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Protein phosphatases play vital roles in phosphorylation-mediated cellular signaling. Although there are 11 serine/threonine protein kinases in Mycobacterium tuberculosis, only one serine/threonine phosphatase, PstP, has been identified. Although PstP has been biochemically characterized and multiple in vitro substrates have been identified, its physiological role has not yet been elucidated. In this study, we have investigated the impact of PstP on cell growth and survival of the pathogen in the host. Overexpression of PstP led to elongated cells and partially compromised survival. We find that depletion of PstP is detrimental to cell survival, eventually leading to cell death. PstP depletion results in elongated multiseptate cells, suggesting a role for PstP in regulating cell division events. Complementation experiments performed with PstP deletion mutants revealed marginally compromised survival, suggesting that all of the domains, including the extracellular domain, are necessary for complete rescue. On the other hand, the catalytic activity of PstP is absolutely essential for the in vitro growth. Mice infection experiments establish a definitive role for PstP in pathogen survival within the host. Depletion of PstP from established infections causes pathogen clearance, indicating that the continued presence of PstP is necessary for pathogen survival. Taken together, our data suggest an important role for PstP in establishing and maintaining infection, possibly via the modulation of cell division events.

Robert Koch utilized animal model systems to put forward his postulates while discovering the etiological agents of anthrax and tuberculosis, Bacillus anthracis and Mycobacterium tuberculosis, respectively. After more than 130 years, we have achieved limited success towards understanding these two pestilences, which have propagated as scourge against humans. B. anthracis and M. tuberculosis are diverse organisms, which share a common evolutionary path in tropics. They adapt unique strategies to overcome unfavorable conditions and surpass the host defense mechanisms. B. anthracis is an endospore forming bacteria that primarily acts by releasing toxins in the host cells.. M. tuberculosis is an intracellular bacteria that resides within the host macrophages by blocking phagosome-lysosome fusion events and ensuring its own survival. The bacterium can remain dormant for long periods, and when activated, it spreads in lungs and other extrapulmonary sites leading to formation of necrotic granulomas. The two diseases are immunologically distinct examples of inducing primarily either humoral or cell mediated immunity. Natural immune response to the two diseases probably explains early success achieved with the anthrax vaccine, while the hunt for successful tuberculosis prevention is still on. For comprehensive understanding of these diseases, model systems are of utmost importance that can alleviate detailed assessment of disease etiology and introductory treatment regimes. In this review, we discuss the various in vitro and in vivo model systems used to study these two diseases, discussing their contributions and recent themes.

Dual specificity protein kinases (DSPKs) are unique enzymes that can execute multiple functions in the cell, which are otherwise performed exclusively by serine/threonine and tyrosine protein kinases. In this study, we have characterized the protein kinases Bas2152 (PrkD) and Bas2037 (PrkG) from Bacillus anthracis. Transcriptional analyses of these kinases showed that they are expressed in all phases of growth. In a serendipitous discovery, both kinases were found to be DSPKs. PrkD was found to be similar to the eukaryotic dual specificity Tyr phosphorylation-regulated kinase class of dual specificity kinases, which autophosphorylates on Ser, Thr, and Tyr residues and phosphorylates Ser and Thr residues on substrates. PrkG was found to be a bona fide dual specificity protein kinase that mediates autophosphorylation and substrate phosphorylation on Ser, Thr, and Tyr residues. The sites of phosphorylation in both of the kinases were identified through mass spectrometry. Phosphorylation on Tyr residues regulates the kinase activity of PrkD and PrkG. PrpC, the only known Ser/Thr protein phosphatase, was also found to possess dual specificity. Genistein, a known Tyr kinase inhibitor, was found to inhibit the activities of PrkD and PrkG and affect the growth of B. anthracis cells, indicating a possible role of these kinases in cell growth and development. In addition, the glycolytic enzyme pyruvate kinase was found to be phosphorylated by PrkD on Ser and Thr residues but not by PrkG. Thus, this study provides the first evidence of DSPKs in B. anthracis that belong to different classes and have different modes of regulation.

