Dual phosphorylation in response regulator protein PrrA is crucial for intracellular survival of mycobacteria consequent upon transcriptional activation

2017 ◽  
Vol 474 (24) ◽  
pp. 4119-4136 ◽  
Author(s):  
Alok K. Mishra ◽  
Shivraj M. Yabaji ◽  
Rikesh K. Dubey ◽  
Ekta Dhamija ◽  
Kishore K. Srivastava
Keyword(s):  
Transcriptional Activation ◽  
Response Regulator ◽  
Regulatory Protein ◽  
Peritoneal Macrophages ◽  
Physical Interaction ◽  
Fast Growing ◽  
Regulatory Circuit ◽  
Regulator Protein ◽  
Promoter Dna

The remarkable ability of Mycobacterium tuberculosis (Mtb) to survive inside human macrophages is attributed to the presence of a complex sensory and regulatory network. PrrA is a DNA-binding regulatory protein, belonging to an essential two-component system (TCS), PrrA/B, which is required for early phase intracellular replication of Mtb. Despite its importance, the mechanism of PrrA/B-mediated signaling is not well understood. In the present study, we demonstrate that the binding of PrrA on the promoter DNA and its consequent activation is cumulatively controlled via dual phosphorylation of the protein. We have further characterized the role of terminal phospho-acceptor domain in the physical interaction of PrrA with its cognate kinase PrrB. The genetic deletion of prrA/B in Mycobacterium smegmatis was possible only in the presence of ectopic copies of the genes, suggesting the essentiality of this TCS in fast-growing mycobacterial strains as well. The overexpression of phospho-mimetic mutant (T6D) altered the growth of M. smegmatis in an in vitro culture and affected the replication of Mycobacterium bovis BCG in mouse peritoneal macrophages. Interestingly, the Thr6 site was found to be conserved in Mtb complex, whereas it was altered in some fast-growing mycobacterial strains, indicating that this unique phosphorylation might be predominant in employing the regulatory circuit in M. bovis BCG and presumably also in Mtb complex.

scholarly journals Regulators of the Bacillus subtilis cydABCD Operon: Identification of a Negative Regulator, CcpA, and a Positive Regulator, ResD

2007 ◽  
Vol 189 (9) ◽  
pp. 3348-3358 ◽  
Author(s):  
Ankita Puri-Taneja ◽  
Matthew Schau ◽  
Yinghua Chen ◽  
F. Marion Hulett
Keyword(s):  
Bacillus Subtilis ◽  
Response Regulator ◽  
Regulatory Protein ◽  
Negative Regulator ◽  
Regulatory Proteins ◽  
Regulation Of Respiration ◽  
Regulator Protein ◽  
Promoter Fusion

ABSTRACT The cydABCD operon of Bacillus subtilis encodes products required for the production of cytochrome bd oxidase. Previous work has shown that one regulatory protein, YdiH (Rex), is involved in the repression of this operon. The work reported here confirms the role of Rex in the negative regulation of the cydABCD operon. Two additional regulatory proteins for the cydABCD operon were identified, namely, ResD, a response regulator involved in the regulation of respiration genes, and CcpA, the carbon catabolite regulator protein. ResD, but not ResE, was required for full expression of the cydA promoter in vivo. ResD binding to the cydA promoter between positions −58 and −107, a region which includes ResD consensus binding sequences, was not enhanced by phosphorylation. A ccpA mutant had increased expression from the full-length cydA promoter during stationary growth compared to the wild-type strain. Maximal expression in a ccpA mutant was observed from a 3′-deleted cydA promoter fusion that lacked the Rex binding region, suggesting that the effect of the two repressors, Rex and CcpA, was cumulative. CcpA binds directly to the cydA promoter, protecting the region from positions −4 to −33, which contains sequences similar to the CcpA consensus binding sequence, the cre box. CcpA binding was enhanced upon addition of glucose-6-phosphate, a putative cofactor for CcpA. Mutation of a conserved residue in the cre box reduced CcpA binding 10-fold in vitro and increased cydA expression in vivo. Thus, CcpA and ResD, along with the previously identified cydA regulator Rex (YdiH), affect the expression of the cydABCD operon. Low-level induction of the cydA promoter was observed in vivo in the absence of its regulatory proteins, Rex, CcpA, and ResD. This complex regulation suggests that the cydA promoter is tightly regulated to allow its expression only at the appropriate time and under the appropriate conditions.

