Jufang Wang

A chimeric toxin vaccine protects against primary and recurrent Clostridium difficile infection.

ABSTRACT The global emergence of Clostridium difficile infection (CDI) has contributed to the recent surge in severe antibiotic-associated diarrhea and colonic inflammation. C. difficile produces two homologous glucosylating exotoxins, TcdA and TcdB, both of which are pathogenic and require neutralization to prevent disease occurrence. However, because of their large size and complex multifunctional domain structures, it has been a challenge to produce native recombinant toxins that may serve as vaccine candidates. Here, we describe a novel chimeric toxin vaccine that retains major neutralizing epitopes from both toxins and confers complete protection against primary and recurrent CDI in mice. Using a nonpathogenic Bacillus megaterium expression system, we generated glucosyltransferase-deficient holotoxins and demonstrated their loss of toxicity. The atoxic holotoxins induced potent antitoxin neutralizing antibodies showing little cross-immunogenicity or protection between TcdA and TcdB. To facilitate simultaneous protection against both toxins, we generated an active clostridial toxin chimera by switching the receptor binding domain of TcdB with that of TcdA. The toxin chimera was fully cytotoxic and showed potent proinflammatory activities. This toxicity was essentially abolished in a glucosyltransferase-deficient toxin chimera, cTxAB. Parenteral immunization of mice or hamsters with cTxAB induced rapid and potent neutralizing antibodies against both toxins. Complete and long-lasting disease protection was conferred by cTxAB vaccinations against both laboratory and hypervirulent C. difficile strains. Finally, prophylactic cTxAB vaccination prevented spore-induced disease relapse, which constitutes one of the most significant clinical issues in CDI. Thus, the rational design of recombinant chimeric toxins provides a novel approach for protecting individuals at high risk of developing CDI.

Preparative Scale Cell-free Production and Quality Optimization of MraY Homologues in Different Expression Modes

Background: Functional MraY translocases can be cell-free expressed in high levels. Results: Bacillus subtilis MraY activity is stable and robust, whereas Escherichia coli MraY depends on lipids. Conclusion: Activity of MraY can be modulated by cell-free expression modes. Artificial hydrophobic environments have a strong impact on the MraY sample quality. Significance: New strategy for the efficient production and analysis of drug targets. MraY translocase catalyzes the first committed membrane-bound step of bacterial peptidoglycan synthesis leading to the formation of lipid I. The essential membrane protein therefore has a high potential as target for drug screening approaches to develop antibiotics against Gram-positive as well as Gram-negative bacteria. However, the production of large integral membrane proteins in conventional cellular expression systems is still very challenging. Cell-free expression technologies have been optimized in recent times for the production of membrane proteins in the presence of detergents (D-CF), lipids (L-CF), or as precipitates (P-CF). We report the development of preparative scale production protocols for the MraY homologues of Escherichia coli and Bacillus subtilis in all three cell-free expression modes followed by their subsequent quality evaluation. Although both proteins can be cell-free produced at comparable high levels, their requirements for optimal expression conditions differ markedly. B. subtilus MraY was stably folded in all three expression modes and showed highest translocase activities after P-CF production followed by defined treatment with detergents. In contrast, the E. coli MraY appears to be unstable after post- or cotranslational solubilization in detergent micelles. Expression kinetics and reducing conditions were identified as optimization parameters for the quality improvement of E. coli MraY. Most remarkably, in contrast to B. subtilis MraY the E. coli MraY has to be stabilized by lipids and only the production in the L-CF mode in the presence of preformed liposomes resulted in stable and translocase active protein samples.

Antibody-enhanced, Fc gamma receptor-mediated endocytosis of Clostridium difficile toxin A.

