TRIM14 Is a Key Regulator of the Type I IFN Response during Mycobacterium tuberculosis Infection.

Tripartite motif-containing proteins (TRIMs) play a variety of recently described roles in innate immunity. Although many TRIMs regulate type I IFN expression following cytosolic nucleic acid sensing of viruses, their contribution to innate immune signaling and gene expression during bacterial infection remains largely unknown. Because Mycobacterium tuberculosis is an activator of cGAS-dependent cytosolic DNA sensing, we set out to investigate a role for TRIM proteins in regulating macrophage responses to M. tuberculosis In this study, we demonstrate that TRIM14, a noncanonical TRIM that lacks an E3 ubiquitin ligase RING domain, is a critical negative regulator of the type I IFN response in Mus musculus macrophages. We show that TRIM14 interacts with both cGAS and TBK1 and that macrophages lacking TRIM14 dramatically hyperinduce IFN stimulated gene (ISG) expression following M. tuberculosis infection, cytosolic nucleic acid transfection, and IFN-ß treatment. Consistent with a defect in resolution of the type I IFN response, Trim14 knockout macrophages have more phospho-Ser754 STAT3 relative to phospho-Ser727 and fail to upregulate the STAT3 target Socs3, which is required to turn off IFNAR signaling. These data support a model whereby TRIM14 acts as a scaffold between TBK1 and STAT3 to promote phosphorylation of STAT3 at Ser727 and resolve ISG expression. Remarkably, Trim14 knockout macrophages hyperinduce expression of antimicrobial genes like Nos2 and are significantly better than control cells at limiting M. tuberculosis replication. Collectively, these data reveal an unappreciated role for TRIM14 in resolving type I IFN responses and controlling M. tuberculosis infection.

Environmental Assessment of Enzyme Production and Purification.

The importance of bioprocesses has increased in recent decades, as they are considered to be more sustainable than chemical processes in many cases. E factors can be used to assess the sustainability of processes. However, it is noticeable that the contribution of enzyme synthesis and purification is mostly neglected. We, therefore, determined the E factors for the production and purification of 10 g enzymes. The calculated complete E factor including required waste and water is 37,835 gwaste·genzyme-1. This result demonstrates that the contribution of enzyme production and purification should not be neglected for sustainability assessment of bioprocesses.

Purification and biochemical characterisation of GlmU from Yersinia pestis.

Antibiotic resistance has emerged as a real threat to mankind, rendering many compounds ineffective in the fight against bacterial infection, including for significant diseases such as plague caused by Yersinia pestis. Essential genes have been identified as promising targets for inhibiting with new classes of compounds. Previously, the gene encoding the bifunctional UDP-N-acetylglucosamine pyrophosphorylase/glucosamine-1-phosphate N-acetyltransferase enzyme GlmU was confirmed as an essential gene in Yersinia. As a step towards exploiting this target for antimicrobial screening, we undertook a biochemical characterisation of the Yersinia GlmU. Effects of pH and magnesium concentration on the acetyltransferase and uridyltransferase activities were analysed, and kinetic parameters were determined. The acetyltransferase activity, which is strongly increased in the presence of reducing agent, was shown to be susceptible to oxidation and thiol-specific reagents.

Purification and functional characterization of diadenosine 5',5‴-P(1),P(4)-tetraphosphate phosphorylases from Mycobacterium smegmatis and Mycobacterium avium.

We recently demonstrated that the Rv2613c protein from Mycobacterium tuberculosis H37Rv is a novel diadenosine 5',5‴-P(1),P(4)-tetraphosphate (Ap4A) phosphorylase (MtAPA) that forms a tetramer. Mycobacterium avium and Mycobacterium smegmatis express proteins named MAV_3489 and MSMEG_2932, respectively, that are homologous to MtAPA. Here we showed that the MAV_3489 and MSMEG_2932 proteins possess Ap4A phosphorylase activity and enzymatic properties similar to those of MtAPA. Furthermore, gel-filtration column chromatography revealed that MAV_3489 and MSMEG_2932 assembled into homotetramers in solution, indicating that they may also form unique Ap4A-binding sites composed of tetramers.

Expression, Purification and Biophysical Characterisation of Klebsiella Pneumoniae Protein Adenylyltransferase: A Systematic Integration of Empirical and Computational Modelling Approaches.