scholarly journals Helicobacter pylori FlhA Binds the Sensor Kinase and Flagellar Gene Regulatory Protein FlgS with High Affinity

2015 ◽  
Vol 197 (11) ◽  
pp. 1886-1892 ◽  
Author(s):  
Jennifer Tsang ◽  
Takanori Hirano ◽  
Timothy R. Hoover ◽  
Jonathan L. McMurry
Keyword(s):  
Helicobacter Pylori ◽  
Response Regulator ◽  
Regulatory Protein ◽  
Kinase Domain ◽  
Optical Biosensing ◽  
Content Type ◽  
High Affinity ◽  
Flagellar Assembly ◽  
Flagellar Biogenesis

ABSTRACTFlagellar biogenesis is a complex process that involves multiple checkpoints to coordinate transcription of flagellar genes with the assembly of the flagellum. InHelicobacter pylori, transcription of the genes needed in the middle stage of flagellar biogenesis is governed by RpoN and the two-component system consisting of the histidine kinase FlgS and response regulator FlgR. In response to an unknown signal, FlgS autophosphorylates and transfers the phosphate to FlgR, initiating transcription from RpoN-dependent promoters. In the present study, export apparatus protein FlhA was examined as a potential signal protein. Deletion of its N-terminal cytoplasmic sequence dramatically decreased expression of two RpoN-dependent genes,flaBandflgE. Optical biosensing demonstrated a high-affinity interaction between FlgS and a peptide consisting of residues 1 to 25 of FlhA (FlhANT). TheKD(equilibrium dissociation constant) was 21 nM and was characterized by fast-on (kon= 2.9 × 104M−1s−1) and slow-off (koff= 6.2 × 10−4s−1) kinetics. FlgS did not bind peptides consisting of smaller fragments of the FlhANTsequence. Analysis of binding to purified fragments of FlgS demonstrated that the C-terminal portion of the protein containing the kinase domain binds FlhANT. FlhANTbinding did not stimulate FlgS autophosphorylationin vitro, suggesting that FlhA facilitates interactions between FlgS and other structures required to stimulate autophosphorylation.IMPORTANCEThe high-affinity binding of FlgS to FlhA characterized in this study points to an additional role for FlhA in flagellar assembly. Beyond its necessity for type III secretion, the N-terminal cytoplasmic sequence of FlhA is required for RpoN-dependent gene expression via interaction with the C-terminal kinase domain of FlgS.

scholarly journals MACROPHAGE-LYMPHOCYTE CLUSTERS IN THE IMMUNE RESPONSE TO SOLUBLE PROTEIN ANTIGEN IN VITRO

1974 ◽  
Vol 140 (5) ◽  
pp. 1245-1259 ◽  
Author(s):  
Ole Werdelin ◽  
Otto Brændstrup ◽  
Eskild Pedersen
Keyword(s):  
Immune Response ◽  
Lymph Node ◽  
Cluster Formation ◽  
Peritoneal Macrophages ◽  
Physical Interaction ◽  
Antigen Binding ◽  
Purified Protein Derivative ◽  
Cell Clusters ◽  
Immune Lymphocytes

We have studied the physical interaction between macrophages and lymphocytes during the immune response to purified protein derivative of tuberculin (PPD) in vitro. Mixtures of peritoneal macrophages and lymph node lymphocytes from guinea pigs immunized with tubercle bacilli formed cell clusters during 20 h of culture with PPD. The number of clusters produced was correlated to the number of immune lymphocytes in the cultures. Peritoneal macrophages which had been pulsed with PPD and untreated lymph node lymphocytes produced cell clusters in the absence of free PPD in numbers equivalent to those produced by the same cells in the presence of free PPD. In cultures containing a mixture of PPD-pulsed macrophages, not-pulsed macrophages, and immune lymphocytes with no free PPD, cell clusters developed mainly between the antigen-pulsed macrophages and lymphocytes. Cluster formation was antigen-specific with the specificity residing in the lymphocytes, mainly or exclusively in the T lymphocytes. These data indicate that in the process of cell cluster formation macrophages serve as antigen-binding (or -processing) cells, while a subpopulation of lymphocytes interact physically and specifically with the macrophages.

scholarly journals RR06 Activates Transcription of spr1996 and cbpA in Streptococcus pneumoniae

