Escherichia coli K1 induces pterin production for enhanced expression of Fcγ receptor I to invade RAW 264.7 macrophages

Macrophages serve as permissive niches for Escherichia coli (E. coli) K1 to attain high grade bacteremia in the pathogenesis of meningitis in neonates. Although pterin levels are a diagnostic marker for immune activation, the role of macrophages in pterin production and in the establishment of meningitis is unknown. Here, we demonstrate that macrophages infected with E. coli K1 produce both neopterin and biopterin through increased expression of GTP-cyclohydrolase 1 (GCH1). Of note, increased production of biopterin enhances the expression of Fc-gamma receptor I (CD64), which in turn, aided the entry of E. coli K1 in macrophages while increased neopterin suppresses reactive oxygen species (ROS), thereby aiding bacterial survival. Inhibition of GCH1 by 2, 4-Diamino-6-hydroxypyrimidine (DAHP) prevented the E. coli K1 induced expression of CD64 in macrophages in vitro and the development of bacteremia in a newborn mouse model of meningitis. These studies suggest that targeting GCH1 could be therapeutic strategy for preventing neonatal meningitis by E. coli K1.     

Co-evolution of cytochrome c 6 and plastocyanin, mobile proteins transferring electrons from cytochrome b 6f to photosystem I

 Cytochrome c ₆ and plastocyanin are soluble metalloproteins that act as mobile carriers transferring electrons between the two membrane-embedded photosynthetic complexes cytochrome b ₆ f and photosystem I (PSI). First, an account of recent data on structural and functional features of these two membrane complexes is presented. Afterwards, attention is focused on the mobile heme and copper proteins – and, in particular, on the structural factors that allow recognition and confer molecular specificity and control the rates of electron transfer from and to the membrane complexes. The interesting question of why plastocyanin has been chosen over the ancient heme protein is discussed to place emphasis on the evolutionary aspects. In fact, cytochrome c ₆ and plastocyanin are presented herein as an excellent case study of biological evolution, which is not only convergent (two different structures but the same physiological function), but also parallel (two proteins adapting themselves to vary accordingly to each other within the same organism). Received: 4 July 1996 / Accepted: 16 September 1996     

Charged nylon membrane substrate for convenient and versatile high resolution microscopic analysis of Escherichia coli & mammalian cells in suspension culture

Preparation of isolated cells and microorganisms for ultrastructural examination always provides a challenge in terms of adequate immobilization of the cells and prevention of subsequent sample loss and damage during various steps of sample processing. Using a positively charged nylon membrane substrate we demonstrate that it is possible to easily immobilize and retain a sample of isolated cells in culture for a wide variety of microscopy-based techniques. Radiolabelled E. coli cells when immobilized on the charged membrane were seen to be highly resistant to detachment when subjected to the normal sample processing procedures associated with microscopy. In contrast cells on regular millipore membranes were rapidly lost during sample preparation. We demonstrate the utility of charged nylon membranes for a wide variety of microscopy based analysis including scanning and transmission electron microscopy (SEM and TEM), atomic force microscopy (AFM), TEM based immunogold labelling, laser confocal microscopy and SEM based elemental analysis.     

Generation and characterization of an inter-generic bivalent alpha domain fusion protein αCS from Clostridium perfringens and Staphylococcus aureus for concurrent diagnosis and therapeutic applications

Aim: To evaluate an inter‐generic recombinant alpha domain fusion protein for simultaneous detection and neutralization of Clostridium perfringens and Staphylococcus aureus alpha toxins. Methods and Results: Truncated portions of clostridial and staphylococcal alpha haemolysin genes were PCR amplified and linked to each other through a hydrophilic flexible Glycine linker sequence using overlap‐extension PCR to form a chimeric gene αCS. The recombinant αCS fusion protein was expressed and characterized for its toxicity, cell binding capacity and haemolysis inhibition properties. The fusion protein was nontoxic and effectively retarded staphylococcal alpha haemolysis, probably by competitively interacting with putative staphylococcal alpha haemolysin receptors on erythrocytes. Murine hyperimmune polysera raised against r‐αCS specifically detected 42‐kDa and 33‐kDa proteins when culture supernatants of Cl. perfringens (clostridial alpha toxin) and Staph. aureus (staphylococcal alpha toxin), respectively, were analysed in Western blot. The polyclonal antisera effectively diminished the haemolytic action of both the wild‐type toxins in vitro. Conclusions: The r‐αCS fusion protein was nontoxic competitive inhibitor of staphylococcal alpha haemolysin. The protein elicited specific immune response against Cl. perfringens and Staph. aureus alpha toxins. The antisera also neutralized the toxicities of both the native wild‐type toxins in vitro. Significance of the Study: The bivalent recombinant αCS protein could be a novel intervention in the field of diagnostics and therapeutics against Cl. perfringens and Staph. aureus infections, particularly, in case of co‐infections like gangrenous ischaemia, gangrenous mastitis, etc.     

