**3. Participation of cAMP in mycobacterial pathogenic processes**

Little is known about the role of cAMP in mycobacteria, although it is found in both pathogenic and non-pathogenic species. Ingestion of live *M. microti* or *M. bovis* BCG (but not *M. lepraemurium*) increased macrophage intracellular cAMP levels, whereas no change occurred in cells engulfing dead bacilli, latex beads or colloidal gold (Lowrie *et al.*, 1979). The rise in cAMP levels appears directly related to mycobacterial capacity to interfere with phagolysosome formation, evidence suggesting that these microorganisms modify cAMPdependent signalling pathways as a manner to control virulence and infection (Lowrie *et al.*, 1975, Lowrie et al., 1979). Elevated cAMP levels were correlated with reduced phagolysosome fusion during mycobacterial infection of macrophages (Kalamidas *et al.*, 2006). Increased cAMP levels inside phagocytes were shown to negatively modulate actindependent processes, including chemotactic movement and phagocytosis. Macrophage passage was found to have a stimulatory effect on cAMP production by mycobacteria, as cAMP levels within macrophage-passaged mycobacteria were ~50-fold higher than cAMP levels within bacteria incubated in the tissue culture medium alone (Bai et al., 2009).

Evidences exist that cAMP regulates gene expression in mycobacteria during bacterial growth *in vitro* (Stapleton et al., 2010, Dass et al., 2008), and during macrophage infection (Rickman et al., 2005) where some studies identified Cmr as a transcription factor that regulates cAIGs (cAMP-iduced genes) within macrophages, and suggest that multiple factors affect cAMP-associated gene regulation in tuberculosis-complex mycobacteria (Gazdik et al., 2009). Even during phagocytosis, expression may be down regulated in response to high cAMP or NO levels inside the macrophage environment, providing a mechanism to integrate the transcriptional response to two important signals associated with infection (Smith *et al.*, 2010). Generally, increases in cAMP levels compromise the bactericidal activity of the host immune system.

It is likely that each cyclase is associated with a distinct signaling pathway. It is expected that specific cyclases are activated to modify cAMP levels in response to different physiological conditions as for example hypoxia, intramacrophage enviroment or pH changes. cAMP receptor protein (CRP) Rv3676 was found to exist as dimmer and exhibited cAMP binding in a concentration-dependent manner and could bind specifically to the putative CRP/FNR nucleotide sequence elements in response to hypoxia (Akhter et al., 2008). The protein itself is composed of three distinct regions of the polypeptide: a large Nterminal domain that binds cAMP, a long -helix (termed the C-helix) that mediates most of

Mycobacterium Tuberculosis Signaling via c-AMP 113

the inter monomer interactions, and a small C-terminal DNA-binding domain. It is also capable of binding to two, three, or four cAMP molecules, but the specificity of recognition sequentially diminishes beyond two cAMP molecules bound to CRP, although the physiological importance of this molecular interactions are not yet known (Stapleton et al., 2010). Data available at Tuberculosis Database (http://www.tbdb.org/, Table 3) might help

Currently, few studies have addressed the participation of cAMP signalling in *M. tuberculosis* pathogenesis *in vivo*. Rickman *et al.* (Rickman et al., 2005) found that a mutant in *Rv3676* (CRP) had diminished bacterial burden in lung and spleen after intravenous infection of BALB/c mice, compared to wild type bacteria. On the other hand, Agarwal *et al.* (Agarwal *et al.*, 2009) found that a *Rv0386 M. tuberculosis* mutant was affected in its capacity to replicate in BALB/c or C57BL/6 mice lungs, following aerosol infection. Neither

In addition to determining bacterial burden in infected lungs Agarwal et al. (Agarwal et al., 2009) demonstrated that cAMP produced by *M. tuberculosis* Rv0386 during J774.16 macrophage infection was a substrate for protein kinase A, in order to control the amount of phosphorylated CREB (CREB-P), by using specific chemical inhibitors of selected signalling transduction pathways. They found CREB-P induced TNF- production, and led to an unregulated host inflammatory response, which favoured bacterial survival. To date, this is the first indication of how adenylyl cyclase action helps sustaining an infection by

Abdel Motaal, A., I. Tews, J. E. Schultz & J. U. Linder, (2006) Fatty acid regulation of

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scale portrait of cAMP-receptor protein (CRP) regulons in mycobacteria points to

BCG cyclic AMP receptor-like protein is a functional DNA binding protein in vitro and in vivo, but its activity differs from that of its M. tuberculosis ortholog, Rv3676.

suggesting particular conditions where each AC gene may be required.

publication mentioned the capacity of either strain to kill mice.

their role in pathogenesis. *Gene* 407: 148-158.

pathogenic mycobacteria.

4219-4228.

2749-2756.

