Publications by authors named "Yongrong Zhang"

24 Publications

  • Page 1 of 1

Structures and Chromogenic Ion-Pair Recognition of a Catechol-Functionalized 1,8-Anthraquinone Macrocycle in Dimethyl Sulfoxide.

Inorg Chem 2021 Apr 11;60(7):5042-5053. Epub 2021 Mar 11.

Department of Chemistry, Shanghai University, Shanghai 200444, China.

A lariat anthraquinone macrocycle functionalized with catechol (HL) was synthesized via the Mannich reaction. The Mannich base HL can be partially decomposed into L1·3HO and HL1·NO·2HO in the presence of tetrabutylammonium hydroxide/Al(NO)·9HO in dimethyl sulfoxide (DMSO). Free L1·3HO is essentially coplanar, while protonated HL1·NO·2HO is highly distorted. Dark-green FeCl·HL·2HO powder and Fe(HL)Cl crystal can be isolated from ethanol (CHOH) in high/low HL concentration. Anthraquinone in HL is essentially coplanar but distorted in Fe(HL)Cl. The Fe(III) ion in Fe(HL)Cl adopts a less common five-coordination with three catecholate O and two Cl atoms in the dimer. The distortion of inbound C═O is much higher than that of outbound C═O in anthraquinone in all of these compounds. HL responds to chlorides of Li, Na, K, Cs, Mg, Ca, Sr, Ba, Fe, Cu, Zn, and Al in a DMSO solution, which can be observed by differential pulse voltammetry, UV-vis, and H NMR. All of these metal ions shift of anthraquinone to positive, especially the second reduction peak of anthraquinone. Fe, Zn, and Al change the reduction of catechol fundamentally. HL (0.50 mM) shows a chromogenic response to FeCl and Fe(NO) to form uncommon 2:1 and 3:2 (HL/Fe) complexes, both peaking at 748 nm in DMSO. In the presence of 2 equiv of sodium hydroxide (NaOH), the 748 nm absorbance shifts to 777 nm, identical with Fe(HL)Cl in DMSO. Different from the fast reaction between HL and FeCl, Fe(NO) reacts with HL rather slowly in DMSO. Catechol can coordinate to FeCl without any deprotonation in CHOH and DMSO. HL also shows a chromogenic response to fluorides and hydroxides, which peak at 670 and 684 nm, respectively, in DMSO. The binding ratio between HL and F/OH is 1:2. In a higher concentration of hydroxides, a 684 nm greenish-blue 1:2 complex forms immediately, which gradually transforms to a red complex and peaks at ∼530 nm in minutes at room temperature. No color change can be observed in an CHOH solution in the presence of OH.
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http://dx.doi.org/10.1021/acs.inorgchem.1c00083DOI Listing
April 2021

A probiotic yeast-based immunotherapy against infection.

Sci Transl Med 2020 10;12(567)

Department of Microbial Pathogenesis, University of Maryland School of Dentistry, Baltimore, MD 21201, USA.

Antibiotic-resistant is an anaerobic Gram-positive bacterium that colonizes the colon and is responsible for more than 29,000 deaths in the United States each year. Hence, infection (CDI) poses an urgent threat to public health. Antibody-mediated neutralization of TcdA and TcdB toxins, the major virulence factors of CDI, represents an effective strategy to combat the disease without invoking antibiotic resistance. However, current antitoxin approaches are mostly based on parenteral infusion of monoclonal antibodies that are costly, narrow spectrum, and not optimized against the intestinal disease. Here, we engineered probiotic to constitutively secrete a single tetra-specific antibody that potently and broadly neutralized both toxins and demonstrated protection against primary and recurrent CDI in both prophylactic and therapeutic mouse models of disease. This yeast immunotherapy is orally administered, can be used concurrently with antibiotics, and may have potential as a prophylactic against CDI risk and as a therapeutic for patients with CDI.
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http://dx.doi.org/10.1126/scitranslmed.aax4905DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7692727PMC
October 2020

Structure of the full-length Clostridium difficile toxin B.

Nat Struct Mol Biol 2019 08 15;26(8):712-719. Epub 2019 Jul 15.

Department of Physiology and Biophysics, University of California, Irvine, Irvine, CA, USA.