Lipid–protein nanodiscs for cell-free production of integral membrane proteins in a soluble and folded state: Comparison with detergent micelles,bicelles and liposomes

Production of integral membrane proteins (IMPs) in a folded state is a key prerequisite for their functional and structural studies. In cell-free (CF) expression systems membrane mimicking components could be added to the reaction mixture that promotes IMP production in a soluble form. Here lipid–protein nanodiscs (LPNs) of different lipid compositions (DMPC, DMPG, POPC, POPC/DOPG) have been compared with classical membrane mimicking media such as detergent micelles, lipid/detergent bicelles and liposomes by their ability to support CF synthesis of IMPs in a folded and soluble state. Three model membrane proteins of different topology were used: homodimeric transmembrane (TM) domain of human receptor tyrosine kinase ErbB3 (TM-ErbB3, 1TM); voltage-sensing domain of K⁺ channel KvAP (VSD, 4TM); and bacteriorhodopsin from Exiguobacterium sibiricum (ESR, 7TM). Structural and/or functional properties of the synthesized proteins were analyzed. LPNs significantly enhanced synthesis of the IMPs in a soluble form regardless of the lipid composition. A partial disintegration of LPNs composed of unsaturated lipids was observed upon co-translational IMP incorporation. Contrary to detergents the nanodiscs resulted in the synthesis of ~ 80% active ESR and promoted correct folding of the TM-ErbB3. None of the tested membrane mimetics supported CF synthesis of correctly folded VSD, and the protocol of the domain refolding was developed. The use of LPNs appears to be the most promising approach to CF production of IMPs in a folded state. NMR analysis of ¹⁵N-Ile-TM-ErbB3 co-translationally incorporated into LPNs shows the great prospects of this membrane mimetics for structural studies of IMPs produced by CF systems.     

Expression,purification, and characterization of N-terminal His-tagged proteins with mutations in zinc finger 3 of zinc finger protein ZNF191(243–368)

Abstract

Zinc finger protein ZNF191(243–368), the zinc finger region of ZNF191, is potentially associated with cell proliferation in hepatocellular carninoma. A His-tag expression system was used to express and purify proteins with mutations in the zinc finger 3 of ZNF191(243–368) for analysis of protein properties, structure, and functions. The purification of the His-tag fusion proteins was simpler and faster than that of the ZNF191(243–368) inclusion bodies. The properties and structures of the His-tag fusion mutant proteins were investigated using spectrographic techniques and DNA hydrolysis experiment. The His₆-tag system could be used to express ZNF191(243–368). The presence of the His₆-tag at the N-terminus of ZNF191(243–368) did not evidently affect its properties and structure. However, the site-directed mutations in zinc finger 3 affected the structure of the protein. The DNA hydrolase activity of His₆-ZF-F3/H4 suggested that four histidines in zinc finger 3 might form a structure similar to that of the active center in a hydrolase. This work reports that continuous histidines need to form a certain structure for specific functions, and provides new insights into the design of an artificial nuclease.     

Expression,purification, and evaluation of in vivo anti-fibrotic activity for soluble truncated TGF-β receptor II as a cleavable His-SUMO fusion protein

