preferentially under starvation conditions. This protein is homologous to a family of proteins called Dps (DNA binding Protein from Starved cells) that are known to protect DNA under various kinds of environmental stresses. Although Dps kind of proteins have, so far, not been yet reported in mycobacteria, it is highly possible that similar system exists. Using comparative modelling it was possible to demonstrate that Dps from *Mycobacterium smegmatis* could form a dodecamer structure similar to the Dps from *Escherichia coli* (Gupta et al., 2002). The intriguing properties related to protein stability, DNA binding property and protection was further revisited to consolidate the structure function attribute of this unique protein (Ceci et al., 2005). These studies put emphasis on the need of global proteome analysis not only for actively growing cells but also for cells under dormant conditions. The proteomics can also

concentration of the vital proteins in the cell and ensure survival of the bacteria.

be useful to identify the proteins related to starvation response of the pathogen.

**5. Future perspective and therapeutic interventions** 

Applying a systems biology approach the contemporary knowledge reveals approximately 4000 genes in mycobacterium genome and proteomic studies on the persistence reveals only very few which are differentially expressed during dormancy. A big proportion of these correspond to stress response proteins and metabolic enzymes (Cho et al., 2006). Unfortunately, there seems to be no exact correlation between the transcriptiome and the proteome data, which would otherwise be very useful (Mattow et al., 2006). The research group led by KVS Rao at ICGEB, New Delhi came up with an innovative strategy to comprehend the host key players that are involved in the onset and persistence of mycobacterium. They employed exhaustive genome-wide small interfering RNA (Si RNA) and identified 275 key molecules that are intricately associated in a network. This has led to a better understanding of host intracellular adaptation as result of bacterial diversification and host-pathogen interactions (Kumar et al., 2010). A bird's eye view from this aspect is bound to yield innovative insights in the pathophysiology of host pathogen interactions.

There has been a great interest in looking for better alternatives and treatment modules throughout the globe to address the issue of this deadly pathogen. The focus has further intensified because of its proven role in immunodeficiency syndromes such as AIDS. The Global TB Alliance estimates that the disease will be a financial burden with expenditure ranging from \$ 1-3 trillion in coming decades for poor countries (www.tballiance.org). Developing countries are at the hotspots where 94% of TB cases and 98% of death takes place. The present treatment employs the strategies being used since decades with longer treatment regimen. The standard treatment regimen of six to nine month was based on clinical practices dating almost half a century back. The puzzle is that the exact molecular mechanism still needs to be deciphered with evidence that Isoniazid one of the key cocktail medicine only acts on actively growing form (Fox et al., 1999) and not against the anaerobic occult form (Thadepalli et al., 1979) makes situation draconian. The first real rescue for tuberculosis came in the form of Streptomycin in 1943 that was found to bind to 16S rRNA and block the initiation of translation (Waksman & Schatz, 1943). As early as in 1948 reports of streptomycin resistance hit the scene where streptomycin seems no more effective at all (1948). It was in 1950's that the field had many new drugs hitting the market and the situation looked quite under control. The most recent drug to hit the space is still 35 years old. Modern therapy relies on a combination of potent bactericidal agents, such as isoniazid, rifampicin and pyrazinamide, in a treatment with six month duration. Isoniazid and Rifampicin are the two most effective drugs in the treatment bouquet that is administered throughout the treatment (Zhang, 2005). These are complemented with Ethambutol and Pyrazinamide in the early phase of treatment. Since there has been no new drugs developed in the last four decades and with excessive use of antibiotics there has emerged a new family of multidrug and extremely drug resistance strains leading to what we call MDR and XDR-TB. MDR refers to a resistance against the frontline drugs i.e. Isoniazid and Rifampicin whereas XDR is a complex form which is coupled to MDR and resistant against second line of potent antibiotics like Amikacin, Kanamycin and Capreomycin etc. (Organization, 2010). The perennial problem of non-compliance led the WHO to come up with universal treatment adherence programs, through a process currently known as the directly observed treatment short-course (DOTS).

The complex milieu of *Mycobacterium tuberculosis* and its intelligent survival instinct by virtue of adaptive diversification in tuberculosis disease have been a major obstacle that hinders the development of shorter treatment regimens to eradicate the disease. Currently the TB Alliance, a global initiative in newer TB drug development is trying to come up with affordable, shorter, safer and effective alternatives to target tuberculosis. At present they have 20 promising projects in the pipeline which includes three crucial drug candidates and a novel treatment regimen under phase three clinical trials (REMOX TB) that would considerably shorten the treatment span by at least two months without compromising efficacy. Another blue- eyed boy is a potential compound TMC207 that promises to be active on both drug sensitive and drug resistance form is under phase two trials. A novel cocktail NC001 is also under clinical trial which would shorten treatment regimen and alternatively provide affordable treatment without compromise (Alliance, 2010 ).

On the vaccination front BCG still occupies the central position being one of the most widely used vaccine against TB to be administered globally without any serious side effects. Its low cost of production ability to confer lifelong protection without booster dose in single immunization, availability of oral alternatives makes it popular in infant immunization programs. BCG has been effective to prevent meningeal TB in children, but it does not confer immunity to pulmonary TB in adults (Orme, 2001b). An interesting investigation in India during the 1960's by WHO in order to establish the efficacy of the BCG vaccine on two separate groups comprising 375,000 people in the province of Madras where one group was vaccinated against TB and the other group was not finally concluded that: "The efficacy of the TB vaccine is 0%" (www.whale.to/a/tb\_q.html). Since then in last decade most of the European countries has removed it from their immunization programme. The result is further substantiated by the argument that contracting TB doesn't provide any immunity against a second infection; and if a natural infection doesn't provide protection then a vaccination certainly won't provide protection either. As of now no promising vaccine has come in to the scene so far for tuberculosis in spite of various attempts in coming up with a DNA vaccine (Young et al., 1988; Lowrie et al., 1999; Orme, 2001b). The vaccine development has again come to picture after lacunae of several decades where the

*Mycobacterium tuberculosis*: Dormancy, Persistence and Survival in the Light of Protein Synthesis 233

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**8. References** 

development pipeline now includes seven vaccine candidates that are being tested in humans. Two non- replicating viral vector vaccines have very recently entered the first phase efficacy trial in infants (the first such trial in 80 years) and in human immunodeficiency virus–infected adults (Beresford & Sadoff, 2010). Yet we have a long way to go for an effective vaccine which would take care of all the thriving forms of this enigmatic bacterium.