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**4. References** 


Table 3. Growth conditions afecting expression of mycobacterial cyclase domain containing genes

Purified surfactant lipids

DETA/NO

Hydrogen peroxide Tioridazine Nicotinamide

Gene Induction Repression

*Rv0891c* Oleic acid DETA/NO

*Rv1264* Carbonyl cyanide chlorophenylhydrazone Palmitic acid

Palmitic acid Acetate *Rv1319c* Hypoxia Arachidonic acid

*Rv1359* Econazole Arachidonic acid Macrophages Palmitic acid

*Rv1625c* Clofazimine + S-nitrosoglutathione Palmitic acid Arachidonic acid Oleic acid

*Rv1647* Linoleic acid Streptomyicin

*Rv1900c* Non-replicative persistence Tetracycline

*Rv2345* Starvation Thioridazine

*Rv2488c* Arachidonic acid Tetracycline

Acetate Palmitic acid

Palmitic acid Acetate *Rv2212* Tetracycline 5-chloro-pyrazinamide

Palmitic acid Arachidonic acid Oleic acid DETA/NO

PA 824 S-nitrosogluthatione + Chlorpromazine

*Rv3645* Oleic acid Oligopeptide permease (*Rv3662c-Rv3665c*) mutant

Table 3. Growth conditions afecting expression of mycobacterial cyclase domain containing

DETA/NO

Acetate Ceramide

Hydrogen peroxide

*Rv1320c* Acetate

genes

Iron

Thioridazine

*Rv0386* Palmitic acid 7H9 medium shaking Tetracyclin Streptomyicin

*Rv1120c* Pulmonary surfactant Non-replicative persistence

*Rv1318c* Oligopeptide permease (Rv3662c-Rv3665c) mutant Pulmonary surfactant protein A (human)

the inter monomer interactions, and a small C-terminal DNA-binding domain. It is also capable of binding to two, three, or four cAMP molecules, but the specificity of recognition sequentially diminishes beyond two cAMP molecules bound to CRP, although the physiological importance of this molecular interactions are not yet known (Stapleton et al., 2010). Data available at Tuberculosis Database (http://www.tbdb.org/, Table 3) might help suggesting particular conditions where each AC gene may be required.

Currently, few studies have addressed the participation of cAMP signalling in *M. tuberculosis* pathogenesis *in vivo*. Rickman *et al.* (Rickman et al., 2005) found that a mutant in *Rv3676* (CRP) had diminished bacterial burden in lung and spleen after intravenous infection of BALB/c mice, compared to wild type bacteria. On the other hand, Agarwal *et al.* (Agarwal *et al.*, 2009) found that a *Rv0386 M. tuberculosis* mutant was affected in its capacity to replicate in BALB/c or C57BL/6 mice lungs, following aerosol infection. Neither publication mentioned the capacity of either strain to kill mice.

In addition to determining bacterial burden in infected lungs Agarwal et al. (Agarwal et al., 2009) demonstrated that cAMP produced by *M. tuberculosis* Rv0386 during J774.16 macrophage infection was a substrate for protein kinase A, in order to control the amount of phosphorylated CREB (CREB-P), by using specific chemical inhibitors of selected signalling transduction pathways. They found CREB-P induced TNF- production, and led to an unregulated host inflammatory response, which favoured bacterial survival. To date, this is the first indication of how adenylyl cyclase action helps sustaining an infection by pathogenic mycobacteria.

#### **4. References**


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**6** 

*Mycobacterium tuberculosis*

**RD-1 Secreted Antigens as** 

**Protective and Risk Factors** 

Niladri Ganguly1,2 and Pawan Sharma1

*2Wellcome Trust Biocentre, College of Life Sciences,* 

*1International Centre for Genetic Engineering and Biotechnology,* 

*Mycobacterium tuberculosis* (Mtb) infects about 8 million people every year and causes death of about 2-3 million (Raviglione, 2003). In recent times, there has been a wider spread of tuberculosis, mainly due to emergence of multi drug resistance (MDR) bacilli and enhanced susceptibility to the disease by patients infected with human immunodeficiency virus (HIV) (Elliot et al., 1995; Chintu and Mwinga, 1999). Transmission of the infection by Mtb bacilli is air borne and occurs through inhalation of aerosol containing the bacilli exhaled by coughing, sneezing or spitting by patients suffering from pulmonary tuberculosis. The inhaled bacilli are engulfed by the alveolar macrophages, where the bacilli are able to persist successfully in a latent or proliferating state. This persistence is achieved by modulation of several intracellular signaling pathways in order to create a suitable environment for the bacilli. The interplay of mycobacteria with host signaling pathways is a complex and dynamic process that is not clearly understood. Mtb secretes several molecules that modulate the signaling pathways (Koul et al., 2004). Most of these molecules commonly target macrophages, which helps the bacilli to evade innate immune response and

The proteins secreted by Mtb have gained attention in recent years as putative vaccine and diagnostic candidates (Harboe et al., 1996; Colangeli et al., 2000). But there have been recent reports about their role in modulation of macrophage signaling pathways leading to compromise of macrophage functions (Trajkovic et al., 2002; Pym et al., 2003; Guinn et al., 2004). Thus the secretory proteins can act as risk or virulent factors too. This notion is also supported by the fact that only live but not dead bacilli can down regulate macrophage functions (Malik et al., 2001). In this chapter, we focus our discussion on the role of the

propagate throughout the system (Rosenberger and Finlay, 2003).

**1. Introduction** 

**for Tuberculosis** 

*University of Dundee, Dundee,* 

*Aruna Asaf Ali Marg,* 

*New Delhi,* 

*1India 2UK* 

