**8. References**

232 Understanding Tuberculosis – Deciphering the Secret Life of the Bacilli

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

The geography of tuberculosis has achieved a global dimension with transmigration across the boundaries as a result TB pandemic has taken the world in its clutches thus making it a true global concern. The TB Alliance, a global initiative has embarked an integrated innovative approach to combat and sabotage one of the oldest, deadly, and most resilient enemies of the mankind. At the level of individual it impacts them in the most productive phase of their life thereby sucking their income and decreasing the productivity and thus inflicting a great loss in human capital. On average one person infects fifteen others before finally getting successfully treated; the death statistics is alarming with one death every twenty second. The right to health and hope is one of the fundamental rights that is robbed by this enemy of the mankind. Bill Gates propounded at the World Health Assembly in 2005: "Today, we have tuberculosis drugs you have to take for 9 months. Why can't we find

The research arena has also received momentum with the deciphering of the Mycobacterial genome in 1998 coupled with advanced molecular biology tools, structural genomics, target based drug design, high throughput screening, *in silico* experiments, whole cell screening and advanced imaging technologies to study real time changes and system biology platforms coming under one umbrella. The idea now is to leverage on the existing portfolio having more than two dozens of potent molecules and drug regimen in pipeline at various stages of clinical trials. The TB development has galvanized further by the coming together of WHO, TB Alliance and DNDi (Drug for Neglected Disease initiative) sharing a common podium. TDR for research on diseases of poverty has been working under the joint conglomeration of WHO, UNICEF, WORLD BANK and UNDP to address this concern globally. Nelson Mandela commented that "we cannot win the battle against AIDS, if we do not also fight TB; TB is too often a death sentence for people with AIDS". The WHO targets to treat 80% of the MDR-TB patients by 2015 with an estimated cost of \$15 billion. With initiatives at full bloom the scenario looks promising and hopeful in global attempts to

The conquest of the Mycobacterium to the mankind needs to be the priority of the synergistic efforts by the scientific community. In nutshell the enemy of humanity needs to be taken in to clutches by innovative approaches from drug development that includes quest for effective molecular scaffolds and their derivatives both old and new as well as reengineering delivery strategies for the drug to penetrate the recalcitrant stubborn microbes. On one hand there are challenges to finding safer, cheap, less toxic, shorter regimen and compatible drugs quickly while on the other the socio economic feasibilities that can deliver these magic bullets to the neediest ones have to be ensured. Revamping the health care system with Government, business houses and NGO's is the need of the hour to combat these perpetrators of human misery from the globe. Wave of optimism exists as we

enigmatic bacterium.

address tuberculosis.

**6. The odyssey ahead** 

one that works in 3 days?" this is still a dream.


*Mycobacterium tuberculosis*: Dormancy, Persistence and Survival in the Light of Protein Synthesis 235

Gupta, S., Pandit, S. B., Srinivasan, N. and Chatterji, D. (2002). Proteomics analysis of

Gyanu Lamichhane, M. Z., Natalie J. Blades, Deborah E. Geiman, Annette Dougherty,

Application to Mycobacterium tuberculosis. *PNAS*, Vol. 100 (12), No. pp. Hautzel, R., Anke, H. and Sheldrick, W. S. (1990). Mycenon, a new metabolite from a

Hobbie, S. N., Bruell, C., Kalapala, S., Akshay, S., Schmidt, S., Pfister, P. and Bottger, E. C.

Hobbie, S. N., Kalapala, S. K., Akshay, S., Bruell, C., Schmidt, S., Dabow, S., Vasella, A.,

Hobbie, S. N., Pfister, P., Bruell, C., Sander, P., Francois, B., Westhof, E. and Bottger, E. C.

Hobbie, S. N., Pfister, P., Brull, C., Westhof, E. and Bottger, E. C. (2005). Analysis of the

Honer Zu Bentrup, K., Miczak, A., Swenson, D. L. and Russell, D. G. (1999).