Clostridium difficile is an opportunistic pathogen that establishes in the colon when the gut microbiota are disrupted by antibiotics or disease. C. difficile infection (CDI) is largely caused by two virulence factors, TcdA and TcdB. Here, we report a 3.87-Å-resolution crystal structure of TcdB holotoxin that captures a unique conformation of TcdB at endosomal pH. Complementary biophysical studies suggest that the C-terminal combined repetitive oligopeptides (CROPs) domain of TcdB is dynamic and can sample open and closed conformations that may facilitate modulation of TcdB activity in response to environmental and cellular cues during intoxication. Furthermore, we report three crystal structures of TcdB-antibody complexes that reveal how antibodies could specifically inhibit the activities of individual TcdB domains. Our studies provide novel insight into the structure and function of TcdB holotoxin and identify intrinsic vulnerabilities that could be exploited to develop new therapeutics and vaccines for the treatment of CDI.
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http://dx.doi.org/10.1038/s41594-019-0268-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6684407PMC
August 2019

Selection and characterization of ultrahigh potency designed ankyrin repeat protein inhibitors of C. difficile toxin B.

PLoS Biol 2019 06 24;17(6):e3000311. Epub 2019 Jun 24.

Department of Microbial Pathogenesis and Immunology, Texas A&M University Health Science Center, College Station, Texas, United States of America.

Clostridium difficile infection (CDI) is a major nosocomial disease associated with significant morbidity and mortality. The pathology of CDI stems primarily from the 2 C. difficile-secreted exotoxins-toxin A (TcdA) and toxin B (TcdB)-that disrupt the tight junctions between epithelial cells leading to the loss of colonic epithelial barrier function. Here, we report the engineering of a series of monomeric and dimeric designed ankyrin repeat proteins (DARPins) for the neutralization of TcdB. The best dimeric DARPin, DLD-4, inhibited TcdB with a half maximal effective concentration (EC50) of 4 pM in vitro, representing an approximately 330-fold higher potency than the Food and Drug Administration (FDA)-approved anti-TcdB monoclonal antibody bezlotoxumab in the same assay. DLD-4 also protected mice from a toxin challenge in vivo. Cryo-electron microscopy (cryo-EM) studies revealed that the 2 constituent DARPins of DLD-4-1.4E and U3-bind the central and C-terminal regions of the delivery domain of TcdB. Competitive enzyme-linked immunosorbent assay (ELISA) studies showed that the DARPins 1.4E and U3 interfere with the interaction between TcdB and its receptors chondroitin sulfate proteoglycan 4 (CSPG4) and frizzled class receptor 2 (FZD2), respectively. Our cryo-EM studies revealed a new conformation of TcdB (both apo- and DARPin-bound at pH 7.4) in which the combined repetitive oligopeptides (CROPS) domain points away from the delivery domain. This conformation of the CROPS domain is in stark contrast to that seen in the negative-stain electron microscopy (EM) structure of TcdA and TcdB at the same pH, in which the CROPS domain bends toward and "kisses" the delivery domain. The ultrapotent anti-TcdB molecules from this study serve as candidate starting points for CDI drug development and provide new biological tools for studying the pathogenicity of C. difficile. The structural insights regarding both the "native" conformation of TcdB and the putative sites of TcdB interaction with the FZD2 receptor, in particular, should help accelerate the development of next-generation anti-C. difficile toxin therapeutics.
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http://dx.doi.org/10.1371/journal.pbio.3000311DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6590788PMC
June 2019

Cysteine Protease-Mediated Autocleavage of Toxins Regulates Their Proinflammatory Activity.

Cell Mol Gastroenterol Hepatol 2018 9;5(4):611-625. Epub 2018 Feb 9.

Department of Microbial Pathogenesis, School of Dentistry, University of Maryland, Baltimore, Maryland.

Background & Aims: toxin A (TcdA) and toxin toxin B (TcdB), the major virulence factors of the bacterium, cause intestinal tissue damage and inflammation. Although the 2 toxins are homologous and share a similar domain structure, TcdA is generally more inflammatory whereas TcdB is more cytotoxic. The functional domain of the toxins that govern the proinflammatory activities of the 2 toxins is unknown.

Methods: Here, we investigated toxin domain functions that regulate the proinflammatory activity of toxins. By using a mouse ilea loop model, human tissues, and immune cells, we examined the inflammatory responses to a series of chimeric toxins or toxin mutants deficient in specific domain functions.

Results: Blocking autoprocessing of TcdB by mutagenesis or chemical inhibition, while reducing cytotoxicity of the toxin, significantly enhanced its proinflammatory activities in the animal model. Furthermore, a noncleavable mutant TcdB was significantly more potent than the wild-type toxin in the induction of proinflammatory cytokines in human colonic tissues and immune cells.

Conclusions: In this study, we identified a novel mechanism of regulating the biological activities of toxins in that cysteine protease-mediated autoprocessing regulates toxins' proinflammatory activities. Our findings provide new insight into the pathogenesis of infection and the design of therapeutics against the disease.
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http://dx.doi.org/10.1016/j.jcmgh.2018.01.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6009800PMC
February 2018

Mice with Inflammatory Bowel Disease are Susceptible to Clostridium difficile Infection With Severe Disease Outcomes.

