Andaleeb Sajid

Forkhead-associated Domain-containing Protein Rv0019c and Polyketide-associated Protein PapA5, from Substrates of Serine/Threonine Protein Kinase PknB to Interacting Proteins of Mycobacterium tuberculosis

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 Thr36 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.

Protein Phosphatases of Pathogenic Bacteria: Role in Physiology and Virulence

The role of protein phosphatases in pathogenic bacteria has been studied extensively over the last two decades. Ser/Thr and Tyr phosphatases are associated with growth and virulence of many bacteria. These phosphatases control kinase-mediated functions and return the proteins to their unmodified state. Biochemical, structural, and functional studies, in addition to extensive genetic characterization, have highlighted the importance of phosphatases in bacteria. However, questions remain regarding the mechanisms driving localization of secretory phosphatases to cellular compartments, identification of receptor phosphatase sensory signals, and a possible role of cofactors and ligands in their functions. This review focuses on the role of Ser/Thr- and Tyr-specific phosphatases present in pathogenic bacteria, with an emphasis on the regulation of basic cellular processes and virulence. Furthermore, we highlight their clinical importance and analyze the development of drugs targeting protein phosphatases.

HupB, a Nucleoid-Associated Protein of Mycobacterium tuberculosis, Is Modified by Serine/Threonine Protein Kinases In Vivo

HU, a widely conserved bacterial histone-like protein, regulates many genes, including those involved in stress response and virulence. Whereas ample data are available on HU-DNA communication, the knowledge on how HU perceives a signal and transmit it to DNA remains limited. In this study, we identify HupB, the HU homolog of the human pathogen Mycobacterium tuberculosis, as a component of serine/threonine protein kinase (STPK) signaling. HupB is extracted in its native state from the exponentially growing cells of M. tuberculosis H37Ra and is shown to be phosphorylated on both serine and threonine residues. The STPKs capable of modifying HupB are determined in vitro and the residues modified by the STPKs are identified for both in vivo and the in vitro proteins through mass spectrometry. Of the identified phosphosites, Thr(65) and Thr(74) in the DNA-embracing β-strand of the N-terminal domain of HupB (N-HupB) are shown to be crucial for its interaction with DNA. In addition, Arg(55) is also identified as an important residue for N-HupB-DNA interaction. N-HupB is shown to have a diminished interaction with DNA after phosphorylation. Furthermore, hupB is shown to be maximally expressed during the stationary phase in M. tuberculosis H37Ra, while HupB kinases were found to be constitutively expressed (PknE and PknF) or most abundant during the exponential phase (PknB). In conclusion, HupB, a DNA-binding protein, with an ability to modulate chromatin structure is proposed to work in a growth-phase-dependent manner through its phosphorylation carried out by the mycobacterial STPKs.

Unveiling the Novel Dual Specificity Protein Kinases in Bacillus anthracis IDENTIFICATION OF THE FIRST PROKARYOTIC DUAL SPECIFICITY TYROSINE PHOSPHORYLATION-REGULATED KINASE (DYRK)-LIKE KINASE

Background: Characterization of Ser/Thr protein kinases present in Bacillus anthracis genome. Results: Dual specificity protein kinases were identified, of which one is similar to the eukaryotic DYRK superfamily. Conclusion: B. anthracis has lost key tyrosine kinases and gained novel dual specificity kinases. Significance: Reporting the first prokaryotic enzyme similar to DYRKs shows that this class of enzymes is not restricted to eukaryotes. 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.

Regulation of homocysteine metabolism by Mycobacterium tuberculosis S-adenosylhomocysteine hydrolase

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.

Expression profiling of lymph nodes in tuberculosis patients reveal inflammatory milieu at site of infection.

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.

Zinc regulates the activity of kinase-phosphatase pair (BasPrkC/BasPrpC) in Bacillus anthracis

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 Zn2+ metal ions can alter their activities. Zn2+ promotes BasPrkC kinase activity while inhibits the BasPrpC phosphatase activity. Concentration of Zn2+ in growing B. anthracis cells was found to vary with growth phase. Zn2+ was found to be lowest in log phase cells while it was highest in spores. This variation in Zn2+ 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 Zn2+ 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.

Serine/threonine protein phosphatase PstP of Mycobacterium tuberculosis is necessary for accurate cell division and survival of pathogen

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.

Ser/Thr protein kinase PrkC-mediated regulation of GroEL is critical for biofilm formation in Bacillus anthracis

PrkC is a conserved Ser/Thr protein kinase encoded in Bacillus anthracis genome. PrkC is shown to be important for B. anthracis pathogenesis, but little is known about its other functions and phosphorylated substrates. Systemic analyses indicate the compelling role of PrkC in phosphorylating multiple substrates, including the essential chaperone GroEL. Through mass spectrometry, we identified that PrkC phosphorylates GroEL on six threonine residues that are distributed in three canonical regions. Phosphorylation facilitates the oligomerization of GroEL to the physiologically active tetradecameric state and increases its affinity toward the co-chaperone GroES. Deletion of prkC in B. anthracis abrogates its ability to form biofilm. Overexpression of native GroEL recovers the biofilm-forming ability of prkC deletion strain. Similar overexpression of GroEL phosphorylation site mutants (Thr to Ala) does not augment biofilm formation. Further analyses indicate the phosphorylation of GroEL in diverse bacterial species. Thus, our results suggest that PrkC regulates biofilm formation by modulating the GroEL activity in a phosphorylation-dependent manner. The study deciphers the molecular signaling events that are important for biofilm formation in B. anthracis.Anthrax bacteria: a step in the pathway to biofilmsAn enzyme that adds phosphate groups to other proteins, PrkC, mediates molecular signaling events that allow anthrax bacteria to form biofilms. Bacillus anthracis is widely used as a model to explore the formation of biofilms that allows many bacterial infections to resist immune defenses. An international research team led by Yogendra Singh and Andaleeb Sajid at the CSIR-Institute of Genomics and Integrative Biology in Delhi, India, studied the bacterial protein kinase PrkC. The researchers found that PrkC phosphorylates a “chaperone” protein that assist the assembly and disassembly of other protein-based structures. This signaling protein and the chaperone help in biofilm formation. Establishing this link in the signaling chain leading to biofilms will guide future research to combat the role of biofilms in disease.

Identification of Ser/Thr kinase and forkhead associated domains in Mycobacterium ulcerans: characterization of novel association between protein kinase Q and MupFHA.

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.