scholarly journals Protein kinase B controls Mycobacterium tuberculosis growth via phosphorylation of the global transcriptional regulator Lsr2

2019 ◽  
Author(s):  
Kawther Alqaseer ◽  
Obolbek Turapov ◽  
Philippe Barthe ◽  
Heena Jagatia ◽  
Angélique De Visch ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Protein Kinase ◽  
Dna Binding ◽  
Protein Kinase B ◽  
Transcriptional Regulator ◽  
The Body ◽  
Altered Expression ◽  
Chip Sequencing ◽  
Essential Protein ◽  
Wide Range

ABSTRACTMycobacterium tuberculosis is able to persist in the body through months of multi-drug therapy. Mycobacteria possess a wide range of regulatory proteins, including the essential protein kinase B (PknB), that control transitions between growth states. Here, we establish that depletion of PknB in replicating M. tuberculosis results in transcriptional adaptations that implicate the DNA-binding protein Lsr2 in coordinating these changes. We show that Lsr2 is phosphorylated by PknB, and that phosphorylation of Lsr2 at threonine 112 is important for M. tuberculosis growth and survival under hypoxic conditions. Fluorescence anisotropy and electrophoretic mobility shift assays demonstrate that phosphorylation reduces Lsr2 binding to DNA, and ChIP-sequencing confirms increased DNA binding of a phosphoablative (T112A) Lsr2 mutant in M. tuberculosis. Altered expression of target genes in T112A Lsr2 compared to wild type Lsr2 M. tuberculosis offers further evidence that phosphorylation mediates expression of the Lsr2 regulon. Structural studies reveal increased dynamics of the Lsr2 DNA binding domain from a T112D phosphomimetic Lsr2 mutant, providing a molecular basis for decreased DNA binding by phosphorylated Lsr2. Our findings suggest that, the essential protein kinase, PknB controls M. tuberculosis growth and adaptations to the changing host environment by phosphorylating the global transcriptional regulator Lsr2.

scholarly journals Protein kinase B controls Mycobacterium tuberculosis growth via phosphorylation of the transcriptional regulator Lsr2 at threonine 112

2019 ◽  
Vol 112 (6) ◽  
pp. 1847-1862
Author(s):  
Kawther Alqaseer ◽  
Obolbek Turapov ◽  
Philippe Barthe ◽  
Heena Jagatia ◽  
Angélique De Visch ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Protein Kinase ◽  
Protein Kinase B ◽  
Transcriptional Regulator

scholarly journals Mycobacterium tuberculosis Ser/Thr Protein Kinase B Mediates an Oxygen-Dependent Replication Switch

PLoS Biology ◽  
2014 ◽  
Vol 12 (1) ◽  
pp. e1001746 ◽  
Author(s):  
Corrie Ortega ◽  
Reiling Liao ◽  
Lindsey N. Anderson ◽  
Tige Rustad ◽  
Anja R. Ollodart ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Protein Kinase ◽  
Protein Kinase B

CsA-sensitive purine-box transcriptional regulator in bronchial epithelial cells contains NF45, NF90, and Ku

1998 ◽  
Vol 275 (6) ◽  
pp. L1164-L1172 ◽  
Author(s):  
Yosuke Aoki ◽  
Guohua Zhao ◽  
Daoming Qiu ◽  
Lingfang Shi ◽  
Peter N. Kao
Keyword(s):  
Protein Kinase ◽  
Dna Binding ◽  
Dna Sequence ◽  
Transcriptional Activation ◽  
Nuclear Dna ◽  
Transcriptional Regulator ◽  
Catalytic Subunit ◽  
Dependent Protein Kinase ◽  
Bronchial Epithelial ◽  
Dependent Protein

Human bronchial epithelial (HBE) cells express interleukin (IL)-2 [Y. Aoki, D. Qiu, A. Uyei, and P. N. Kao. Am. J. Physiol. 272 ( Lung Cell. Mol. Physiol. 16): L276–L286, 1997]. 16HBE-transformed cells contain constitutive and inducible nuclear DNA-binding activity for the purine-box/nuclear factor (NF) of activated T cell (NFAT) target DNA sequence in the human IL-2 enhancer. Transcriptional activation through the purine-box DNA sequence requires stimulation with phorbol 12-myristate 13-acetate + ionomycin, and this activation is inhibited by cyclosporin A. Immunohistochemical staining of 16HBE cells demonstrates nuclear expression of the purine-box DNA-binding proteins NF45 and NF90 and no expression of NFATp or NFATc. NF90 and NF45 associate with the DNA-dependent protein kinase catalytic subunit and the DNA-targeting subunits Ku80 and Ku70 (N. S. Ting, P. N. Kao, D. W. Chan, L. G. Lintott, and S. P. Lees-Miller. J. Biol. Chem. 273: 2136–2145, 1998). Antibodies to Ku potently inhibit the purine-box DNA-binding complex. The purine-box transcriptional regulator in 16HBE cells likely comprises NF45, NF90, Ku80, Ku70, and the DNA-dependent protein kinase catalytic subunit.