Inflamm Bowel Dis 2018 02;24(3):573-582

Department of Microbial Pathogenesis, University of Maryland School of Dentistry, Baltimore, Maryland, USA.

Background: Over the past several decades, there has been a significant increase in the incidence of Clostridium difficile infection (CDI) in patients suffering from inflammatory bowel disease (IBD). However, a wild-type animal model is not available to study these comorbid diseases.

Methods: We evaluated the susceptibility to CDI of mice with dextran sulfate sodium salt (DSS)-induced colitis (IBD mice) with or without antibiotic exposure; we examined the histopathology and cytokine response in the concomitant diseases after the model was created.

Results: No CDI occurs in healthy control mice, wherease the incidence of CDI in IBD mice is 40%; however, in IBD mice that received antibiotics, the incidence of CDI is 100% and the disease is accompanied by high levels of toxins in the mouse feces and sera. Compared to IBD and CDI alone, those IBD mice infected with C. difficile have more severe symptoms, toxemia, histopathological damage, and higher mortality. Moreover, several proinflammatory cytokines and chemokines are significantly elevated in the colon tissues from IBD mice infected with C. difficile.

Conclusions: We, for the first time, demonstrate in an animal model that mice with dextran sulfate sodium induced-inflammatory bowel disease are significantly more susceptible to C. difficile infection, and that the bacterial infection led to more severe disease and death. These findings are consistent with clinical observations, thus, the animal model will permit us to study the pathogenesis of these concurrent diseases and to develop therapeutic strategies against the comorbidity of IBD and CDI.
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http://dx.doi.org/10.1093/ibd/izx059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5936643PMC
February 2018

A Continuous Identity Authentication Scheme Based on Physiological and Behavioral Characteristics.

Sensors (Basel) 2018 Jan 10;18(1). Epub 2018 Jan 10.

School of Electronic Science, National University of Defense Technology, Changsha 410073, China.

Wearable devices have flourished over the past ten years providing great advantages to people and, recently, they have also been used for identity authentication. Most of the authentication methods adopt a one-time authentication manner which cannot provide continuous certification. To address this issue, we present a two-step authentication method based on an own-built fingertip sensor device which can capture motion data (e.g., acceleration and angular velocity) and physiological data (e.g., a photoplethysmography (PPG) signal) simultaneously. When the device is worn on the user's fingertip, it will automatically recognize whether the wearer is a legitimate user or not. More specifically, multisensor data is collected and analyzed to extract representative and intensive features. Then, human activity recognition is applied as the first step to enhance the practicability of the authentication system. After correctly discriminating the motion state, a one-class machine learning algorithm is applied for identity authentication as the second step. When a user wears the device, the authentication process is carried on automatically at set intervals. Analyses were conducted using data from 40 individuals across various operational scenarios. Extensive experiments were executed to examine the effectiveness of the proposed approach, which achieved an average accuracy rate of 98.5% and an F1-score of 86.67%. Our results suggest that the proposed scheme provides a feasible and practical solution for authentication.
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http://dx.doi.org/10.3390/s18010179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5796290PMC
January 2018

The role of purified Clostridium difficile glucosylating toxins in disease pathogenesis utilizing a murine cecum injection model.

Anaerobe 2017 Dec 12;48:249-256. Epub 2017 Oct 12.

Department of Microbial Pathogenesis, University of Maryland School of Dentistry, Baltimore, MD, USA. Electronic address:

Most pathogenic Clostridium difficile produce two major exotoxins TcdA and TcdB, in the absence of which the bacterium is non-pathogenic. While it is important to investigate the role of each toxin in the pathogenesis of C. difficile infection (CDI) using isogenic strains, it is impossible to precisely control the expression levels of individual toxins and exclude bacterial factors that may contribute to the toxins' effects during infection. In this study, we utilized an acute intestinal disease model by injecting purified toxins directly into mouse cecum after a midline laparotomy. We evaluated the physical condition of mice by clinical score and survival, and the intestinal tissue damage and inflammation by histology. Depending on the dose of the toxins, mice developed mild to severe colitis, experienced diarrhea or rapidly died. We found that both purified TcdA and TcdB were able to induce clinical disease, intestinal inflammation, and tissue damage that resembled CDI. TcdA was significantly faster in inducing intestinal inflammation and tissue damage, and was approximately five times more potent than TcdB in terms of inducing severe gut disease and death outcomes in mice. Moreover, we found that the two toxins had significant synergistic effects on disease induction. Comparison of the in vivo toxicity of TcdB from clinical strains revealed that TcdB from an epidemic RT 027 strain was more toxic than the others. Our study thus demonstrates that both TcdA and TcdB, independent of other factors from C. difficile bacterium, are able to cause disease that resembles CDI and highlights the importance of targeting both toxins for vaccines and therapeutics against the disease.
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http://dx.doi.org/10.1016/j.anaerobe.2017.10.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5792825PMC
December 2017

A Multiparticulate Delivery System for Potential Colonic Targeting Using Bovine Serum Albumin as a Model Protein : Theme: Formulation and Manufacturing of Solid Dosage Forms Guest Editors: Tony Zhou and Tonglei Li.