Infections that are acquired due to a prolonged hospital stay and manifest 2 days following the admission of a patient to a health-care institution can be classified as hospital-acquired infections. Klebsiella pneumoniae (K. pneumoniae) has become a critical pathogen, posing serious concern globally due to the rising incidences of hypervirulent and carbapenem-resistant strains. Glutaredoxin is a redox protein that protects cells from oxidative stress as it associates with glutathione to reduce mixed disulfides. Protein adenylyltransferase (PrAT) is a pseudokinase with a proposed mechanism of transferring an AMP group from ATP to glutaredoxin. Inducing oxidative stress to the bacterium by inhibiting the activity of PrAT is a promising approach to combating its contribution to hospital-acquired infections. Thus, this study aims to overexpress, purify, and analyse the effects of ATP and Mg2+ binding to Klebsiella pneumoniae PrAT (KpPrAT). The pET expression system and nickel affinity chromatography were effective in expressing and purifying KpPrAT. Far-UV CD spectroscopy demonstrates that the protein is predominantly α-helical, even in the presence of Mg2+. Extrinsic fluorescence spectroscopy with ANS indicates the presence of a hydrophobic pocket in the presence of ATP and Mg2+, while mant-ATP studies allude to the potential nucleotide binding ability of KpPrAT. The presence of Mg2+ increases the thermostability of the protein. Isothermal titration calorimetry provides insight into the binding affinity and thermodynamic parameters associated with the binding of ATP to KpPrAT, with or without Mg2+. Conclusively, the presence of Mg2+ induces a conformation in KpPrAT that favours nucleotide binding.

Expression, purification, crystallization and preliminary X-ray analysis of ribitol-5-phosphate cytidylyltransferase from Bacillus subtilis.

TarI is a ribitol-5-phosphate cytidylyltransferase that catalyzes the formation of CDP-ribitol, which is involved in the biosynthesis of wall teichoic acids, from CTP and ribitol 5-phosphate. TarI from Bacillus subtilis (BsTarI) was purified and crystallized using the sitting-drop vapour-diffusion method. The crystals diffracted to a resolution of 1.78 Šand belonged to the monoclinic space group C2, with unit-cell parameters a = 103.74, b = 60.97, c = 91.80 Å, ß = 113.48°. The initial structural model indicated that the crystals of BsTarI contained a dimer in the asymmetric unit.

Comparative kinetic analysis of glycerol 3-phosphate cytidylyltransferase from Enterococcus faecalis and Listeria monocytogenes.

BACKGROUND: Glycerol 3-phosphate cytidylyltransferase (GCT) is an enzyme central to the synthesis of teichoic acids, components of the cell wall in gram positive bacteria. Catalysis by GCT from Enterococcus faecalis and Listeria monocytogenes has been investigated and catalytic properties compared. MATERIAL/METHODS: The genes encoding GCT were cloned from genomic DNA and recombinant proteins expressed in E. coli and purified. Enzyme assays were used to determine kinetic constants kcat and Km. Chemical crosslinking provided a means to assess quaternary structure of each GCT. RESULTS: Recombinant Enterococcus faecalis GCT had an apparent kcat value of 1.51 s⁻¹ and apparent Km values of 2.42 mM and 4.03 mM with respect to substrates cytidine 5'-triphosphate (CTP) and glycerol phosphate. Listeria monocytogenes GCT had an apparent kcat value of 4.15 s⁻¹ and apparent Km values of 1.52 mM and 6.56 mM with respect to CTP and glycerol phosphate. This resulted in kcat/Km values of 0.62 s⁻¹mM⁻¹ and 0.37 s⁻¹mM⁻¹ for E. faecalis GCT and 2.73 s⁻¹mM⁻¹ and 0.63 s⁻¹mM⁻¹ for L. monocytogenes GCT with respect to CTP and glycerol phosphate, respectively. CONCLUSIONS: The genome of both Enterococcus faecalis and Listeria monocytogenes contain a gene that encodes a functional GCT. The genes are 67% identical at the nucleotide level and the encoded proteins exhibit a 63% amino acid identity. The purified, recombinant enzymes each appear to be dimeric and display similar kinetic characteristics. Studying the catalytic characteristics of GCT isoforms from pathogenic bacteria provides information important for the future development of potential antibacterial agents.