ABSTRACT Toxin A (TcdA) and toxin B (TcdB) are major virulence factors of Clostridium difficile. These two toxins intoxicate cultured cells by similar mechanisms, and TcdB generally is more potent than TcdA in cultured cells. The exact reason for this difference is unclear. Here, we report that the cellular effects of TcdA can be substantially enhanced via an opsonizing antibody through Fc gamma receptor I (FcγRI)-mediated endocytosis. A TcdA-specific monoclonal antibody, A1H3, was found to significantly enhance the cytotoxicity of TcdA to macrophages and monocytes. The A1H3-dependent enhancement of glucosyltransferase activity, cytoskeleton disruption, and tumor necrosis factor alpha production induced by TcdA was further demonstrated using RAW 264.7 cells. Subsequent experiments indicated that the interaction of FcγRI with A1H3 underlays the antibody-dependent enhancement of the cellular effects of TcdA. While blocking FcγRII and FcγRIII with anti-CD16/32 antibodies did not affect the TcdA-mediated glucosylation of Rac1 in RAW 264.7 cells, presaturation of FcγRI with anti-CD64 antibodies in THP1 cells significantly reduced this activity. Incubation of a TcdA-A1H3 immune complex with recombinant mouse CD64 completely abrogated the A1H3-mediated enhancement of the glucosyltransferase activity of TcdA in RAW 264.7 cells. Moreover, expression of FcγRI in CHO cells strikingly enhanced the sensitivity of these cells to TcdA complexed with A1H3. We showed that the presence of A1H3 facilitated cell surface recruitment of TcdA, contributing to the antibody-dependent, FcγRI-mediated enhancement of TcdA activity. Finally, studies using chlorpromazine and endosomal acidification inhibitors revealed an important role of the endocytic pathway in the A1H3-dependent enhancement of TcdA activity.

pH-responsive unimolecular micelle-gold nanoparticles-drug nanohybrid system for cancer theranostics

The development of an in situ formed pH-responsive theranostic nanocomposite for anticancer drug delivery and computed tomography (CT) imaging was reported. β-cyclodextrin-{poly(lactide)-poly(2-(dimethylamino) ethyl methacrylate)-poly[oligo(2-ethyl-2-oxazoline)methacrylate]}21 [β-CD-(PLA-PDMAEMA-PEtOxMA)21] unimolecular micelles served as a template for the in situ formation of gold nanoparticles (GNPs) and the subsequent encapsulation of doxorubicin (DOX). The formation of unimolecular micelles, microstructures and the distributions of GNPs and DOX were investigated through the combination of experiments and dissipative particle dynamics (DPD) simulations. β-CD-(PLA-PDMAEMA-PEtOxMA)21 formed spherical unimolecular micelles in aqueous solution within a certain range of polymer concentrations. GNPs preferentially distributed in the PDMAEMA area. The maximum wavelength (λmax) and the size of GNPs increased with increasing concentration of HAuCl4. DOX preferentially distributed in the PDMAEMA mesosphere, but penetrated the inner PLA core with increasing DOX concentration. DOX-loaded micelles with 41-61% entrapment efficiency showed fast release (88% after 102h) under acidic tumor conditions. Both in vitro and in vivo experiments revealed superior anticancer efficacy and effective CT imaging properties for β-CD-(PLA-PDMAEMA-PEtOxMA)21/Au/DOX. We conclude that the reported unimolecular micelles represent a class of versatile smart nanocarriers for theranostic application.nnnSTATEMENT OF SIGNIFICANCEnDeveloping polymeric nanoplatforms as integrated theranostic vehicles for improving cancer diagnostics and therapy is an emerging field of much importance. This article aims to develop an in situ formed pH-responsive theranostic nanocomposite for anticancer drug delivery and computed tomography (CT) imaging. Specific emphases is on structure-properties relationship. There is a sea of literature on polymeric drug nanocarriers, and a couple of polymer-stabilized gold nanoparticles (GNPs) systems for cancer diagnosis are also known. However, to our knowledge, there has been no report on polymeric unimolecular micelles capable of dual loading of GNPs without external reducing agents and anticancer drugs for cancer diagnosis and treatment. To this end, the target of the current work was to develop an in situ formed nanocarrier, which actively dual wrapped CT contrast agent GNPs and hydrophobic anticancer drug doxorubicin (DOX), achieving high CT imaging and antitumor efficacy under in vitro and in vivo acid tumor condition. Meanwhile, by taking advantage of dissipative particle dynamics (DPD) simulation, we further obtained the formation process and mechanism of unimolecular micelles, and detailed distributions and microstructures of GNPs and DOX on unimolecular micelles. Taken together, our results here provide insight and guidance for the design of more effective nanocarriers for cancer theranostic application.