Pharm Res 2017 Dec 14;34(12):2663-2674. Epub 2017 Aug 14.

Department of Pharmaceutical Sciences, University of Maryland, 20 N. Pine St, Baltimore, Maryland, 21201, USA.

Purpose: There are many important diseases whose treatment could be improved by delivering a therapeutic protein to the colon, for example, Clostridium difficile infection, ulcerative colitis and Crohn's Disease. The goal of this project was to investigate the feasibility of colonic delivery of proteins using multiparticulate beads.

Methods: In this work, bovine serum albumin (BSA) was adopted as a model protein. BSA was spray layered onto beads, followed by coating of an enteric polymer EUDRAGIT® FS 30 D to develop a colonic delivery system. The secondary and tertiary structure change and aggregation of BSA during spray layering process was examined. The BSA layered beads were then challenged in an accelerated stability study using International Council for Harmonization (ICH) conditions. The in vitro release of BSA from enteric coated beads was examined using United States Pharmacopeia (USP) dissolution apparatus 1.

Results: No significant changes in the secondary and tertiary structure or aggregation profile of BSA were observed after the spray layering process. Degradation of BSA to different extents was detected after storing at 25°C and 40°C for 38 days. Enteric coated BSA beads were intact in acidic media while released BSA in pH 7.4 phosphate buffer.

Conclusion: We showed the feasibility of delivering proteins to colon in vitro using multiparticulate system.
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http://dx.doi.org/10.1007/s11095-017-2237-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5738263PMC
December 2017

Newly identified bacteriolytic enzymes that target a wide range of clinical isolates of Clostridium difficile.

Biotechnol Bioeng 2016 12 20;113(12):2568-2576. Epub 2016 Jun 20.

School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, 950 Atlantic Drive, Atlanta, 30332, Georgia.

Clostridium difficile has emerged as a major cause of infectious diarrhea in hospitalized patients, with increasing mortality rate and annual healthcare costs exceeding $3 billion. Since C. difficile infections are associated with the use of antibiotics, there is an urgent need to develop treatments that can inactivate the bacterium selectively without affecting commensal microflora. Lytic enzymes from bacteria and bacteriophages show promise as highly selective and effective antimicrobial agents. These enzymes often have a modular structure, consisting of a catalytic domain and a binding domain. In the current work, using consensus catalytic domain and cell-wall binding domain sequences as probes, we analyzed in silico the genome of C. difficile, as well as phages infecting C. difficile. We identified two genes encoding cell lytic enzymes with possible activity against C. difficile. We cloned the genes in a suitable expression vector, expressed and purified the protein products, and tested enzyme activity in vitro. These newly identified enzymes were found to be active against C. difficile cells in a dose-dependent manner. We achieved a more than 4-log reduction in the number of viable bacteria within 5 h of application. Moreover, we found that the enzymes were active against a wide range of C. difficile clinical isolates. We also characterized the biocatalytic mechanism by identifying the specific bonds cleaved by these enzymes within the cell wall peptidoglycan. These results suggest a new approach to combating the growing healthcare problem associated with C. difficile infections. Biotechnol. Bioeng. 2016;113: 2568-2576. © 2016 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/bit.26029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5367918PMC
December 2016

Intravenous adenovirus expressing a multi-specific, single-domain antibody neutralizing TcdA and TcdB protects mice from Clostridium difficile infection.

Pathog Dis 2016 10 7;74(7). Epub 2016 Aug 7.

Department of Microbial Pathogenesis, University of Maryland School of Dentistry, Baltimore, MD 21201, USA

Clostridium difficile infection (CDI) is the most common cause of antibiotic-associated diarrhea and colitis in developed countries. The disease is mainly mediated via two major exotoxins TcdA and TcdB secreted by the bacterium. We have previously developed a novel, potently neutralizing, tetravalent and bispecific heavy-chain-only single domain (VHH) antibody to both TcdA and TcdB (designated as ABA) that reverses fulminant CDI in mice. Since ABA has a short serum half-life, in this study a replication-deficient recombinant adenovirus expressing ABA was generated and the long-lasting expression of functional ABA was demonstrated in vitro and in vivo Mice transduced with one dose of the adenovirus displayed high levels of serum ABA for more than1 month and were fully protected against systemic toxin challenges. More importantly, the ABA delivered by the adenovirus protected mice from both primary and recurrent CDI. Thus, replication-deficient adenoviral vector may be used to deliver neutralizing antibodies against the toxins in order to prevent CDI and recurrence.
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http://dx.doi.org/10.1093/femspd/ftw078DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5985491PMC
October 2016

Pathogenic effects of glucosyltransferase from Clostridium difficile toxins.