2007 ◽  
Vol 189 (6) ◽  
pp. 2497-2509 ◽  
Author(s):  
Zhuo Ma ◽  
Jing-Ren Zhang
Keyword(s):  
Streptococcus Pneumoniae ◽  
Transcriptional Activation ◽  
Protective Antigen ◽  
Response Regulator ◽  
Phosphorylation Site ◽  
Promoter Regions ◽  
Conserved Sequence ◽  
Antibody Staining ◽  
Regulator Protein ◽  
Bacterial Replication

ABSTRACT Streptococcus pneumoniae colonizes at the nasopharynx of humans and is able to disseminate and cause various infections. The hallmark of pneumococcal disease is rapid bacterial replication in different tissue sites leading to intense inflammation. The genetic basis of pneumococcal adaptation to different host niches remains sketchy. In this study, we investigated the regulatory effect of RR06, a response regulator protein, on gene expression of S. pneumoniae. Microarray and Northern blot analyses showed that RR06 is specifically required for transcription of spr1996 and cbpA. While the function of Spr1996 is unknown, CbpA has been well characterized as a surface-exposed protective antigen and a virulence factor of S. pneumoniae. A recombinant form of RR06 was able to bind to a 19-bp conserved sequence shared by the spr1996 and cbpA promoter regions. Furthermore, inactivation of rr06 resulted in loss of CbpA expression as detected by antibody staining and bacterial adhesion. CbpA expression was restored in trans by the intact rr06 gene. However, a mutant, RR06(D51A), with a point mutation in the aspartate residue at position 51 (a predicted major phosphorylation site) of RR06, completely abolished the CbpA expression, suggesting that RR06 phosphorylation is required for transcriptional activation of spr1996 and cbpA. Finally, inactivation of rr06 in additional pneumococcal strains also led to the loss of CbpA expression. These data implicate that RR06 activates the expression of spr1996 and cbpA in many other pneumococcal strains.

scholarly journals The biology of nematode- and IL4Rα-dependent murine macrophage polarization in vivo as defined by RNA-Seq and targeted lipidomics

Blood ◽  
2012 ◽  
Vol 120 (25) ◽  
pp. e93-e104 ◽  
Author(s):  
Graham D. Thomas ◽  
Dominik Rückerl ◽  
Benjamin H. Maskrey ◽  
Phillip D. Whitfield ◽  
Mark L. Blaxter ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Expression Profiles ◽  
Macrophage Polarization ◽  
Peritoneal Macrophages ◽  
Mitochondrial Metabolism ◽  
Alternative Activation ◽  
Alternatively Activated Macrophages ◽  
Transcription Start Sites

Abstract Alternatively activated macrophages (AAMφ) are a major component of the response to helminth infection; however, their functions remain poorly defined. To better understand the helminth-induced AAMφ phenotype, we performed a systems-level analysis of in vivo derived AAMφ using an established mouse model. With next-generation RNA sequencing, we characterized the transcriptomes of peritoneal macrophages from BALB/c and IL4Rα−/− mice elicited by the nematode Brugia malayi, or via intraperitoneal thioglycollate injection. We defined expression profiles of AAMφ-associated cytokines, chemokines, and their receptors, providing evidence that AAMφ contribute toward recruitment and maintenance of eosinophilia. Pathway analysis highlighted complement as a potential AAMφ-effector function. Up-regulated mitochondrial genes support in vitro evidence associating mitochondrial metabolism with alternative activation. We mapped macrophage transcription start sites, defining over-represented cis-regulatory motifs within AAMφ-associated promoters. These included the binding site for PPAR transcription factors, which maintain mitochondrial metabolism. Surprisingly PPARγ, implicated in the maintenance of AAMφ, was down-regulated on infection. PPARδ expression, however, was maintained. To explain how PPAR-mediated transcriptional activation could be maintained, we used lipidomics to quantify AAMφ-derived eicosanoids, potential PPAR ligands. We identified the PPARδ ligand PGI2 as the most abundant AAMφ-derived eicosanoid and propose a PGI2-PPARδ axis maintains AAMφ during B malayi implantation.

scholarly journals Subcellular Clustering of the Phosphorylated WspR Response Regulator Protein Stimulates Its Diguanylate Cyclase Activity

mBio ◽  
2013 ◽  
Vol 4 (3) ◽  
Author(s):  
Varisa Huangyutitham ◽  
Zehra Tüzün Güvener ◽  
Caroline S. Harwood
Keyword(s):  
Signal Transduction ◽  
Biofilm Formation ◽  
Response Regulator ◽  
Cluster Formation ◽  
Cyclase Activity ◽  
Diguanylate Cyclase ◽  
Regulator Protein ◽  
Response Regulator Protein

