Publications by authors named "Hanping Feng"

66 Publications

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

Therapeutic Mechanism of Macrophage Inflammatory Protein 1 α Neutralizing Antibody (CCL3) in Clostridium difficile Infection in Mice.

J Infect Dis 2020 04;221(10):1623-1635

Vatche and Tamar Manoukian Division of Digestive Diseases, David Geffen School of Medicine at the University of California Los Angeles, Los Angeles, California, USA.

Background: Clostridium difficile infection (CDI) causes diarrhea and colitis. We aimed to find a common pathogenic pathway in CDI among humans and mice by comparing toxin-mediated effects in human and mouse colonic tissues.

Method: Using multiplex enzyme-linked immunosorbent assay, we determined the cytokine secretion of toxin A- and B-treated human and mouse colonic explants.

Results: Toxin A and toxin B exposure to fresh human and mouse colonic explants caused different patterns of cytokine secretion. Toxin A induced macrophage inflammatory protein (MIP) 1α secretion in both human and mouse explants. Toxin A reduced the expression of chloride anion exchanger SLC26A3 expression in mouse colonic explants and human colonic epithelial cells. Patients with CDI had increased colonic MIP-1 α expression and reduced colonic SLC26A3 (solute carrier family 26, member 3) compared with controls. Anti-MIP-1 α neutralizing antibody prevented death, ameliorated colonic injury, reduced colonic interleukin 1β (IL-1β) messenger RNA expression, and restored colonic SLC26a3 expression in C. difficile-infected mice. The anti-MIP-1 α neutralizing antibody prevented CDI recurrence. SLC26a3 inhibition augmented colonic IL-1 β messenger RNA expression and abolished the protective effect of anti-MIP-1 α neutralizing antibody in mice with CDI.

Conclusion: MIP-1 α is a common toxin A-dependent chemokine in human and mouse colon. MIP-1 α mediates detrimental effects by reducing SLC26a3 and enhancing IL-1 β expression in the colon.
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http://dx.doi.org/10.1093/infdis/jiz640DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7184920PMC
April 2020

Designed Ankyrin Repeat Protein (DARPin) Neutralizers of TcdB from Clostridium difficile Ribotype 027.

mSphere 2019 10 2;4(5). Epub 2019 Oct 2.

Department of Microbial Pathogenesis and Immunology, Texas A&M University Health Science Center, College Station, Texas, USA

infection (CDI) is a leading cause of hospital-acquired diarrhea. In recent decades, the emergence of the "hypervirulent" BI/NAP1/027 strains of significantly increased the morbidity and mortality of CDI. The pathogenesis of CDI is primarily mediated by the action of two toxins, TcdA and TcdB, with TcdB being the major virulent factor in humans. In this report, we describe the engineering of a panel of designed ankyrin repeat proteins (DARPins) that potently neutralize TcdB from the BI/NAP1/027 strains (e.g., TcdB). The most effective DARPin, D16, inhibits TcdB with a 50% effective concentration (EC) of 0.5 nM, which is >66-fold lower than that of the FDA-approved anti-TcdB antibody bezlotoxumab (EC, ∼33 nM). Competitive enzyme-linked immunosorbent assays (ELISAs) showed that D16 blocks interactions between TcdB and its receptor, chondroitin sulfate proteoglycan 4 (CSPG4). The dimeric DARPin U3D16, which pairs D16 with DARPin U3, a disrupter of the interaction of TcdB with Frizzled 1/2/7 receptor, exhibits 10-fold-to-20-fold-enhanced neutralization potency against TcdB from strains VPI 10463 (laboratory strain) and M68 (CF/NAP9/017) but identical activity against TcdB relative to D16. Subsequent ELISAs revealed that TcdB did not significantly interact with Frizzled 1/2/7. Computation modeling revealed 4 key differences at the Frizzled 1/2/7 binding interface which are likely responsible for the significantly reduced binding affinity. We report the engineering and characterization of designed ankyrin proteins as potent neutralizers of TcdB toxin secreted by a hypervirulent ribotype 027 strain of We further show that although TcdB toxins from both ribotype 027 and VPI 10461 interact efficiently with TcdB receptors CSPG4 and Pvrl3, TcdB lacks significant ability to bind the only known physiologically relevant TcdB receptor, Frizzled 1/2/7.
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http://dx.doi.org/10.1128/mSphere.00596-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6796971PMC
October 2019