Pathog Dis 2016 Jun 4;74(4):ftw024. Epub 2016 Apr 4.

Department of Microbial Pathogenesis, University of Maryland Baltimore, 650 W. Baltimore Street, Baltimore, MD 21201, USA

The glucosyltransferase domain ofClostridium difficiletoxins modifies guanine nucleotide-binding proteins of Rho family. It is the major virulent domain of the holotoxins. Various pathogenic effects ofC. difficiletoxins in response to Rho glucosylation have been investigated including cytoskeleton damage, cell death and inflammation. The most recent studies have revealed some significant characteristics of the holotoxins that are independent of glucosylating activity. These findings arouse discussion about the role of glucosyltransferase activity in toxin pathogenesis and open up new insights for toxin mechanism study. In this review, we summarize the pathogenic effects of glucosyltransferase domain of the toxins in the past years.
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http://dx.doi.org/10.1093/femspd/ftw024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5985493PMC
June 2016

Glucosyltransferase activity of Clostridium difficile Toxin B is essential for disease pathogenesis.

Gut Microbes 2015 Jul 19;6(4):221-4. Epub 2015 Jun 19.

a Department of Microbial Pathogenesis ; University of Maryland Dental School ; Baltimore , MD USA.

Clostridium difficile TcdB harbors a glucosyltransferase that targets host Rho GTPases. However, the role of the enzyme activity in the induction of host intestinal disease has not been demonstrated. In this study, we established a mouse acute intestinal disease model by cecum injection of wild type and glucosyltransferase-deficient TcdB and a chronic model by delivering toxin intraluminally via engineered surrogate host Bacillus megaterium. We demonstrated, for the first time, that the glucosyltransferase activity of TcdB is essential for inducing disease symptoms and intestinal pathological responses that resemble human disease, highlighting the importance of targeting toxin glucosyltransferase activity for future therapy.
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http://dx.doi.org/10.1080/19490976.2015.1062965DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4615278PMC
July 2015

Masking autoprocessing of Clostridium difficile toxin A by the C-terminus combined repetitive oligo peptides.

Biochem Biophys Res Commun 2015 Apr 26;459(2):259-263. Epub 2015 Feb 26.

Department of Microbial Pathogenesis, University of Maryland Dental School, Baltimore, MD 21201, USA. Electronic address:

Clostridium difficile toxin A and B (TcdA and TcdB) are the major virulence factors of the bacterium, both of which consist of two enzymatic domains: an effector glucosyltransferase domain (GTD) and a cysteine protease domain (CPD) responsible for autocleavage and release of GTD. Although the CPDs from both toxins share a similar structure and mechanism of hexakisphosphate (InsP6)-induced activation, TcdA is substantially less sensitive to the autocleavage as compared with TcdB. In this study, we provided evidence of inter-domain regulation of CPD activity of TcdA and its autoprocessing. The C-terminus combined repetitive oligo peptides (CROPs) of TcdA reduced the accessibility of TcdB CPD to its substrate in a chimeric toxin TxB-Ar, consequently blocking autoprocessing. Moreover, interference of antibodies with the CROPs of full-length TcdA efficiently enhanced its GTD release. In conclusion, by utilizing chimeric toxins and specific antibodies, we identified that the CROPs of TcdA plays a crucial role in controlling the InsP6-mediated activation of CPD and autocleavage of GTD. Our data provides insights on the molecular mode of action of the C. difficile toxins.
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http://dx.doi.org/10.1016/j.bbrc.2015.02.095DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4426850PMC
April 2015

Resveratrol induces cell cycle arrest via a p53-independent pathway in A549 cells.

Mol Med Rep 2015 Apr 16;11(4):2459-64. Epub 2014 Dec 16.

Laboratory of Hepatobiliary Surgery, Zhanjiang Key Laboratory of Hepatobiliary Diseases, Affiliated Hospital of Guangdong Medical College, Zhanjiang, Guangdong 524001, P.R. China.