Role of key residues at the flavin mononucleotide (FMN):adenylyltransferase catalytic site of the bifunctional riboflavin kinase/flavin adenine dinucleotide (FAD) Synthetase from Corynebacterium ammoniagenes.

In mammals and in yeast the conversion of Riboflavin (RF) into flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) is catalysed by the sequential action of two enzymes: an ATP:riboflavin kinase (RFK) and an ATP:FMN adenylyltransferase (FMNAT). However, most prokaryotes depend on a single bifunctional enzyme, FAD synthetase (FADS), which folds into two modules: the C-terminal associated with RFK activity and the N-terminal associated with FMNAT activity. Sequence and structural analysis suggest that the 28-HxGH-31, 123-Gx(D/N)-125 and 161-xxSSTxxR-168 motifs from FADS must be involved in ATP stabilisation for the adenylylation of FMN, as well as in FAD stabilisation for FAD phyrophosphorolysis. Mutants were produced at these motifs in the Corynebacterium ammoniagenes FADS (CaFADS). Their effects on the kinetic parameters of CaFADS activities (RFK, FMNAT and FAD pyrophosphorilase), and on substrates and product binding properties indicate that H28, H31, N125 and S164 contribute to the geometry of the catalytically competent complexes at the FMNAT-module of CaFADS.

Heterologous production, purification and characterization of enzymatically active Sindbis virus nonstructural protein nsP1.

Alphavirus nonstructural protein nsP1 possesses distinct methyltransferase (MTase) and guanylyltransferase (GTase) activities involved in the capping of viral RNAs. In alphaviruses, the methylation of GTP occurs before RNA transguanylation and nsP1 forms a covalent complex with m(7)GMP unlike the host mRNA guanylyltransferase which forms GMP-enzyme complex. In this study, full length SINV nsP1 was expressed in a soluble form with an N-terminal histidine tag in Escherichia coli and purified to homogeneity. The purified protein is enzymatically active and contains both MTase and GTase activity indicating that SINV nsP1 does not require membrane association for its enzymatic function. Biochemical analysis shows that detergents abolish nsP1 GTase activity, whereas nonionic detergents do not affect MTase activity. Furthermore, SINV nsP1 contains the metal-ion dependent GTase, whereas MTase does not require a metal ion. Circular dichroism spectroscopic analysis of purified protein indicate that nsP1 has a mixed α/ß structure and is in the folded native conformation.

Expression, purification, crystallization and preliminary X-ray crystallographic analysis of the hyperthermophilic nucleotidyltransferase TTHA1015 from Thermus thermophilus HB8.

The TTHA1015 gene from Thermus thermophilus HB8 encodes a hyperthermophilic nucleotidyltransferase. TTHA1015 has high homology to proteins belonging to two related families: the nucleotidyltransferase-domain superfamily and the DNA polymerase ß-like family. However, no crystal structures of these proteins have been reported. Determination of the crystal structure of TTHA1015 will help in elucidation of its function and will be useful for understanding the relationship between the structure and the function of these homologous proteins. In this study, TTHA1015 was expressed, purified and crystallized. X-ray diffraction data were collected to 1.70 Šresolution. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a=65.5, b=34.7, c=42.4 Å, ß=119.1°. There was one molecule per asymmetric unit, giving a Matthews coefficient of 1.86 Å3 Da(-1) and an approximate solvent content of 34%.

Archaeal RibL: a new FAD synthetase that is air sensitive.