ABSTRACT WspR is a hybrid response regulator-diguanylate cyclase that is phosphorylated by the Wsp signal transduction complex in response to growth of Pseudomonas aeruginosa on surfaces. Active WspR produces cyclic di-GMP (c-di-GMP), which in turn stimulates biofilm formation. In previous work, we found that when activated by phosphorylation, yellow fluorescent protein (YFP)-tagged WspR forms clusters that are visible in individual cells by fluorescence microscopy. Unphosphorylated WspR is diffuse in cells and not visible. Thus, cluster formation is an assay for WspR signal transduction. To understand how and why WspR forms subcellular clusters, we analyzed cluster formation and the enzymatic activities of six single amino acid variants of WspR. In general, increased cluster formation correlated with increased in vivo and in vitro diguanylate cyclase activities of the variants. In addition, WspR specific activity was strongly concentration dependent in vitro, and the effect of the protein concentration on diguanylate cyclase activity was magnified when WspR was treated with the phosphor analog beryllium fluoride. Cluster formation appears to be an intrinsic property of phosphorylated WspR (WspR-P). These results support a model in which the formation of WspR-P subcellular clusters in vivo in response to a surface stimulus is important for potentiating the diguanylate cyclase activity of WspR. Subcellular cluster formation appears to be an additional means by which the activity of a response regulator protein can be regulated. IMPORTANCE Bacterial sensor proteins often phosphorylate cognate response regulator proteins when stimulated by an environmental signal. Phosphorylated response regulators then mediate an appropriate adaptive cellular response. About 6% of response regulator proteins have an enzymatic domain that is involved in producing or degrading cyclic di-GMP (c-di-GMP), a molecule that stimulates bacterial biofilm formation. In this work, we examined the in vivo and in vitro behavior of the response regulator-diguanylate cyclase WspR. When phosphorylated in response to a signal associated with surface growth, WspR has a tendency to form oligomers that are visible in cells as subcellular clusters. Our results show that the formation of phosphorylated WspR (WspR-P) subcellular clusters is important for potentiating the diguanylate cyclase activity of WspR-P, making it more active in c-di-GMP production. We conclude that oligomer formation visualized as subcellular clusters is an additional mechanism by which the activities of response regulator-diguanylate cyclases can be regulated.

scholarly journals nSec-1 (munc-18) interacts with both primed and unprimed syntaxin 1A and associates in a dimeric complex on adrenal chromaffin granules

1999 ◽  
Vol 342 (3) ◽  
pp. 707-714 ◽  
Author(s):  
Lee P. HAYNES ◽  
Alan MORGAN ◽  
Robert D. BURGOYNE
Keyword(s):  
Fusion Protein ◽  
Regulatory Protein ◽  
Physical Interaction ◽  
Dimeric Complex ◽  
Chromaffin Granules ◽  
Chromaffin Granule ◽  
Attachment Protein ◽  
Syntaxin 1A

The target-SNARE syntaxin 1A is an essential component of the core machinery required for regulated exocytosis (where SNARE is the soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor). Syntaxin 1A interacts with a variety of other proteins, two of which, N-ethylmaleimide-sensitive fusion protein (NSF) and α-soluble NSF attachment protein (α-SNAP) have been suggested to impart a conformational rearrangement on this protein during a reaction referred to as priming. We have studied the effect of the primed state on the binding properties of syntaxin 1A and we have confirmed that primed syntaxin 1A no longer associated with α-SNAP or its cognate vesicle-SNARE, vesicle-associated membrane protein (VAMP). Under such conditions, however, it retained the ability to bind to nSec-1. It has been demonstrated that nSec-1, a regulatory protein also involved in neuronal exocytosis, binds syntaxin 1A with high affinity in vitro, although evidence for this physical interaction occurring in vivo has proven elusive. We analysed the subcellular distribution of these two proteins in fractions from bovine adrenal medulla and detected syntaxin 1A and nSec-1 in both plasma membrane and chromaffin-granule fractions. Using a cross-linking approach with chromaffin-granule membranes we detected a putative dimeric complex composed of approx. 54% total granule membrane nSec-1 and approx. 30% total syntaxin 1A. The results of this study therefore suggest the possibility of nSec-1 interactions with primed syntaxin 1A and demonstrate a potentially significant interaction of syntaxin 1A and nSec-1 on the membranes of chromaffin granules.