Resveratrol, a non‑flavone polyphenol compound, has a chemopreventive and chemotherapeutic effect against the progression of multiple types of cancer, including lung cancer. However, the molecular mechanism underlying the effects of resveratrol on cancer remain to be elucidated. In the present study, using an MTT assay, it was demonstrated that resveratrol inhibited cell proliferation in a concentration‑ and time‑dependent manner. In addition, morphological features were observed in the A549, human lung cancer cell line, which included cell shrinkage, cells became distorted, certain cells became rounded and there was a concentration‑dependent increase in the number of sloughed cells. Cell cycle analysis revealed that resveratrol may induce cell cycle arrest in the G0/G1 phase by downregulating the expression levels of cyclin D1, cyclin‑dependent kinase (CDK)4 and CDK6, and upregulating the expression levels of the CDK inhibitors, p21 and p27. The immunofluorescence and western blot analysis results revealed that resveratrol upregulated the nuclear expression of p53 in A549 cells. Further studies have demonstrated that p53 downregulation did not contribute to the G0/G1 cell cycle arrest induced by resveratrol. In addition, resveratrol had no effect on the expression of p21, through use of the p53 inhibitor, pifithrin‑α. The present study may offer a scientific basis for the further in‑depth evaluation of resveratrol in the association of p53 and cell cycle arrest.
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http://dx.doi.org/10.3892/mmr.2014.3100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4337473PMC
April 2015

Mechanisms of protection against Clostridium difficile infection by the monoclonal antitoxin antibodies actoxumab and bezlotoxumab.

Infect Immun 2015 Feb 8;83(2):822-31. Epub 2014 Dec 8.

Department of Microbial Pathogenesis, University of Maryland Dental School, Baltimore, Maryland, USA

Clostridium difficile infection (CDI) represents the most prevalent cause of antibiotic-associated gastrointestinal infections in health care facilities in the developed world. Disease symptoms are caused by the two homologous exotoxins, TcdA and TcdB. Standard therapy for CDI involves administration of antibiotics that are associated with a high rate of disease recurrence, highlighting the need for novel treatment paradigms that target the toxins rather than the organism itself. A combination of human monoclonal antibodies, actoxumab and bezlotoxumab, directed against TcdA and TcdB, respectively, has been shown to decrease the rate of recurrence in patients treated with standard-of-care antibiotics. However, the exact mechanism of antibody-mediated protection is poorly understood. In this study, we show that the antitoxin antibodies are protective in multiple murine models of CDI, including systemic and local (gut) toxin challenge models, as well as primary and recurrent models of infection in mice. Systemically administered actoxumab-bezlotoxumab prevents both the damage to the gut wall and the inflammatory response, which are associated with C. difficile in these models, including in mice challenged with a strain of the hypervirulent ribotype 027. Furthermore, mutant antibodies (N297Q) that do not bind to Fcγ receptors provide a level of protection similar to that of wild-type antibodies, demonstrating that the mechanism of protection is through direct neutralization of the toxins and does not involve host effector functions. These data provide a mechanistic basis for the prevention of recurrent disease observed in CDI patients in clinical trials.
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http://dx.doi.org/10.1128/IAI.02897-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4294251PMC
February 2015

Critical roles of Clostridium difficile toxin B enzymatic activities in pathogenesis.

Infect Immun 2015 Feb 17;83(2):502-13. Epub 2014 Nov 17.

Department of Microbial Pathogenesis, University of Maryland Dental School, Baltimore, Maryland, USA

TcdB is one of the key virulence factors of Clostridium difficile that is responsible for causing serious and potentially fatal colitis. The toxin contains at least two enzymatic domains: an effector glucosyltransferase domain for inactivating host Rho GTPases and a cysteine protease domain for the delivery of the effector domain into host cytosol. Here, we describe a novel intrabody approach to examine the role of these enzymes of TcdB in cellular intoxication. By screening a single-domain heavy chain (V(H)H) library raised against TcdB, we identified two V(H)H antibodies, 7F and E3, that specifically inhibit TcdB cysteine protease and glucosyltransferase activities, respectively. Cytoplasmic expression of 7F intrabody in Vero cells inhibited TcdB autoprocessing and delayed cellular intoxication, whereas E3 intrabody completely blocked the cytopathic effects of TcdB holotoxin. These data also demonstrate for the first time that toxin autoprocessing occurs after cysteine protease and glucosyltransferase domains translocate into the cytosol of target cells. We further determined the role of the enzymatic activities of TcdB in in vivo toxicity using a sensitive systemic challenge model in mice. Consistent with these in vitro results, a cysteine protease noncleavable mutant, TcdB-L543A, delayed toxicity in mice, whereas glycosyltransferase-deficient TcdB demonstrated no toxicity up to 500-fold of the 50% lethal dose (LD50) when it was injected systemically. Thus, glucosyltransferase but not cysteine protease activity is critical for TcdB-mediated cytopathic effects and TcdB systemic toxicity, highlighting the importance of targeting toxin glucosyltransferase activity for future therapy.
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http://dx.doi.org/10.1128/IAI.02316-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4294253PMC
February 2015

A segment of 97 amino acids within the translocation domain of Clostridium difficile toxin B is essential for toxicity.

