Molecular structure of EmbR, a response element of Ser/Thr kinase signaling in
            <i>Mycobacterium</i>
            <i>tuberculosis</i>

Alderwick, Luke J.; Molle, Virginie; Kremer, Laurent; Cozzone, Alain J.; Dafforn, Timothy R.; Besra, Gurdyal S.; Fütterer, Klaus

doi:10.1073/pnas.0507766103

Cited by 68 publications

(84 citation statements)

References 50 publications

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“…These results clearly indicated at the necessity of threonine residues (Thr 171 and Thr 173 ) in the activation loop of PknB for effective phosphorylation of 19c⌬T. The activation loop threonine residues were previously shown to be critical for the phosphorylation of GarA by PknB (40), whereas similar residues in PknH played no role in its interaction with the FHA domain containing protein EmbR (34).…”

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confidence: 67%

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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

Gupta

Sajid

Arora

et al. 2009

Journal of Biological Chemistry

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Mycobacterium tuberculosis profoundly exploits protein phosphorylation events carried out by serine/threonine protein kinases (STPKs) for its survival and pathogenicity. Forkheadassociated 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.

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confidence: 67%

“…FHA domains of mycobacterial proteins with known x-ray or NMR structure (34,29) shows the conservation of six of the seven hallmark residues for FHA domains in Rv0019c (Fig. 1B).…”

Section: Domain Architecture and Genomic Organization Of Rv0019c-mentioning

confidence: 99%

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

Gupta

Sajid

Arora

et al. 2009

Journal of Biological Chemistry

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“…Eight proteins, which we found up-regulated at R24, were Rv3058c, EmbR, Rv2175c, Rv1019, Rv2258c, HrcA, DevS, and DevR. EmbR (Rv1267c), a putative transcriptional regulator of arabinan cell wall metabolism (60), and Rv3058c, a probable transcriptional factor that belongs to TetR family (61), were detected only in the control and R24 (five-fold increase at R24). Under unfavorable conditions, bacteria shift their metabolic activities by stringent response (62).…”

Section: Validation Of Quantitative Proteomic Data By Qpcr-tomentioning

confidence: 99%

Profiling the Proteome of Mycobacterium tuberculosis during Dormancy and Reactivation

Gopinath

Raghunandanan

Gomez

et al. 2015

Molecular & Cellular Proteomics

114

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Tuberculosis, caused by Mycobacterium tuberculosis, still remains a major global health problem. The main obstacle in eradicating this disease is the ability of this pathogen to remain dormant in macrophages, and then reactivate later under immuno-compromised conditions. The physiology of hypoxic nonreplicating M. tuberculosis is well-studied using many in vitro dormancy models. However, the physiological changes that take place during the shift from dormancy to aerobic growth (reactivation) have rarely been subjected to a detailed investigation. In this study, we developed an in vitro reactivation system by re-aerating the virulent laboratory strain of M. tuberculosis that was made dormant employing Wayne's dormancy model, and compared the proteome profiles of dormant and reactivated bacteria using label-free onedimensional LC/MS/MS analysis. The proteome of dormant bacteria was analyzed at nonreplicating persistent stage 1 (NRP1) and stage 2 (NRP2), whereas that of reactivated bacteria was analyzed at 6 and 24 h post reaeration. Proteome of normoxially grown bacteria served as the reference. In total, 1871 proteins comprising 47% of the M. tuberculosis proteome were identified, and many of them were observed to be expressed differentially or uniquely during dormancy and reactivation. The number of proteins detected at different stages of dormancy (764 at NRP1, 691 at NRP2) and reactivation (768 at R6 and 983 at R24) was very low compared with that of the control (1663). The number of unique proteins identified during normoxia, NRP1, NRP2, R6, and R24 were 597, 66, 56, 73, and 94, respectively. We analyzed various biological functions during these conditions. Fluctuation in the relative quantities of proteins involved in energy metabolism during dormancy and reactivation was the most significant observation we made in this study. Proteins that are up-regulated or uniquely expressed during reactivation from dormancy offer to be attractive targets for therapeutic intervention to