Excessive production of transforming growth factor-β1 (TGF-β1) and its binding to transforming growth factor-β receptor type II (TGF-βRII) promotes fibrosis by activation of the TGF-β1-mediated signaling pathway. Thus, the truncated extracellular domain of TGF-βRII (tTβRII) is a promising anti-fibrotic candidate, as it lacks the signal transduction domain. In this work, the native N-terminal tTβRII was prepared as a His-SUMO fusion protein (termed His-SUMO-tTβRII) in Escherichia coli strain BL21 (DE3). His-SUMO-tTβRII was expressed as a soluble protein under optimal conditions (6 h of induction with 0.5 mM IPTG at 37 °C). His-SUMO-tTβRII was purified by Ni–NTA resin chromatography, and then cleaved with SUMO protease to release native tTβRII, which was re-purified using a Ni–NTA column. Approximately 12 mg of native tTβRII was obtained from a one liter fermentation culture with no less than 95% purity. In vivo studies demonstrated that tTβRII prevented CCl₄-induced liver fibrosis, as evidenced by the inhibition of fibrosis-related Col I and α-SMA protein expression in C57BL/6 mice. In addition, tTβRII downregulated phosphorylation of SMAD2/3, which partly repressed TGF-β1-mediated signaling. These data indicate that the His-SUMO expression system is an efficient approach for preparing native tTβRII that possesses anti-liver fibrotic activity, allowing for the large-scale production of tTβRII, which potentially could serve as an anti-fibrotic candidate for treatment of TGF-β1-related diseases.     

A new membrane-bound OprI lipoprotein expression vector: High production of heterologous fusion proteins in Gram (−) bacteria and the implications for oral vaccination

We have previously described the development of cloning vectors for the production of OprI-based outer membrane fusion proteins in E. coli (Cornelis et al., 1996) and now describe the construction of a new vector, containing a lacI^q gene, resulting in tight repression of the promotor and allowing its use in other Gram (−) bacteria. The new pVUB3 expression vector encodes a truncated but active LacI^q′(341) repressor which binds to the single operator in the vector. A high repression of the trc promotor was observed, resulting in a very low basal leakage of expression and very high production levels of OprI or derivatives after IPTG induction in E. coli. Bacterial viability was not affected under uninduced conditions, but the number of viable cell counts decreased after production of large amounts of the outer membrane-bound OprI lipoprotein and its derivatives, both in E. coli and Salmonella typhimurium. This highly repressible system allows us to extend the use of OprI vectors in other Gram (−) bacteria, resulting in the production of outer membrane-bound lipid-modified molecules, opening the possibility for its application in the design of potential live Salmonella-based subunit vaccines.     

Evaluation of a double synergy differential test (DSDT) for differential detection of ESBL and AmpC-type β-lactamases in Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis

This work describes a simple and practical double synergy differential test (DSDT) that couples the detection of ESBLs and AmpC-type enzymes by means of a combo-disk approach using cefotaxime and ceftazidime as indicator substrates, and clavulanate and boronic acid as enzyme inhibitors. The DSDT was tested with a collection of 118 Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis strains with different beta-lactamase profiles, and proved to be highly sensitive and specific for the detection of ESBL and AmpC-producing isolates.     

Engineering Escherichia coli BL21 (DE3) for high-yield production of germacrene A,a precursor of β-elemene via combinatorial metabolic engineering strategies

β-elemene is one of the most commonly used antineoplastic drugs in cancer treatment. As a plant-derived natural chemical, biologically engineering microorganisms to produce germacrene A to be converted to β-elemene harbors great expectations since chemical synthesis and plant isolation methods come with their production deficiencies. In this study, we report the design of an Escherichia coli cell factory for the de novo production of germacrene A to be converted to β-elemene from a simple carbon source. A series of systematic approaches of engineering the isoprenoid and central carbon pathways, translational and protein engineering of the sesquiterpene synthase, and exporter engineering yielded high-efficient β-elemene production. Specifically, deleting competing pathways in the central carbon pathway ensured the availability of acetyl-coA, pyruvate, and glyceraldehyde-3-phosphate for the isoprenoid pathways. Adopting lycopene color as a high throughput screening method, an optimized NS^Y305N was obtained via error-prone polymerase chain reaction mutagenesis. Further overexpression of key pathway enzymes, exporter genes, and translational engineering produced 1161.09 mg/L of β-elemene in a shake flask. Finally, we detected the highest reported titer of 3.52 g/L of β-elemene and 2.13 g/L germacrene A produced by an E. coli cell factory in a 4-L fed-batch fermentation. The systematic engineering reported here generally applies to microbial production of a broader range of chemicals. This illustrates that rewiring E. coli central metabolism is viable for producing acetyl-coA-derived and pyruvate-derived molecules cost-effectively.     