Folic acid grafted and tertiary amino based pH-responsive pentablock polymeric micelles for targeting anticancer drug delivery

Increasing target to tumor sites and reducing accumulation at normal tissue sites of anticancer drugs are essential to improve the cancer chemotherapy efficiency. In this study, we have developed a novel pentablock polymeric poly(ethylene glycol)-b-(poly(2-(diethylamino) ethyl methacrylate)-b-poly (hydroxyethyl methacrylate)-g-folic acid)2 [PEG-b-(PDEAEMA-b-PHEMA-g-FA)2] micelles as anticancer drug nanocarrier. The carriers could target tumor cells rapidly, and response to the tumor sites pH to control drug release. The critical micelle concentration (CMC) of the intermediates copolymers was 4.37-7.08mg/L, which indicated that the self-assembled micelles had comparatively good internal circulation stability. The drug loaded micelles were prepared using dialysis method, resulting in an average particle size of below 120nm, and the drug loading content and entrapment efficiency were 21% and 48% respectively. The pH-responsiveness and in vitro drug release of the micelles were studied, and the results showed a higher doxorubicin (DOX) cumulative amount at pH5.0 (~90%) compared to pH7.4 (~20%) owing to the protonation of the tertiary amino groups. In vitro cytotoxicity and endocytosis experiments showed that the tumor-suppressing effect of drug-loaded micelles was close to those of free DOX. The loaded DOX could be delivered into the cancer cells in a short time, and about 80% of the tumor cells were killed after 48h incubation. The results indicate that the pentablock polymeric micelles have the potential to be applied for targeting anticancer drug delivery and control release.

PDEAEMA-based pH-sensitive amphiphilic pentablock copolymers for controlled anticancer drug delivery

The synthesis of a series of PDEAEMA-based pH-sensitive amphiphilic pentablock copolymers poly(methyl methacrylate)-b-poly(2-(diethylamino)ethyl methacrylate)-b-poly(ethylene glycol)-b-poly(2-(diethylamino)ethyl methacrylate)-b-poly(methyl methacrylate) [PEG-b-(PDEAEMA-b-PMMA)2] with different compositions proceeded via the combination of a bromination reaction and continuous activators regenerated by electron transfer atom transfer radical polymerization (ARGET ATRP). All the copolymers were characterized by 1H NMR and gel permeation chromatography (GPC). The amphiphilic copolymers can self-assemble into micelles in aqueous solution, and the CMC values were comparatively low (2.40–2.80 mg L−1). The pKb buffering region, particle sizes, zeta potentials and optical transmittance were measured to investigate the pH-sensitivity of the polymeric micelles. The size and morphology of the self-assembled blank and DOX-loaded micelles were determined by transmission electron microscopy (TEM) and dynamic light scattering (DLS). The in vitro release rate was sharply increased by decreasing the pH from 7.4 to 5.0, due to the swelling of micelles at lower pH caused by the protonation of tertiary amine groups of PDEAEMA. The in vitro cytotoxicity of DOX-loaded micelles against Hela cells were measured and compared with free DOX, suggesting that the blank micelles provide low cytotoxicity and the DOX-loaded micelles provided high cytotoxicity nearly that of free DOX. The results indicate that this new kind of amphiphilic copolymers could serve as promising nanocarriers for controlled anticancer drug delivery.

High Efficient Expression, Purification, and Functional Characterization of Native Human Epidermal Growth Factor in Escherichia coli

Human epidermal growth factor (hEGF) is a small, mitotic growth polypeptide that promotes the proliferation of various cells and is widely applied in clinical practices. However, high efficient expression of native hEGF in Escherichia coli has not been successful, since three disulfide bonds in monomer hEGF made it unable to fold into correct 3D structure using in vivo system. To tackle this problem, we fused Mxe GyrA intein (Mxe) at the C-terminal of hEGF followed by small ubiquitin-related modifier (SUMO) and 10x His-tag to construct a chimeric protein hEGF-Mxe-SUMO-H10. The fusion protein was highly expressed at the concentration of 281u2009mg/L and up to 59.5% of the total cellular soluble proteins. The fusion protein was purified by affinity chromatography and 29.4u2009mg/L of native hEGF can be released by thiol induced N-terminal cleavage without any proteases. The mitotic activity in Balb/c 3T3 cells is proliferated by commercial and recombinant hEGF measured with methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay which indicated that recombinant hEGF protein stimulates the cell proliferation similar to commercial protein. This study significantly improved the yield and reduced the cost of hEGF in the recombinant E. coli system and could be a better strategy to produce native hEGF for pharmaceutical development.