PLoS One 2013 6;8(3):e58634. Epub 2013 Mar 6.

School of Bioengineering, East China University of Science and Technology, Shanghai, China.

Clostridium difficile toxin B (TcdB) intoxicates target cells by glucosylating Rho GTPases. TcdB (269 kDa) consists of at least 4 functional domains including a glucosyltransferase domain (GTD), a cysteine protease domain (CPD), a translocation domain (TD), and a receptor binding domain (RBD). The function and molecular mode of action of the TD, which is the largest segment of TcdB and comprises nearly 50% of the protein, remain largely unknown. Here we show that a 97-amino-acid segment (AA1756 - 1852, designated as ?97 or D97), located in the C-terminus of the TD and adjacent to the RBD, is essential for the cellular activity of TcdB. Deletion of this segment in TcdB (designated as TxB-D97), did not adversely alter toxin enzymatic activities or its cellular binding and uptake capacity. TxB-D97 bound to and entered cells in a manner similar to TcdB holotoxin. Both wild type and mutant toxins released their GTDs similarly in the presence of inositol hexakisphosphate (InsP6), and showed a similar glucosyltransferase activity in a cell-free glucosylating assay. Despite these similarities, the cytotoxic activity of TxB-D97 was reduced by more than 5 logs compared to wild type toxin, supported by the inability of TxB-D97 to glucosylate Rac1 of target cells. Moreover, the mutant toxin failed to elicit tumor necrosis factor alpha (TNF-α) in macrophages, a process dependent on the glucosyltransferase activity of the toxin. Cellular fractionation of toxin-exposed cells revealed that TxB-D97 was unable to efficiently release the GTD into cytosol. Thereby, we conclude the 97-amino-acid region of the TD C-terminus of TcdB adjacent to the RBD, is essential for the toxicity of TcdB.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0058634PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3590123PMC
September 2013

Immunotherapy using IL-2 and GM-CSF is a potential treatment for multidrug-resistant Mycobacterium tuberculosis.

Sci China Life Sci 2012 Sep 27;55(9):800-6. Epub 2012 Sep 27.

State Key Laboratory of Bioreactor Engineering, School of Bioengineering, East China University of Science and Technology, Shanghai 200237, China.

This study investigated the therapeutic effects of interleukin (IL)-2 and granulocyte-macrophage colony-stimulating factor (GM-CSF) co-administrated with antibacterial agents isoniazid (INH) and rifampin (RIF) to treat a mouse model of tuberculosis (TB) infection. A drug-susceptible TB strain, H37Rv was used to infect mice and the effectiveness of IL-2 and GM-CSF was initially evaluated based on survival rate, bacterial counts in lungs and spleens and the pathological condition of the lungs. Next, the therapeutic effect of the immunotherapy regimen was assessed in multidrug-resistant strain OB35-infected mice. In the H37Rv infection model, IL-2 and GM-CSF monotherapies reduced bacterial numbers in the lungs by 0.82 (P<0.01) and 0.58 (P<0.05) lg colony-forming units (CFU), respectively, and in the spleens by 1.42 (P<0.01) and 1.22 (P<0.01) lg CFU, respectively, compared with the untreated group. Mice receiving immunotherapy developed fewer lesions in the lungs compared with mice receiving antibacterial therapy alone. In the OB35 infection model, immunotherapy with either cytokine resulted in a significant reduction of bacterial load in the lungs and spleens and less severe lesions in the lungs compared with the untreated or antibacterial therapy treated mice. Notably, mice receiving immunotherapy with both cytokines had a 30% survival rate which was higher than that in other treated groups, and had significantly less CFUs in the lungs and spleens (1.02 and 1.34 lg CFU) compared with antibacterial therapy alone (P<0.01). This study demonstrated that immunotherapy with both IL-2 and GM-CSF may be useful to treat multidrug resistant tuberculosis (MDR-TB).
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http://dx.doi.org/10.1007/s11427-012-4368-xDOI Listing
September 2012

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

Infect Immun 2012 Aug 21;80(8):2678-88. Epub 2012 May 21.

School of Bioscience and Biotechnology, South China University of Technology, Guangzhou, China.

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.
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http://dx.doi.org/10.1128/IAI.00215-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3434558PMC
August 2012

Systemic dissemination of Clostridium difficile toxins A and B is associated with severe, fatal disease in animal models.

J Infect Dis 2012 Feb 5;205(3):384-91. Epub 2011 Dec 5.