Honore, N. and Cole, S. T. (1994). Streptomycin resistance in mycobacteria. *Antimicrob* 

Hoyt, J. C., Johnson, K. E. and Reeves, H. C. (1991). Purification and characterization of

Hoyt, J. C., Robertson, E. F., Berlyn, K. A. and Reeves, H. C. (1988). Escherichia coli isocitrate lyase: properties and comparisons. *Biochim Biophys Acta*, Vol. 966, No. 1, pp. 30-35 Hu, Y. M., Butcher, P. D., Sole, K., Mitchison, D. A. and Coates, A. R. (1998). Protein

Kanyok, T. P., Reddy, M. V., Chinnaswamy, J., Danziger, L. H. and Gangadharam, P. R.

oxygen or heat shock. *FEMS Microbiol Lett*, Vol. 158, No. 1, pp. 139-145

Inderlied, C. B. (1991). *Antibiotics in Laboratory Medicine*, Williams & Wilkins,

changing environment. *Trends Microbiol*, Vol. 9, No. 12, pp. 597-605

pp. 11554-11559

6086-6093

6848

4, pp. 1489-1496

*Eng*, Vol. 15, No. 6, pp. 503-512

*Antibiot (Tokyo)*, Vol. 43, No. 10, pp. 1240-1244

decoding site. *Biochimie*, Vol. 88, No. 8, pp. 1033-1043

*Antimicrob Agents Chemother*, Vol. 49, No. 12, pp. 5112-5118

*Agents Chemother*, Vol. 38, No. 2, pp. 238-242

*Chemother*, Vol. 38, No. 2, pp. 170-173

capture of transcribed sequences (SCOTS). *Proc Natl Acad Sci U S A*, Vol. 96, No. 20,

carbon-starved Mycobacterium smegmatis: induction of Dps-like protein. *Protein* 

Jacques Grosset, Karl W. Broman, and William R. Bishai (2003). A postgenomic method for predicting essential genes at subsaturation levels of mutagenesis:

Mycena species TA 87202 (basidiomycetes) as an inhibitor of isocitrate lyase. *J* 

(2006a). A genetic model to investigate drug-target interactions at the ribosomal

Sander, P. and Bottger, E. C. (2007). Engineering the rRNA decoding site of eukaryotic cytosolic ribosomes in bacteria. *Nucleic Acids Res*, Vol. 35, No. 18, pp.

(2006b). Binding of neomycin-class aminoglycoside antibiotics to mutant ribosomes with alterations in the A site of 16S rRNA. *Antimicrob Agents Chemother*, Vol. 50, No.

contribution of individual substituents in 4,6-aminoglycoside-ribosome interaction.

Characterization of activity and expression of isocitrate lyase in Mycobacterium avium and Mycobacterium tuberculosis. *J Bacteriol*, Vol. 181, No. 23, pp. 7161-7167 Honer zu Bentrup, K. and Russell, D. G. (2001). Mycobacterial persistence: adaptation to a

Acinetobacter calcoaceticus isocitrate lyase. *J Bacteriol*, Vol. 173, No. 21, pp. 6844-

synthesis is shutdown in dormant Mycobacterium tuberculosis and is reversed by

(1994). In vivo activity of paromomycin against susceptible and multidrug-resistant Mycobacterium tuberculosis and M. avium complex strains. *Antimicrob Agents* 


Ceci, P., Ilari, A., Falvo, E., Giangiacomo, L. and Chiancone, E. (2005). Reassessment of

Cho, S. H., Goodlett, D. and Franzblau, S. (2006). ICAT-based comparative proteomic

Cole, S. T. (1999). Learning from the genome sequence of Mycobacterium tuberculosis

Daniel, J., Maamar, H., Deb, C., Sirakova, T. D. and Kolattukudy, P. E. (2011).