FAD synthetases catalyze the transfer of the AMP portion of ATP to FMN to produce FAD and pyrophosphate (PP(i)). Monofunctional FAD synthetases exist in eukaryotes, while bacteria have bifunctional enzymes that catalyze both the phosphorylation of riboflavin and adenylation of FMN to produce FAD. Analyses of archaeal genomes did not reveal the presence of genes encoding either group, yet the archaea contain FAD. Our recent identification of a CTP-dependent archaeal riboflavin kinase strongly indicated the presence of a monofunctional FAD synthetase. Here we report the identification and characterization of an archaeal FAD synthetase. Methanocaldococcus jannaschii gene MJ1179 encodes a protein that is classified in the nucleotidyl transferase protein family and was previously annotated as glycerol-3-phosphate cytidylyltransferase (GCT). The MJ1179 gene was cloned and its protein product heterologously expressed in Escherichia coli. The resulting enzyme catalyzes the adenylation of FMN with ATP to produce FAD and PP(i). The MJ1179-derived protein has been designated RibL to indicate that it follows the riboflavin kinase (RibK) step in the archaeal FAD biosynthetic pathway. Aerobically isolated RibL is active only under reducing conditions. RibL was found to require divalent metals for activity, the best activity being observed with Co(2+), where the activity was 4 times greater than that with Mg(2+). Alkylation of the two conserved cysteines in the C-terminus of the protein resulted in complete inactivation. RibL was also found to catalyze cytidylation of FMN with CTP, making the modified FAD, flavin cytidine dinucleotide (FCD). Unlike other FAD synthetases, RibL does not catalyze the reverse reaction to produce FMN and ATP from FAD and PP(i). Also in contrast to other FAD synthetases, PP(i) inhibits the activity of RibL.

Purification and molecular characterization of a novel diadenosine 5',5'''-P(1),P(4)-tetraphosphate phosphorylase from Mycobacterium tuberculosis H37Rv.

In this study, Rv2613c, a protein that is encoded by the open reading frame Rv2613c in Mycobacterium tuberculosis H37Rv, was expressed, purified, and characterized for the first time. The amino acid sequence of Rv2613c contained a histidine triad (HIT) motif consisting of H-phi-H-phi-H-phi-phi, where phi is a hydrophobic amino acid. This motif has been reported to be the characteristic feature of several diadenosine 5',5'''-P(1),P(4)-tetraphosphate (Ap4A) hydrolases that catalyze Ap4A to adenosine 5'-triphosphate (ATP) and adenosine monophosphate (AMP) or 2 adenosine 5'-diphosphate (ADP). However, enzymatic activity analyses for Rv2613c revealed that Ap4A was converted to ATP and ADP, but not AMP, indicating that Rv2613c has Ap4A phosphorylase activity rather than Ap4A hydrolase activity. The Ap4A phosphorylase activity has been reported for proteins containing a characteristic H-X-H-X-Q-phi-phi motif. However, no such motif was found in Rv2613c. In addition, the amino acid sequence of Rv2613c was significantly shorter compared to other proteins with Ap4A phosphorylase activity, indicating that the primary structure of Rv2613c differs from those of previously reported Ap4A phosphorylases. Kinetic analysis revealed that the K(m) values for Ap4A and phosphate were 0.10 and 0.94mM, respectively. Some enzymatic properties of Rv2613c, such as optimum pH and temperature, and bivalent metal ion requirement, were similar to those of previously reported yeast Ap4A phosphorylases. Unlike yeast Ap4A phosphorylases, Rv2613c did not catalyze the reverse phosphorolysis reaction. Taken together, it is suggested that Rv2613c is a unique protein, which has Ap4A phosphorylase activity with an HIT motif.

Archaeal RNA ligase is a homodimeric protein that catalyzes intramolecular ligation of single-stranded RNA and DNA.

RNA ligases participate in repair, splicing and editing pathways that either reseal broken RNAs or alter their primary structure. Here, we report the characterization of an RNA ligase from the thermophilic archaeon, Methanobacterium thermoautotrophicum. The 381-amino acid Methanobacterium RNA ligase (MthRnl) catalyzes intramolecular ligation of 5'-PO(4) single-strand RNA to form a covalently closed circular RNA molecule through ligase-adenylylate and RNA-adenylylate (AppRNA) intermediates. At the optimal temperature of 65 degrees C, AppRNA was predominantly ligated to a circular product. In contrast, at 35 degrees C, phosphodiester bond formation was suppressed and the majority of the AppRNA was deadenylylated. Sedimentation analysis indicates that MthRnl is a homodimer in solution. The C-terminal 127-amino acid segment is required for dimerization, is itself capable of oligomeization and acts in trans to inhibit the ligation activity of native MthRnl. MthRnl can also join single-stranded DNA to form a circular molecule. The lack of specificity for RNA and DNA by MthRnl may exemplify an undifferentiated ancestral stage in the evolution of ATP-dependent ligases.

Efficient production of uridine 5'-diphospho-N-acetylglucosamine by the combination of three recombinant enzymes and yeast cells.