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“…This class also contains EmbR, the regulator of three arabinosyltransferases that are the targets of the front-line tuberculosis drug ethambutol (8). The structure of EmbR has been elucidated, revealing DNA binding, bacterial transcriptional activation (BTA), and forkhead-associated domains (1). While BCG3145 lacks the forkhead-associated domain, the E159G mutation in BCG3145 mutates to glycine a conserved glutamic acid residue located in a tetratricopeptide repeat in the BTA domain (region T3).…”

Section: Growth On Glycerolmentioning

confidence: 99%

A Comprehensive Survey of Single Nucleotide Polymorphisms (SNPs) across Mycobacterium bovis Strains and M. bovis BCG Vaccine Strains Refines the Genealogy and Defines a Minimal Set of SNPs That Separate Virulent M. bovis Strains and M. bovis BCG Strains

Pelayo¹,

Uplekar

Keniry

et al. 2009

Infect Immun

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To further unravel the mechanisms responsible for attenuation of the tuberculosis vaccine Mycobacterium bovis BCG, comparative genomics was used to identify single nucleotide polymorphisms (SNPs) that differed between sequenced strains of Mycobacterium bovis and M. bovis BCG. SNPs were assayed in M. bovis isolates from France and the United Kingdom and from different BCG vaccines in order to identify those that arose during the attenuation process which gave rise to BCG. Informative data sets were obtained for 658 SNPs from 21 virulent M. bovis strains and 13 BCG strains; these SNPs showed phylogenetic clustering that was consistent with the geographical origin of the strains and previous schemes for BCG genealogies. The data revealed a closer relationship between BCG Tice and BCG Pasteur than was previously appreciated, while we were able to position BCG Beijing within a grouping of BCG Denmark-derived strains. Only 186 SNPs were identified between virulent M. bovis strains and all BCG strains, with 115 nonsynonymous SNPs affecting important functions such as global regulators, transcriptional factors, and central metabolism, which might impact on virulence. We therefore refine previous genealogies of BCG vaccines and define a minimal set of SNPs between virulent M. bovis strains and the attenuated BCG strain that will underpin future functional analyses.Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the only vaccine available against tuberculosis and is the most widely used vaccine in the world. It was derived by the repeated subculture of a strain of Mycobacterium bovis on potato slices soaked in glycerol and ox bile (10), leading to the in vitro accumulation of mutations and ultimately attenuation. Despite the widespread use of BCG, the precise genetic lesions that led to attenuation are not defined. Furthermore, the success of BCG led to its distribution from the Institut Pasteur to laboratories around the world, each of which continued the subculturing process, thereby leading to the generation of a number of daughter strains named after their geographical origin (hence BCG Tokyo, BCG Russia, etc.). The protective efficacy of these strains has been shown to vary in both laboratory models and epidemiological studies (6,18,36).As BCG is the only vaccine currently available against tuberculosis, there is a clear need to understand the molecular basis of attenuation and variable efficacy afforded by BCG. The first study that attempted to identify mutations linked to attenuation was performed by Mahairas and colleagues, who identified three deletions, RD1 to RD3, from the genome of BCG strain Connaught (39). The RD1 locus was shown to be deleted from all BCG strains but present in all virulent strains of M. bovis and Mycobacterium tuberculosis studied. Subsequent work has shown that this deletion played a major role in the attenuation of BCG (38, 46). However, complementation of BCG with RD1 does not restore virulence to wild-type levels, suggesting that other attenuating mutations exist. Indeed, all BCG...

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Molecular structure of EmbR, a response element of Ser/Thr kinase signaling in Mycobacterium tuberculosis

Cited by 68 publications

References 50 publications

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

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

Profiling the Proteome of Mycobacterium tuberculosis during Dormancy and Reactivation

A Comprehensive Survey of Single Nucleotide Polymorphisms (SNPs) across Mycobacterium bovis Strains and M. bovis BCG Vaccine Strains Refines the Genealogy and Defines a Minimal Set of SNPs That Separate Virulent M. bovis Strains and M. bovis BCG Strains

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