De Stasio, E. A., Moazed, D., Noller, H. F. and Dahlberg, A. E. (1989). Mutations in 16S

Deb, C., Lee, C. M., Dubey, V. S., Daniel, J., Abomoelak, B., Sirakova, T. D., Pawar, S.,

Dennis, P. P. and Bremer, H. (1974). Differential rate of ribosomal protein synthesis in

Dixon, G. H., Kornberg, H. L. and Lund, P. (1960). Purification and properties of malate

Douglass, J. and Steyn, L. M. (1993). A ribosomal gene mutation in streptomycin-resistant Mycobacterium tuberculosis isolates. *J Infect Dis*, Vol. 167, No. 6, pp. 1505-1506 Fox, W., Ellard, G. A. and Mitchison, D. A. (1999). Studies on the treatment of tuberculosis

Funatsu, G. and Wittmann, H. G. (1972). Ribosomal proteins. 33. Location of amino-acid

Ghosh, J., Larsson, P., Singh, B., Pettersson, B. M., Islam, N. M., Sarkar, S. N., Dasgupta, S.

Giachetti, E. and Vanni, P. (1991). Effect of Mg2+ and Mn2+ on isocitrate lyase, a non-

Gomez, J. E. and McKinney, J. D. (2004). M. tuberculosis persistence, latency, and drug

Gould, T. A., van de Langemheen, H., Munoz-Elias, E. J., McKinney, J. D. and Sacchettini, J.

Mycobacterium tuberculosis. *Mol Microbiol*, Vol. 61, No. 4, pp. 940-947 Graham, J. E. and Clark-Curtiss, J. E. (1999). Identification of Mycobacterium tuberculosis

*Biol Chem*, Vol. 280, No. 41, pp. 34776-34785

H37Rv. *FEBS Lett*, Vol. 452, No. 1-2, pp. 7-10

dormant pathogen. *PLoS One*, Vol. 4, No. 6, pp. e6077

Escherichia coli B-r. *J Mol Biol*, Vol. 85, No. 3, pp. 407-422

synthetase. *Biochim Biophys Acta*, Vol. 41, No. pp. 217-233

streptomycin. *J Mol Biol*, Vol. 68, No. 3, pp. 547-550

tolerance. *Tuberculosis (Edinb)*, Vol. 84, No. 1-2, pp. 29-44

Vol. 106, No. 26, pp. 10781-10786

*PLoS Pathog*, Vol. 7, No. 6, pp. e1002093

1213-1216

pp. S231-279

230)

*(Edinb)*, Vol. 86, No. 6, pp. 445-460

protein stability, DNA binding, and protection of Mycobacterium smegmatis Dps. *J* 

analysis of non-replicating persistent Mycobacterium tuberculosis. *Tuberculosis* 

Mycobacterium tuberculosis Uses Host Triacylglycerol to Accumulate Lipid Droplets and Acquires a Dormancy-Like Phenotype in Lipid-Loaded Macrophages.

ribosomal RNA disrupt antibiotic--RNA interactions. *EMBO J*, Vol. 8, No. 4, pp.

Rogers, L. and Kolattukudy, P. E. (2009). A novel in vitro multiple-stress dormancy model for Mycobacterium tuberculosis generates a lipid-loaded, drug-tolerant,

undertaken by the British Medical Research Council tuberculosis units, 1946-1986, with relevant subsequent publications. *Int J Tuberc Lung Dis*, Vol. 3, No. 10 Suppl 2,

replacements in protein S12 isolated from Escherichia coli mutants resistant to

and Kirsebom, L. A. (2009). Sporulation in mycobacteria. *Proc Natl Acad Sci U S A*,

essentially metal-ion-activated enzyme. A graphical approach for the discrimination of the model for activation. *Biochem J*, Vol. 276 ( Pt 1), No. pp. 223-

C. (2006). Dual role of isocitrate lyase 1 in the glyoxylate and methylcitrate cycles in

RNAs synthesized in response to phagocytosis by human macrophages by selective

capture of transcribed sequences (SCOTS). *Proc Natl Acad Sci U S A*, Vol. 96, No. 20, pp. 11554-11559


*Mycobacterium tuberculosis*: Dormancy, Persistence and Survival in the Light of Protein Synthesis 237

Lowrie, D. B., Tascon, R. E., Bonato, V. L., Lima, V. M., Faccioli, L. H., Stavropoulos, E.,

Mattow, J., Siejak, F., Hagens, K., Becher, D., Albrecht, D., Krah, A., Schmidt, F., Jungblut, P.