P-glycoprotein (P-gp) is an ABC transporter that exports many amphipathic or hydrophobic compounds, including chemically and functionally dissimilar anticancer drugs, from cells. To understand the role of transmembrane helices (TMH) 1 and 7 in drug-binding and transport, we selected six residues from both TMH1 (V53, I59, I60, L65, M68 and F72) and TMH7 (V713, I719, I720, Q725, F728 and F732); and substituted them with alanine by gene synthesis to generate a variant termed "TMH1,7 mutant P-gp". The expression and function of TMH1,7 mutant P-gp with twelve mutations was characterized using the BacMam baculovirus-HeLa cell expression system. The expression and conformation of TMH1,7 mutant P-gp was not altered by the introduction of the twelve mutations, as confirmed by using the human P-gp-specific antibodies UIC2, MRK16 and 4E3. We tested 25 fluorescently-labeled substrates and found that only three substrates, NBD-cyclosporine A, Rhod-2-AM and X-Rhod-1-AM were transported by the TMH1,7 mutant. The basal ATPase activity of TMH1,7 mutant P-gp was lower (40-50%) compared to wild-type (WT) P-gp, despite similar level of expression. Although most of the substrates modulate ATPase activity of P-gp, the activity of TMH1,7 mutant transporter was not significantly modulated by any of the tested substrates. Docking of selected substrates in homology models showed comparable docking scores for the TMH1,7 mutant and WT P-gp, although the binding conformations were different. Both the ATPase assay and in silico docking analyses suggest that the interactions with residues in the drug-binding pocket are altered as a consequence of the mutations. We demonstrate that it is possible to generate a variant of P-gp with a loss of broad substrate specificity and propose that TMH1 and TMH7 play a critical role in the drug efflux function of this multidrug transporter.

A series of 1,4-disubstituted triazoles (II) is synthesized via click chemistry and evaluated as potential antitubercular agents.

Extrapulmonary manifestations constitute 15 to 20% of tuberculosis cases, with lymph node tuberculosis (LNTB) as the most common form of infection. However, diagnosis and treatment advances are hindered by lack of understanding of LNTB biology. To identify host response, Mycobacterium tuberculosis infected lymph nodes from LNTB patients were studied by means of transcriptomics and quantitative proteomics analyses. The selected targets obtained by comparative analyses were validated by quantitative PCR and immunohistochemistry. This approach provided expression data for 8,728 transcripts and 102 proteins, differentially regulated in the infected human lymph node. Enhanced inflammation with upregulation of T-helper1-related genes, combined with marked dysregulation of matrix metalloproteinases, indicates tissue damage due to high immunoactivity at infected niche. This expression signature was accompanied by significant upregulation of an immunoregulatory gene, leukotriene A4 hydrolase, at both transcript and protein levels. Comparative transcriptional analyses revealed LNTB-specific perturbations. In contrast to pulmonary TB-associated increase in lipid metabolism, genes involved in fatty-acid metabolism were found to be downregulated in LNTB suggesting differential lipid metabolic signature. This study investigates the tissue molecular signature of LNTB patients for the first time and presents findings that indicate the possible mechanism of disease pathology through dysregulation of inflammatory and tissue-repair processes.

Synthetic biologists are trying to apply engineering principles in biology to create an artificial world with unlimited possibilities. The guiding principle is to look beyond finding the solutions and to create new ones. Synthetic biologists are now routinely designing synthetic genetic circuits in bacteria and yeast. The major aim is to use microbes as cell factories for the production of bioactive compounds with a particular focus on drugs, biofuels, and biopolymers. In this chapter, we emphasize on understanding the synthetic biology approaches and their role in creating polyhydroxyalkanoate (PHA)-producing microbial factories.

Mycobacterium tuberculosis modulates expression of various metabolism-related genes to adapt in the adverse host environment. The gene coding for M. tuberculosis S-adenosylhomocysteine hydrolase (Mtb-SahH) is essential for optimal growth and the protein product is involved in intermediary metabolism. However, the relevance of SahH in mycobacterial physiology is unknown. In this study, we analyze the role of Mtb-SahH in regulating homocysteine concentration in surrogate host Mycobacterium smegmatis. Mtb-SahH catalyzes reversible hydrolysis of S-adenosylhomocysteine to homocysteine and adenosine and we demonstrate that the conserved His363 residue is critical for bi-directional catalysis. Mtb-SahH is regulated by serine/threonine phosphorylation of multiple residues by M. tuberculosis PknB. Major phosphorylation events occur at contiguous residues Thr219, Thr220 and Thr221, which make pivotal contacts with cofactor NAD+. Consequently, phosphorylation negatively modulates affinity of enzyme towards NAD+ as well as SAH-synthesis. Thr219, Thr220 and Thr221 are essential for enzyme activity, and therefore, responsible for SahH-mediated regulation of homocysteine.