Tufts Cummings School of Veterinary Medicine, North Grafton, Massachusetts, USA.

Background: Clostridium difficile infection (CDI) can cause a wide range of disease, from mild diarrhea to fulminant systemic disease. The incidence of systemic CDI with fatal consequence has increased rapidly in recent years.

Methods: Using an ultrasensitive cytotoxicity assay, we measured C. difficile toxin A (TcdA) and C. difficile toxin B (TcdB) in sera and body fluids of piglets and mice exposed to C. difficile to investigate the relationship between the presence of toxins in body fluids and systemic manifestations of CDI.

Results: We found that both TcdA and TcdB disseminate systemically, with toxins present in the sera and body fluids of infected animals, and toxemia is significantly correlated with the development of systemic CDI. The systemic administration of neutralizing antibodies against both toxins blocked the development of systemic disease in mice. We measured cytokine concentrations in the sera of mice and piglets with systemic and nonsystemic CDI and found that proinflammatory mediators were considerably elevated in animals with systemic CDI.

Conclusion: Our study demonstrates the existence of a strong correlation between toxemia and the occurrence of systemic disease, supporting the hypothesis that systemic CDI is most likely due to the toxicity of TcdA and TcdB and the induction of proinflammatory cytokines by the toxins.
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http://dx.doi.org/10.1093/infdis/jir748DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3256947PMC
February 2012

Retargeting Clostridium difficile Toxin B to Neuronal Cells as a Potential Vehicle for Cytosolic Delivery of Therapeutic Biomolecules to Treat Botulism.

J Toxicol 2012 20;2012:760142. Epub 2011 Sep 20.

Division of Infectious Diseases, Department of Biomedical Sciences, Tufts Cummings School of Veterinary Medicine, 200 Westboro Road, North Grafton, MA 01536, USA.

Botulinum neurotoxins (BoNTs) deliver a protease to neurons which can cause a flaccid paralysis called botulism. Development of botulism antidotes will require neuronal delivery of agents that inhibit or destroy the BoNT protease. Here, we investigated the potential of engineering Clostridium difficile toxin B (TcdB) as a neuronal delivery vehicle by testing two recombinant TcdB chimeras. For AGT-TcdB chimera, an alkyltransferase (AGT) was appended to the N-terminal glucosyltransferase (GT) of TcdB. Recombinant AGT-TcdB had alkyltransferase activity, and the chimera was nearly as toxic to Vero cells as wild-type TcdB, suggesting efficient cytosolic delivery of the AGT/GT fusion. For AGT-TcdB-BoNT/A-Hc, the receptor-binding domain (RBD) of TcdB was replaced by the equivalent RBD from BoNT/A (BoNT/A-Hc). AGT-TcdB-BoNT/A-Hc was >25-fold more toxic to neuronal cells and >25-fold less toxic to Vero cells than AGT-TcdB. Thus, TcdB can be engineered for cytosolic delivery of biomolecules and improved targeting of neuronal cells.
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http://dx.doi.org/10.1155/2012/760142DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3177232PMC
November 2011

Mouse relapse model of Clostridium difficile infection.

Infect Immun 2011 Jul 16;79(7):2856-64. Epub 2011 May 16.

Tufts Cummings School of Veterinary Medicine, North Grafton, Massachusetts 01536, USA.

Clostridium difficile is the causative agent of primary and recurrent antibiotic-associated diarrhea and colitis in hospitalized patients. The disease is caused mainly by two exotoxins, TcdA and TcdB, produced by the bacteria. Recurrent C. difficile infection (CDI) constitutes one of the most significant clinical issues of this disease, occurs in more than 20% of patients after the first episode, and may be increasing in frequency. However, there is no well-established animal model of CDI relapse currently available for studying disease pathogenesis, prevention, and therapy. Here we report the establishment of a conventional mouse model of recurrence/relapse CDI. We found that the primary episode of CDI induced little or no protective antibody response against C. difficile toxins and mice continued shedding C. difficile spores. Antibiotic treatment of surviving mice induced a second episode of diarrhea, while a simultaneous reexposure of animals to C. difficile bacteria or spores elicited a full spectrum of CDI similar to that of the primary infection. Moreover, mice treated with immunosuppressive agents were prone to more severe and fulminant recurrent disease. Finally, utilizing this model, we demonstrated that vancomycin only delayed disease recurrence, whereas neutralizing polysera against both TcdA and TcdB completely protected mice against CDI relapse. In conclusion, we have established a mouse relapse CDI model that allows for future investigations of the role of the host immune response in the disease's pathogenesis and permits critical testing of new therapeutics targeting recurrent disease.
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http://dx.doi.org/10.1128/IAI.01336-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3191975PMC
July 2011