Mc, C. R., Lee, S. H., Deuschle, K. and Mc, D. W. (1957). Ineffectiveness of isoniazid in

McFadden, B. A. and Purohit, S. (1977). Itaconate, an isocitrate lyase-directed inhibitor in

McKinney, J. D., Honer zu Bentrup, K., Munoz-Elias, E. J., Miczak, A., Chen, B., Chan, W. T.,

Mukamolova, G. V., Yanopolskaya, N. D., Kell, D. B. and Kaprelyants, A. S. (1998). On

Munoz-Elias, E. J., Upton, A. M., Cherian, J. and McKinney, J. D. (2006). Role of the

Murphy, D. J. and Brown, J. R. (2007). Identification of gene targets against dormant phase Mycobacterium tuberculosis infections. *BMC Infect Dis*, Vol. 7, No. pp. 84 Neher, S. B., Sauer, R. T. and Baker, T. A. (2003). Distinct peptide signals in the UmuD and

ClpXP protease. *Proc Natl Acad Sci U S A*, Vol. 100, No. 23, pp. 13219-13224

Orme, I. M. (2001a). The latent tubercle bacillus (I'll let you know if I ever meet one). *Int. J.* 

Orme, I. M. (2001b). The search for new vaccines against tuberculosis. *J Leukoc Biol*, Vol. 70,

Pandey, A. K. and Sassetti, C. M. (2008). Mycobacterial persistence requires the utilization of host cholesterol. *Proc Natl Acad Sci U S A*, Vol. 105, No. 11, pp. 4376-4380 Rao, P. K. and Li, Q. (2009). Protein turnover in mycobacterial proteomics. *Molecules*, Vol. 14,

Rao, P. K., Singh, C. R., Jagannath, C. and Li, Q. (2009). A systems biology approach to study

Reddy, T. B., Riley, R., Wymore, F., Montgomery, P., DeCaprio, D., Engels, R., Gellesch, M.,

the phagosomal proteome modulated by mycobacterial infections. *Int J Clin Exp* 

Hubble, J., Jen, D., Jin, H., Koehrsen, M., Larson, L., Mao, M., Nitzberg, M., Sisk, P., Stolte, C., Weiner, B., White, J., Zachariah, Z. K., Sherlock, G., Galagan, J. E., Ball, C.

growth, and virulence. *Mol Microbiol*, Vol. 60, No. 5, pp. 1109-1122

Organization, W. H. (2010). 2010/2011 TUBERCULOSIS GLOBAL FACTS. No. pp.

Pseudomonas indigofera. *J Bacteriol*, Vol. 131, No. 1, pp. 136-144

ribosomal RNA. *Nature*, Vol. 327, No. 6121, pp. 389-394

*Leeuwenhoek*, Vol. 73, No. 3, pp. 237-243

*Tuberc. Lung Dis.*, Vol. 5, No. pp. 589-593

repressor. *Proc Natl Acad Sci U S A*, Vol. 96, No. 22, pp. 12844-12848

pp. 2485-2494

pp. 1106-1109

No. 1, pp. 1-10

No. 9, pp. 3237-3258

*Med*, Vol. 2, No. 3, pp. 233-247

Colston, M. J., Hewinson, R. G., Moelling, K. and Silva, C. L. (1999). Therapy of tuberculosis in mice by DNA vaccination. *Nature*, Vol. 400, No. 6741, pp. 269-271 Manabe, Y. C., Saviola, B. J., Sun, L., Murphy, J. R. and Bishai, W. R. (1999). Attenuation of

virulence in Mycobacterium tuberculosis expressing a constitutively active iron

R., Kaufmann, S. H. and Schaible, U. E. (2006). Proteins unique to intraphagosomally grown Mycobacterium tuberculosis. *Proteomics*, Vol. 6, No. 8,

modifying the phenomenon of microbial persistence. *Am Rev Tuberc*, Vol. 76, No. 6,