Transient metabolic modeling of Escherichia coli MG1655 and MG1655 ΔackA-pta, ΔpoxB Δpppc ppc-p37 for recombinant β-galactosidase production

Escherichia coli is one of the most widely used hosts for the production of recombinant proteins, among other reasons because its genetics are far better characterized than those of any other microorganism. To improve the understanding of recombinant protein synthesis in E. coli, the production of a model recombinant protein, β-galactosidase, was studied in response to the constitutive overexpression of the anaplerotic reaction afforded by PEP carboxylase. To this end, an IPTG wash-in experiment was performed starting from a well-defined steady-state condition for both the wild-type E. coli and a mutant with a defective acetate pathway and a constitutively overexpressed ppc. In order to compare the dynamics of the fluxes over time during the wash-in experiment, a method referred to as transient metabolic flux analysis, which is based on steady-state metabolic flux analysis, was used. This allowed us to track the intracellular changes/fluxes in both strains. It was observed that the flux towards fermentation products was 3.6 times lower in the ppc overexpression mutant compared to the wild-type E. coli. In the former on the other hand, the PPC flux is in general higher. In addition, the flux towards β-galactosidase was higher (12.4 times), resulting in five times more protein activity. These results indicate that by constitutively overexpressing the anaplerotic ppc gene in E. coli, the TCA cycle intermediates are increasingly replenished. The additional supply of these protein precursors has a positive result on recombinant protein production.     

Molecular Cloning and Expression of the hyu Genes from Microbacterium liquefaciens AJ 3912, Responsible for the Conversion of 5-Substituted Hydantoins to α-Amino Acids,in Escherichia coli

A DNA fragment from Microbacterium liquefaciens AJ 3912, containing the genes responsible for the conversion of 5-substituted-hydantoins to α-amino acids, was cloned in Escherichia coli and sequenced. Seven open reading frames (hyuP, hyuA, hyuH, hyuC, ORF1, ORF2, and ORF3) were identified on the 7.5 kb fragment. The deduced amino acid sequence encoded by the hyuA gene included the N-terminal amino acid sequence of the hydantoin racemase from M. liquefaciens AJ 3912. The hyuA, hyuH, and hyuC genes were heterologously expressed in E. coli; their presence corresponded with the detection of hydantoin racemase, hydantoinase, and N-carbamoyl α-amino acid amido hydrolase enzymatic activities respectively. The deduced amino acid sequences of hyuP were similar to those of the allantoin (5-ureido-hydantoin) permease from Saccharomyces cerevisiae, suggesting that hyuP protein might function as a hydantoin transporter.     

Efficient protocol for backbone and side-chain assignments of large, intrinsically disordered proteins: transient secondary structure analysis of 49.2 kDa microtubule associated protein 2c

Microtubule-associated proteins (MAPs) are abundantly present in axons and dendrites, and have been shown to play crucial role during the neuronal morphogenesis. The period of main dendritic outgrowth and synaptogenesis coincides with high expression levels of one of MAPs, the MAP2c, in rats. The MAP2c is a 49.2 kDa intrinsically disordered protein. To achieve an atomic resolution characterization of such a large protein, we have developed a protocol based on the acquisition of two five-dimensional ¹³C-directly detected NMR experiments. Our previously published 5D CACONCACO experiment (Nová?ek et al. in J Biomol NMR 50(1):1–11, 2011) provides the sequential assignment of the backbone resonances, which is not interrupted by the presence of the proline residues in the amino acid sequence. A novel 5D HC(CC-TOCSY)CACON experiment facilitates the assignment of the aliphatic side chain resonances. To streamline the data analysis, we have developed a semi-automated procedure for signal assignments. The obtained data provides the first atomic resolution insight into the conformational state of MAP2c and constitutes a model for further functional studies of MAPs.     

Expression,purification and characterization of soluble human interleukin-6 receptor α-chain and its mutant protein in <Emphasis Type="Italic">Escherichia coli</Emphasis>

To investigate the function of the N-terminal immunoglobulin (Ig)-like domain of the human interleukin-6 receptor α-chain (hIL-6R), we constructed a soluble human interleukin-6 receptor (shIL-6R) (named EC05, amino acids 20–354) and soluble variants of the shIL-6R lacking the Ig-like domain (named EC70, amino acids 105–354). The two extracellular portions of hIL-6R were expressed as soluble fusion proteins with thioredoxin in Escherichia coli and purified by using Ni-NTA agarose. Western blot showed that purified proteins were immunoreactive with the antibody against hIL-6R. They also possessed specific binding activity with human interleukin-6 (hIL-6) in ELISA analysis. Jing Wang and Zhenhui Yang contributed equally to this work.