scholarly journals Transcriptional Activation of the Bacillus subtilis spoIIG Promoter by the Response Regulator Spo0A Is Independent of the C-Terminal Domain of the RNA Polymerase Alpha Subunit

1998 ◽  
Vol 180 (17) ◽  
pp. 4760-4763 ◽  
Author(s):  
Dean A. Rowe-Magnus ◽  
Mario Mencía ◽  
Fernando Rojo ◽  
Margarita Salas ◽  
George B. Spiegelman
Keyword(s):  
Bacillus Subtilis ◽  
Rna Polymerase ◽  
Transcriptional Activation ◽  
Response Regulator ◽  
In Vitro Transcription ◽  
Alpha Subunit ◽  
Deletion Mutants ◽  
Wild Type ◽  
Initiation Mechanism

ABSTRACT In vitro transcription from the spoIIG promoter byBacillus subtilis RNA polymerase reconstituted with wild-type alpha subunits and with C-terminal deletion mutants of the alpha subunit was equally stimulated by the response regulator Spo0A. Some differences in the structure of open complexes formed by RNA polymerase containing alpha subunit mutants were noted, although the wild-type and mutant polymerases appeared to use the same initiation mechanism.

scholarly journals Physical and functional interaction between the ID1 and p65 for activation of NF-κB

2012 ◽  
Vol 303 (3) ◽  
pp. C267-C277 ◽  
Author(s):  
Xiao Peng ◽  
Yuna Wang ◽  
Swapna Kolli ◽  
Junpeng Deng ◽  
Li Li ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Transcriptional Activation ◽  
Nuclear Translocation ◽  
Luciferase Assay ◽  
Physical Interaction ◽  
Site Mutation ◽  
Transcriptional Activation Domain ◽  
Apoptosis Protein

Inhibitor of differentiation or DNA binding-1 (ID1) is an important helix-loop-helix (HLH) transcription factor involved in diverse biological functions including cell differentiation, proliferation, apoptosis, and senescence. Recently, it was reported that ID1 can activate the NF-κB signaling pathway in a variety of cancer cells and a T cell line, but the mechanisms involved in ID1-mediated transactivation of NF-κB are not clear. In this study, we demonstrate by both in vitro pull-down assays and a cell-based in vivo two-hybrid system that ID1-mediated NF-κB activation is due to its physical interaction with p65. We have identified that the transcriptional activation domain (TAD) in p65 and the HLH domain in ID1 are vital for their interaction. Interestingly, a single site mutation (Leu76) in the HLH domain of ID1 protein drastically decreased its ability to bind with p65. Using a dual-luciferase assay, we demonstrated that the interaction between ID1 and p65 modulates activation of the NF-κB signaling pathway in vivo. In addition, we demonstrated that, by affecting the nuclear translocation of p65, ID1 is essential in regulating TNF-α-induced p65 recruitment to its downstream target, the cellular inhibitor of apoptosis protein 2 (cIAP2) promoter.

scholarly journals Bacillus subtilis Aconitase Is Required for Efficient Late-Sporulation Gene Expression

2006 ◽  
Vol 188 (17) ◽  
pp. 6396-6405 ◽  
Author(s):  
Alisa W. Serio ◽  
Kieran B. Pechter ◽  
Abraham L. Sonenshein
Keyword(s):  
Bacillus Subtilis ◽  
Transcriptional Activation ◽  
Rna Binding ◽  
Regulatory Protein ◽  
Binding Activity ◽  
High Catalytic Activity ◽  
Spore Coat ◽  
Mobility Shift ◽  
Gel Mobility Shift

ABSTRACT Bacillus subtilis aconitase, encoded by the citB gene, is homologous to the bifunctional eukaryotic protein IRP-1 (iron regulatory protein 1). Like IRP-1, B. subtilis aconitase is both an enzyme and an RNA binding protein. In an attempt to separate the two activities of aconitase, the C-terminal region of the B. subtilis citB gene product was mutagenized. The resulting strain had high catalytic activity but was defective in sporulation. The defect was at a late stage of sporulation, specifically affecting expression of σK-dependent genes, many of which are important for spore coat assembly and require transcriptional activation by GerE. Accumulation of gerE mRNA and GerE protein was delayed in the aconitase mutant strain. Pure B. subtilis aconitase bound to the 3′ untranslated region of gerE mRNA in in vitro gel mobility shift assays, strongly suggesting that aconitase RNA binding activity may stabilize gerE mRNA in order to allow efficient GerE synthesis and proper timing of spore coat assembly.

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