Cell-free expression of human glucosamine 6-phosphate N-acetyltransferase (HsGNA1) for inhibitor screening

Glucosamine 6-phosphate N-acetyltransferase (GNA1; EC 2.3.1.4) is required for the de novo synthesis of N-acetyl-d-glucosamine-6-phosphate (GlcNAc-6P), which is an essential precursor in Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) biosynthesis pathway. Therefore, GNA1 is indispensable for the viability of organisms. Here, a novel cell-free expression strategy was developed to efficiently produce large amounts of human GNA1(HsGNA1) and HsGNA1-sGFP for throughput inhibitor screening. The binding site of inhibitor glucose-6-phosphate (G6P) to hGNA was identified by simulated annealing. Subtle differences to the binding site of Aspergillius GNA1(AfGNA1) can be harnessed for inhibitor design. HsGNA1 may be also useful as an antimicrobial and chemotherapeutic target against cancer. Additionally HsGNA1 inhibitors/modulators can possibly be administered with other drugs in the next generation of personalized medicine.

Mutational analysis to identify the residues essential for the acetyltransferase activity of GlmU in Bacillus subtilis

The novel and attractive antimicrobial drug target, glucosamine-1-phosphate acetyltransferase/N-acetylglucosamine-1-phosphate uridyltransferase (GlmU), is a bifunctional enzyme that catalyzes two sequential steps in the biosynthesis of UDP-GlcNAc, essential for both lipopolysaccharide and peptidoglycan in Gram positive and Gram negative bacteria. Glucosamine-1-phosphate acetyltransferase catalyzes the formation of N-acetylglucosamine-1-phosphate and N-acetylglucosamine-1-phosphate uridylyltransferase catalyzes the formation of UDP-GlcNAc. In this study, glmU genes from Escherichia coli and Bacillus subtilis were cloned and the corresponding proteins were produced in an E. coli BL21 (DE3) expression system. The kinetic parameters and reaction conditions, such as initial reaction rate, optimal temperature and pH, and the effect of Mg2+ ion concentration of GlmU enzymes from E. coli (Ec-GlmU) and B. subtilis (Bs-GlmU) were detected. Compared to Ec-GlmU, Bs-GlmU exhibits much lower glucosamine-1-phosphate acetyltransferase activity. Based on the in silico results of a virtual amino acid mutation and molecular docking of Bs-GlmU, five mutants were successfully constructed to verify the predicted alterations of acetyltransferase activity. The result of point mutations indicated that the C405 and A380 residues played key role in catalytic mechanism of acetyltransferase of Bs-GlmU providing new insight into the reaction mechanism of GlmU enzymes and supporting the further development of antimicrobial drugs.

Clostridium difficile toxin B intoxicated mouse colonic epithelial CT26 cells stimulate the activation of dendritic cells

Clostridium difficile causes antibiotic-associated diarrhea and pseudomembranous colitis mainly through two exotoxins TcdA and TcdB that target intestinal epithelial cells. Dendritic cells (DCs) play an important role in regulating intestinal inflammatory responses. In the current study, we explored the interaction of TcdB-intoxicated epithelial cells with mouse bone marrow-derived DCs. TcdB induced cell death and heat shock protein translocation in mouse intestinal epithelial CT26 cells. The intoxicated epithelial cells promoted the phagocytosis and the TNF-α secretion by DCs. Incubation with TcdB-intoxicated CT26 cells stimulated DC maturation. Moreover, TcdB-treated CT26 cells induced DC immigration when they were injected into mice subcutaneously. Taken together, these data demonstrate that TcdB-intoxicated intestinal epithelial cells are able to stimulate DC activation in vitro and attract DCs in vivo, indicating that epithelial cells may be able to regulate DC activation under the exposure of TcdB during C. difficile infection.