Uridine 5'-diphospho N-acetylglucosamine (UDP-GlcNAc) is an important nucleotide sugar in the biochemistry of all living organisms, and it is an important substrate in the synthesis of oligosaccharides. In the present work, three bioactive enzymes, namely, glucokinase (YqgR), GlcNAc-phosphate mutase (Agm1), and N-acetylglucosamine-1-phosphate uridyltransferase (GlmU), were produced effectively as soluble form in recombinant Escherichia coli. These three enzymes and dried yeast together were used to construct a multistep enzymatic system, which could produce UDP-GlcNAc efficiently with N-acetylglucosamine (GlcNAc) as the substrate. After the optimization of various reaction conditions, 31.5 mMUDP-GlcNAc was produced from 50 mMGlcNAc and 50 mMUMP.

Phosphomannose isomerase/GDP-mannose pyrophosphorylase from Pyrococcus furiosus: a thermostable biocatalyst for the synthesis of guanidinediphosphate-activated and mannose-containing sugar nucleotides.

Herein we present an analysis of the chemical function of a recombinant bifunctional phosphomannose isomerase/GDP-mannose pyrophosphorylase (manC) from Pyrococcus furiosus DSM 3638 and its use in the synthesis of guanidinediphospho-hexoses and a range of nucleotidediphospho-mannoses. This enzyme is unusually promiscuous in both its nucleotide triphosphate (NTP) and sugar-1-phosphate acceptance. It accepts all five naturally occurring NTPs (ATP, CTP, GTP, dTTP and UTP) and a range of sugar-1-phosphates (glucose-, mannose-, galactose-, glucosamine-, N-acetylglucosamine- and fucose-1-phosphate). A truncated GDP-mannose pyrophosphorylase domain of the whole length enzyme showed almost 100-fold less sugar nucleotidyltransferase activity with only GTP and mannose 1-phosphate as substrates. The temperature stability and inherently broad substrate tolerance of this archaeal enzyme make it an effective reagent for the rapid chemoenzymatic synthesis of a range of natural and unnatural sugar nucleotides that are challenging to make by chemical means alone.

Transcriptional regulation of human cyclic GMP-AMP synthase gene.

Cyclic GMP-AMP synthase (cGAS, cGAMP synthase) plays crucial roles in autoimmune disease, anti-tumor response, anti-senescence and anti-inflammatory response. Many studies have focused on cGAS-mediated signaling pathway. However, transcriptional mechanisms of cGAS gene have remained largely unknown. Here, we cloned the cGAS promoter region and characterized the molecular mechanisms controlling the cGAS transcriptional activity. By a series of 5' deletion and promoter constructions, we showed that the region (-414 to +76 relatives to the transcription start site) was sufficient for promoter activity. Mutation of Sp1 and CREB binding sites in this promoter region led to an apparent reduction of the cGAS promoter activity. Overexpression of Sp1 and CREB could obviously enhance promoter activity, whereas knocking-down of endogenous Sp1 and CREB markedly restrained the cGAS promoter activity. Sp1 and CREB binding to the cGAS promoter region in vivo was verified by Chromatin immunoprecipitation assay. These results pointed out that transcription factors Sp1 and CREB regulate the transcription of the cGAS gene.

Development of human cGAS-specific small-molecule inhibitors for repression of dsDNA-triggered interferon expression.

Cyclic GMP-AMP synthase (cGAS) is the primary sensor for aberrant intracellular dsDNA producing the cyclic dinucleotide cGAMP, a second messenger initiating cytokine production in subsets of myeloid lineage cell types. Therefore, inhibition of the enzyme cGAS may act anti-inflammatory. Here we report the discovery of human-cGAS-specific small-molecule inhibitors by high-throughput screening and the targeted medicinal chemistry optimization for two molecular scaffolds. Lead compounds from one scaffold co-crystallize with human cGAS and occupy the ATP- and GTP-binding active site. The specificity and potency of these drug candidates is further documented in human myeloid cells including primary macrophages. These novel cGAS inhibitors with cell-based activity will serve as probes into cGAS-dependent innate immune pathways and warrant future pharmacological studies for treatment of cGAS-dependent inflammatory diseases.

Expression, purification and preliminary crystallographic analysis of N-acetylglucosamine-1-phosphate uridylyltransferase from Mycobacterium tuberculosis.