Bacillus anthracis Ser/Thr protein kinase PrkC (BasPrkC) is important for virulence of the bacterium within the host. Homologs of PrkC and its cognate phosphatase PrpC (BasPrpC) are the most conserved mediators of signaling events in diverse bacteria. BasPrkC homolog in Bacillus subtilis regulates critical processes like spore germination and BasPrpC modulates the activity of BasPrkC by dephosphorylation. So far, biochemical and genetic studies have provided important insights into the roles of BasPrkC and BasPrpC; however, regulation of their activities is not known. We studied the regulation of BasPrkC/BasPrpC pair and observed that Zn(2+) metal ions can alter their activities. Zn(2+) promotes BasPrkC kinase activity while inhibits the BasPrpC phosphatase activity. Concentration of Zn(2+) in growing B. anthracis cells was found to vary with growth phase. Zn(2+) was found to be lowest in log phase cells while it was highest in spores. This variation in Zn(2+) concentration is significant for understanding the antagonistic activities of BasPrkC/BasPrpC pair. Our results also show that BasPrkC activity is modulated by temperature changes and kinase inhibitors. Additionally, we identified Elongation Factor Tu (BasEf-Tu) as a substrate of BasPrkC/BasPrpC pair and assessed the impact of their regulation on BasEf-Tu phosphorylation. Based on these results, we propose Zn(2+) as an important regulator of BasPrkC/BasPrpC mediated phosphorylation cascades. Thus, this study reveals additional means by which BasPrkC can be activated leading to autophosphorylation and substrate phosphorylation.

During protein synthesis, translation elongation factor Tu (Ef-Tu) is responsible for the selection and binding of the cognate aminoacyl-tRNA to the acceptor site on the ribosome. The activity of Ef-Tu is dependent on its interaction with GTP. Posttranslational modifications, such as phosphorylation, are known to regulate the activity of Ef-Tu in several prokaryotes. Although a study of the Mycobacterium tuberculosis phosphoproteome showed Ef-Tu to be phosphorylated, the role of phosphorylation in the regulation of Ef-Tu has not been studied. In this report, we show that phosphorylation of M. tuberculosis Ef-Tu ( Mtb Ef-Tu) by PknB reduced its interaction with GTP, suggesting a concomitant reduction in the level of protein synthesis. Overexpression of PknB in Mycobacterium smegmatis indeed reduced the level of protein synthesis. Mtb Ef-Tu was found to be phosphorylated by PknB on multiple sites, including Thr 118 , which is required for optimal activity of the protein. We found that kirromycin, an Ef-Tu-specific antibiotic, had a significant effect on the nucleotide binding of unphosphorylated Mtb Ef-Tu but not on the phosphorylated protein. Our results show that the modulation of the Mtb Ef-Tu–GTP interaction by phosphorylation can have an impact on cellular protein synthesis and growth. These results also suggest that phosphorylation can change the sensitivity of the protein to the specific inhibitors. Thus, the efficacy of an inhibitor can also depend on the posttranslational modification(s) of the target and should be considered during the development of the molecule.

Background: The integrated functions of 11 Ser/Thr protein kinases (STPKs) and one phosphatase manipulate the phosphorylation levels of critical proteins in mycobacterium tuberculosis. In this study, we show that the lone Ser/Thr phosphatase (PstP) is regulated through phosphorylation by STPKs. Principal Findings: PstP is phosphorylated by PknA and PknB and phosphorylation is influenced by the presence of Zn 2+ -ions and inorganic phosphate (Pi). PstP is differentially phosphorylated on the cytosolic domain with Thr 137 , Thr 141 , Thr 174 and Thr 290 being the target residues of PknB while Thr 137 and Thr 174 are phosphorylated by PknA. The Mn 2+ -ion binding residues Asp 38 and Asp 229 are critical for the optimal activity of PstP and substitution of these residues affects its phosphorylation status. Native PstP and its phosphatase deficient mutant PstP c D38G are phosphorylated by PknA and PknB in E. coli and addition of Zn 2+ /Pi in the culture conditions affect the phosphorylation level of PstP. Interestingly, the phosphorylated phosphatase is more active than its unphosphorylated equivalent. Conclusions and Significance: This study establishes the novel mechanisms for regulation of mycobacterial Ser/Thr phosphatase. The results indicate that STPKs and PstP may regulate the signaling through mutually dependent mechanisms. Consequently, PstP phosphorylation may play a critical role in regulating its own activity. Since, the equilibrium between phosphorylated and non-phosphorylated states of mycobacterial proteins is still unexplained, understanding the regulation of PstP may help in deciphering the signal transduction pathways mediated by STPKs and the reversibility of the phenomena.