Swenson, D., Sacchettini, J. C., Jacobs, W. R., Jr. and Russell, D. G. (2000). Persistence of Mycobacterium tuberculosis in macrophages and mice requires the glyoxylate shunt enzyme isocitrate lyase. *Nature*, Vol. 406, No. 6797, pp. 735-738 Moazed, D. and Noller, H. F. (1987). Interaction of antibiotics with functional sites in 16S

resuscitation from the dormant state of Micrococcus luteus. *Antonie Van* 

methylcitrate cycle in Mycobacterium tuberculosis metabolism, intracellular

UmuD' subunits of UmuD/D' mediate tethering and substrate processing by the


Kell, D. B., Kaprelyants, A. S., Weichart, D. H., Harwood, C. R. and Barer, M. R. (1998).

Keren, I., Minami, S., Rubin, E. and Lewis, K. Characterization and Transcriptome Analysis

Keren, I., Minami, S., Rubin, E. and Lewis, K. (2011). Characterization and Transcriptome Analysis of Mycobacterium tuberculosis Persisters. *MBio*, Vol. 2, No. 3, pp. Ko, Y. H. and McFadden, B. A. (1990). Alkylation of isocitrate lyase from Escherichia coli by 3-bromopyruvate. *Arch Biochem Biophys*, Vol. 278, No. 2, pp. 373-380 Ko, Y. H., Vanni, P. and McFadden, B. A. (1989). The interaction of 3-phosphoglycerate and

Kumar, D., Nath, L., Kamal, M. A., Varshney, A., Jain, A., Singh, S. and Rao, K. V. Genome-

Kumar, R. (2009). Glyoxylate Shunt : Combating Mycobacterium at Forefront. *International* 

Kumar, R. and Bhakuni, V. (2008). Mycobacterium tuberculosis isocitrate lyase (MtbIcl): role

Kumar, R. and Bhakuni, V. (2010). A functionally active dimer of mycobacterium tuberculosis malate synthase G. *Eur Biophys J*, Vol. 39, No. 11, pp. 1557-1562 Kumar, R. and Bhakuni, V. (2011). Comparative analysis of malate synthase G from

structural and functional properties. *Int. J. Biol. Macromol.*, Vol. No. pp. Leclercq, R. and Courvalin, P. (1991). Bacterial resistance to macrolide, lincosamide, and

Lee, D., Shin, J., Yoon, K. M., Kim, T. I., Lee, S. H., Lee, H. S. and Oh, K. B. (2008). Inhibition

LI Jun-ming, L. N., ZHU Dao-yin, WAN La-gen, HE Yong-lin , YANG Chun (2008).

Long, K. S., Poehlsgaard, J., Hansen, L. H., Hobbie, S. N., Bottger, E. C. and Vester, B. (2009).

Lorenz, M. C. and Fink, G. R. (2001). The glyoxylate cycle is required for fungal virulence.

practical issues. *Antonie Van Leeuwenhoek*, Vol. 73, No. 2, pp. 169-187 Kelly, B. G., Wall, D. M., Boland, C. A. and Meijer, W. G. (2002). Isocitrate lyase of the

of Mycobacterium tuberculosis Persisters. *MBio*, Vol. 2, No. 3, pp.

isocitrate lyase. *Arch Biochem Biophys*, Vol. 274, No. 1, pp. 155-160

Mycobacterium tuberculosis. *Cell*, Vol. 140, No. 5, pp. 731-743

*Journal of Integrative Biology*, Vol. 7, No. 2, pp. 69-72

*Chinese Medical Journal*, Vol. 121, No. 12, pp.