scholarly journals Characterization of the transcriptional regulator Rv3124 of Mycobacterium tuberculosis identifies it as a positive regulator of molybdopterin biosynthesis and defines the functional consequences of a non-synonymous SNP in the Mycobacterium bovis BCG orthologue

Microbiology ◽  
2010 ◽  
Vol 156 (7) ◽  
pp. 2112-2123 ◽  
Author(s):  
Pablo Mendoza Lopez ◽  
Paul Golby ◽  
Esen Wooff ◽  
Javier Nunez Garcia ◽  
M. Carmen Garcia Pelayo ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Bovis ◽  
Protein Interactions ◽  
Transcriptional Activation ◽  
Transcriptional Regulator ◽  
Proper Function ◽  
Nucleotide Polymorphisms ◽  
Glutamic Acid Residue ◽  
Wide Range ◽  
Functional Consequences

A number of single-nucleotide polymorphisms (SNPs) have been identified in the genome of Mycobacterium bovis BCG Pasteur compared with the sequenced strain M. bovis 2122/97. The functional consequences of many of these mutations remain to be described; however, mutations in genes encoding regulators may be particularly relevant to global phenotypic changes such as loss of virulence, since alteration of a regulator's function will affect the expression of a wide range of genes. One such SNP falls in bcg3145, encoding a member of the AfsR/DnrI/SARP class of global transcriptional regulators, that replaces a highly conserved glutamic acid residue at position 159 (E159G) with glycine in a tetratricopeptide repeat (TPR) located in the bacterial transcriptional activation (BTA) domain of BCG3145. TPR domains are associated with protein–protein interactions, and a conserved core (helices T1–T7) of the BTA domain seems to be required for proper function of SARP-family proteins. Structural modelling predicted that the E159G mutation perturbs the third α-helix of the BTA domain and could therefore have functional consequences. The E159G SNP was found to be present in all BCG strains, but absent from virulent M. bovis and Mycobacterium tuberculosis strains. By overexpressing BCG3145 and Rv3124 in BCG and H37Rv and monitoring transcriptome changes using microarrays, we determined that BCG3145/Rv3124 acts as a positive transcriptional regulator of the molybdopterin biosynthesis moa1 locus, and we suggest that rv3124 be renamed moaR1. The SNP in bcg3145 was found to have a subtle effect on the activity of MoaR1, suggesting that this mutation is not a key event in the attenuation of BCG.

scholarly journals Substituted aminopyrimidine protein kinase B (PknB) inhibitors show activity against Mycobacterium tuberculosis

2012 ◽  
Vol 22 (9) ◽  
pp. 3349-3353 ◽  
Author(s):  
Timothy M. Chapman ◽  
Nathalie Bouloc ◽  
Roger S. Buxton ◽  
Jasveen Chugh ◽  
Kathryn E.A. Lougheed ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Protein Kinase ◽  
Protein Kinase B

The Molecular basis of N-acetylmuramoyl-L-alanine amidase (Rv3915) and Protein Kinase B (PknB) essentiality in Mycobacterium tuberculosis

2019 ◽  
Author(s):  
Ifunanya N Ezechukwu ◽  
Kenechukwu Onyekwelu ◽  
Loreta Nwokolo ◽  
Obolbek Turapov
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Protein Kinase ◽  
Soluble Fraction ◽  
Protein Kinase B ◽  
Sodium Dodecyl ◽  
Coli Strain ◽  
Gene Encoding ◽  
E Coli ◽  
Sds Page

Abstract Background: Protein kinase B (PknB) is critical for the survival of Mycobacterium tuberculosis (M. tuberculosis) in vitro and in hosts. It phosphorylates various enzymes involved in biosynthesis of cell wall and a particular autolysin classified as CwIM or Rv3915 has been recently identified as a PknB substrate. However, in-depth knowledge of this protein is still unknown. The aims of this study were to purify and investigate the activity of Rv3915, as well as monitor the phosphorylation of Rv3915 and the influence of phosphorylation on the activity of this protein. Results: Using the C41 E. coli strain containing a plasmid, with a gene encoding the protein, Rv3915 was either expressed alone or co-expressed with PknB. Sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) was used to observe the amount of Rv3915 purified, anti-poly His and anti-phosphothreonine western blot techniques were used to confirm the presence and phosphorylation of Rv3915 and zymogram assays were run to examine its activity. The results showed that Rv3195 was successfully expressed and was deemed soluble when observed in soluble fraction of E. coli lysate. It was confirmed that Rv3915 is produced as a ~45kDa protein, which does not possess any muralytic activity. However a shorter version of the protein (~25kDA) was active in zymogram, suggesting that Rv3915 is activated by cleavage. Conclusion: Rv3915 was phosphorylated by PknB and phosphorylation apparently controls stability of the protein.

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