Mycobacterium tuberculosis profoundly exploits protein phosphorylation events carried out by serine/threonine protein kinases (STPKs) for its survival and pathogenicity. Forkhead-associated domains (FHA), the phosphorylation-responsive modules, have emerged as prominent players in STPK mediated signaling. In this study, we demonstrate the association of the previously uncharacterized FHA domain-containing protein Rv0019c with cognate STPK PknB. The consequent phosphorylation of Rv0019c is shown to be dependent on the conserved residues in the Rv0019c FHA domain and activation loop of PknB. Furthermore, by creating deletion mutants we identify Thr(36) as the primary phosphorylation site in Rv0019c. During purification of Rv0019c from Escherichia coli, the E. coli protein chloramphenicol acetyltransferase (CAT) specifically and reproducibly copurifies with Rv0019c in a FHA domain-dependent manner. On the basis of structural similarity of E. coli CAT with M. tuberculosis PapA5, a protein involved in phthiocerol dimycocerosate biosynthesis, PapA5 is identified as an interaction partner of Rv0019c. The interaction studies on PapA5, purified as an unphosphorylated protein from E. coli, with Rv0019c deletion mutants reveal that the residues N-terminal to the functional FHA domain of Rv0019c are critical for formation of the Rv0019c-PapA5 complex and thus constitute a previously unidentified phosphoindependent binding motif. Finally, PapA5 is shown to be phosphorylated on threonine residue(s) by PknB, whereas serine/threonine phosphatase Mstp completely reverses the phosphorylation. Thus, our data provides initial clues for a possible regulation of PapA5 and hence the phthiocerol dimycocerosate biosynthesis by PknB, either by direct phosphorylation of PapA5 or indirectly through Rv0019c.

Background Mycobacterium ulcerans, the causative agent of Buruli ulcer in humans, is unique among the members of Mycobacterium genus due to the presence of the virulence determinant megaplasmid pMUM001. This plasmid encodes multiple virulence-associated genes, including mup011, which is an uncharacterized Ser/Thr protein kinase (STPK) PknQ. Methodology/Principal Findings In this study, we have characterized PknQ and explored its interaction with MupFHA (Mup018c), a FHA domain containing protein also encoded by pMUM001. MupFHA was found to interact with PknQ and suppress its autophosphorylation. Subsequent protein-protein docking and molecular dynamic simulation analyses showed that this interaction involves the FHA domain of MupFHA and PknQ activation loop residues Ser170 and Thr174. FHA domains are known to recognize phosphothreonine residues, and therefore, MupFHA may be acting as one of the few unusual FHA-domain having overlapping specificity. Additionally, we elucidated the PknQ-dependent regulation of MupDivIVA (Mup012c), which is a DivIVA domain containing protein encoded by pMUM001. MupDivIVA interacts with MupFHA and this interaction may also involve phospho-threonine/serine residues of MupDivIVA. Conclusions/Significance Together, these results describe novel signaling mechanisms in M. ulcerans and show a three-way regulation of PknQ, MupFHA, and MupDivIVA. FHA domains have been considered to be only pThr specific and our results indicate a novel mechanism of pSer as well as pThr interaction exhibited by MupFHA. These results signify the need of further re-evaluating the FHA domain –pThr/pSer interaction model. MupFHA may serve as the ideal candidate for structural studies on this unique class of modular enzymes., Author Summary Mycobacterium ulcerans is a slow growing pathogen, which is prevalent in many tropical and sub-tropical countries. M. ulcerans possesses unique signaling pathways with only 13 STPK containing genes. This is strikingly different from its closest homolog Mycobacterium marinum and surprisingly closer to the human pathogen, Mycobacterium tuberculosis. PknQ, MupFHA and MupDivIVA are regulatory proteins encoded by the virulence determining plasmid pMUM001 of M. ulcerans. In addition to characterizing the STPK, we focused on deciphering the basis of interaction between the three partner proteins leading to the identification of critical residues. Present study describes the newly identified phosphoserine-based interactions, which is unique amongst the FHA-domain containing proteins. We confirmed our results using structural analysis via specific mutants and their interaction profiles. Importantly, these data highlight the significance of FHA domains and their role in understanding cellular signaling. This work will encourage further studies to elucidate role of M. ulcerans signaling systems. It will also raise questions like how less studied tropical bacterial pathogens acquire eukaryotic-like Ser/Thr protein kinase and exhibit unusual mechanisms to interact with its partner domains.