*Nature*, Vol. 412, No. 6842, pp. 83-86

Vol. 72, No. 3, pp. 892-900

35, No. 7, pp. 1267-1272

No. 1, pp. 48-56

3, pp. 793-798

Viability and activity in readily culturable bacteria: a review and discussion of the

facultative intracellular pathogen Rhodococcus equi. *Microbiology*, Vol. 148, No. Pt

other substrate analogs with the glyoxylate- and succinate-binding sites of

wide analysis of the host intracellular network that regulates survival of

of divalent cations in modulation of functional and structural properties. *Proteins*,

Mycobacterium tuberculosis and E. coli: Role of ionic interaction in modulation of

streptogramin antibiotics by target modification. *Antimicrob Agents Chemother*, Vol.

of Candida albicans isocitrate lyase activity by sesterterpene sulfates from the tropical sponge Dysidea sp. *Bioorg Med Chem Lett*, Vol. 18, No. 20, pp. 5377-5380 Lewis, K. (2007). Persister cells, dormancy and infectious disease. *Nat Rev Microbiol*, Vol. 5,

Isocitrate lyase from Mycobacterium tuberculosis promotes survival of Mycobacterium smegmatis within macrophage by suppressing cell apoptosis.

Single 23S rRNA mutations at the ribosomal peptidyl transferase centre confer resistance to valnemulin and other antibiotics in Mycobacterium smegmatis by perturbation of the drug binding pocket. *Mol Microbiol*, Vol. 71, No. 5, pp. 1218-1227


**13** 

*Russia* 

**Lipid Surrounding of Mycobacteria:** 

*1Lomonosov Moscow State Academy of Fine Chemical Technology 2Biology Department, Lomonosov Moscow State University 3Bach Institute of Biochemistry, Russian Academy of Sciences* 

Inside of the host macrophages *Mycobacterium tuberculosis* cells are supposed to face various hostile conditions as the result of immune response: action of reactive oxygen and nitrogen intermediates, hydrolases, increased acidity, and antimicrobial peptides activities [Russell, 2010]. However mycobacterial cells have developed certain mechanisms to resist these defenses. Transcriptome analysis of *M. tuberculosis* showed that even negligible changes of environmental factors cause considerable alterations in global gene expression profile [Boshoff, 2004; Cole, 1998]. It's important that such alterations were observed both in the presence of chemical agents (respiration inhibitors, antituberculosis drugs (ATD), ATP synthesis inhibitors), and during incubation of mycobacteria in modified conditions (medium рН, nature of nutrient, nutrient depletion and starvation, hypoxia, exposure to nitric oxide). As the result, altered properties of the whole cell enable mycobacteria to resist these effects. For instance, increase of the incubation temperature activated synthesis of heat shock proteins which led to higher thermal resistance of the cells; exposure of mycobacteria to the acid environment induces expression of *aprABC* locus responsible for restructuration of lipids of the mycobacterial cell wall and storage lipids that are required for intraphagosome survival [Abramovitch, 2011; Sung N, 2004]. Low concentrations of antibiotics in cultivation medium, that don't affect the cell growth, activate genes responsible for protein pump synthesis, which provides a removal of the antibiotics from the bacterial cell. This is one of the main mechanisms of the ATD resistance. Substitution of the nutrient, e.g. substitution of glycerol to FA (free FA or as part of phospholipids (PL)), activates genes responsible for synthesis of enzymes that switch metabolism to a different pathway of nutrient utilization. In this case mycobacterial cell involves two forms of isocitrate lyase, and utilization of the nutrient in the tricarboxylic acid cycle goes through the glyoxylate shunt [Munoz-Elias, 2005]. Upregulation of the genes encoding isocitrate lyase was shown for *М. tuberculosis* cells cultivated in anaerobic conditions [Lu, 2005], for cells isolated from human lung granulomas [Fenhalls, 2002] and from infected macrophages [Schnappinger, 2003]. All these data prove glyoxylate shunt to be an essential mechanism for survival of mycobacterial cells in phagosomes inside of the host macrophage, where they

**1. Introduction** 

**Lethal and Resuscitating Effects** 

Alla A. Selishcheva1,2, Galina M. Sorokoumova1

and Evgeniya V. Nazarova1,3

A. and Schoolnik, G. K. (2009). TB database: an integrated platform for tuberculosis research. *Nucleic Acids Res*, Vol. 37, No. Database issue, pp. D499-508