The gene product of open reading frame Rv1018c from Mycobacterium tuberculosis is annotated as encoding a probable N-acetylglucosamine 1-phosphate uridylyltransferase (MtbGlmU), an enzyme that catalyzes the biosynthesis of UDP-N-acetylglucosamine, a precursor common to lipopolysaccharide and peptidoglycan biosynthesis. Following overexpression in Escherichia coli, the enzyme was purified and crystallized using the hanging-drop vapour-diffusion method. Native diffraction data were collected from crystals belonging to space group R32 and processed to a resolution of 2.2 A.

Functional analysis of the Burkholderia cenocepacia J2315 BceAJ protein with phosphomannose isomerase and GDP-D-mannose pyrophosphorylase activities.

The bceA(J) gene from the cystic fibrosis isolate Burkholderia cenocepacia J2315 encodes a 56-kDa bifunctional protein, with phosphomannose isomerase (PMI) and guanosine diphosphate (GDP)-mannose pyrophosphorylase (GMP) activities, a new member of the poorly characterised type II PMI class of proteins. Due to the lack of homology between the type II PMIs and the human PMI, this class of proteins are being regarded as interesting potential targets to develop new antimicrobials. The BceA(J) protein conserves the four typical motifs of type II PMIs: the pyrophosphorylase signature, the GMP active site, the PMI active site and the zinc-binding motif. After overproduction of BceA(J) by Escherichia coli as a histidine tag derivative, the protein was purified to homogeneity by affinity chromatography. The GMP activity is dependent on the presence of Mg(2+) or Ca(2+) as cofactors, while the PMI activity uses a broader range of divalent ions, in the order of activation Mg(2+) > Ca(2+) > Mn(2+) > Co(2+) > Ni(2+). The kinetic parameters K(m), V(max) and K(cat)/K(m) for the PMI and GMP activities were determined. Results suggest that the enzyme favours the formation of GDP-mannose instead of mannose catabolism, thus channelling precursors to the formation of glycoconjugates.

Resistance to Aminoglycosides of Methicillin-Resistant Strains of Staphylococcus aureus, Originating in the Surgical and Transplantation Wards of the Warsaw Clinical Center-A Retrospective Analysis.

INTRODUCTION: Aminoglycoside resistance (AR) is common in health care-associated methicillin-resistant Staphylococcus aureus (HA-MRSA). AR is most often associated with the production of antibiotic modifying enzymes: bidomain AAC(6')-Ie/APH(2″)-Ia acetyltransferase and phosphotransferase, ANT(4')-Ia nucleotidyltransferase, and APH(3″)-IIIa phosphotransferase. AIM: Determination of aminoglycoside sensitivity, presence of genes encoding enzymes, and molecular typing of HA-MRSA strains derived from patients hospitalized in surgical and transplantation wards. MATERIALS AND METHODS: Fifty-four HA-MRSA strains, isolated from various materials from patients in the surgical and transplantation wards of Warsaw's clinical hospital, hospitalized between 1991 and 2007. The MIC values of gentamicin-GEN/tobramycin-TOB/amikacin-AK/netilmicin-NET were determined by the E-test (CLSI/EUCAST). Genes mecA/aacA-aphD/aadD/aph(3″)-IIIa were detected using PCR. SCCmec types were determined according to the Oliveira method and the sequence type (ST)/clonal complex (CC) by the MLST method. RESULTS: Of the isolates tested, 36 (66.7%) showed resistance to at least one aminoglycoside: TOB (57.4%), GEN (53.7%), AK (55.6%), NET (24.1%). The aacA-aphD gene was present in 29 MRSA-GEN-R (most often in combination with aadD, 15/29 or aph(3″)-IIIa, 10/29); the aacA-aphD gene was the only determinant of resistance in 1 isolate. The AR variants mainly belonged to the CC8 clonal complex (ST239/247/241/254/8) and most frequently contained SCCmec type III (3A) cassettes. CONCLUSIONS: Resistance to at least one aminoglycoside was present in 66.7% of HA-MRSA and in more than 22% to all of them. The presence of the aacA-aphD gene was sufficient to express the resistance phenotype to GEN/TOB/AK/NET. Resistant isolates were closely related to each other.