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

Host-targeted niclosamide inhibits C. difficile virulence and prevents disease in mice without disrupting the gut microbiota.

Nat Commun 2018 12 7;9(1):5233. Epub 2018 Dec 7.

Molecular Medicine, Hospital for Sick Children, 686 Bay St., Toronto, ON, M5G 0A4, Canada.

Clostridium difficile is the leading cause of nosocomial diarrhea and colitis in the industrialized world. Disruption of the protective gut microbiota by antibiotics enables colonization by multidrug-resistant C. difficile, which secrete up to three different protein toxins that are responsible for the gastrointestinal sequelae. Oral agents that inhibit the damage induced by toxins, without altering the gut microbiota, are urgently needed to prevent primary disease and break the cycle of antibiotic-induced disease recurrence. Here, we show that the anthelmintic drug, niclosamide, inhibits the pathogenesis of all three toxins by targeting a host process required for entry into colonocytes by each toxin. In mice infected with an epidemic strain of C. difficile, expressing all three toxins, niclosamide reduced both primary disease and recurrence, without disrupting the diversity or composition of the gut microbiota. Given its excellent safety profile, niclosamide may address an important unmet need in preventing C. difficile primary and recurrent diseases.
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http://dx.doi.org/10.1038/s41467-018-07705-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6286312PMC
December 2018

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

TPL2 Is a Key Regulator of Intestinal Inflammation in Clostridium difficile Infection.

Infect Immun 2018 08 23;86(8). Epub 2018 Jul 23.

Department of Molecular Medicine, Morsani School of Medicine, University of South Florida, Tampa, Florida, USA

Tumor progression locus 2 (TPL2), a serine/threonine protein kinase, is a major inflammatory mediator in immune cells. The predominant inflammatory actions of TPL2 depend on the activation of mitogen-activated protein kinases (MAPK) and the upregulated production of the cytokines tumor necrosis factor alpha (TNF-α) and interleukin 1β (IL-1β) in macrophages and dendritic cells in response to lipopolysaccharide (LPS). Significant increases in TNF-α, IL-6, IL-β, and IL-8 levels in patients with infection (CDI) have been reported. Both TNF-α and IL-6 have been postulated to play key roles in the systemic inflammatory response in CDI, and IL-8 is essential for the development of local intestinal inflammatory responses in CDI. The objective of this study was to elucidate the role of TPL2 in the pathogenesis of CDI. We found that TPL2 was significantly activated in human and mouse intestinal tissues upon toxin exposure or CDI. We further demonstrated that TPL2 knockout (TPL2-KO) mice were significantly more resistant to CDI than wild-type mice, with significantly reduced production of TNF-α, IL-6, IL-1β, KC (a mouse homologue of IL-8), and myeloperoxidase (MPO) in the ceca and colons of TPL2-KO mice. Finally, we found that TPL2 inhibition by a specific inhibitor or TPL2 gene ablation significantly reduced TcdB-induced production of TNF-α, IL-6, IL-β, and KC by inhibiting the activation of p38, extracellular signal-regulated kinase (ERK), and c-Jun NH-terminal kinase (JNK). Taken together, our data suggest that TPL2 represents a potential therapeutic target for CDI treatment.
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http://dx.doi.org/10.1128/IAI.00095-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6056850PMC
August 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

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

Cytokines Are Markers of the Clostridium difficile-Induced Inflammatory Response and Predict Disease Severity.

Clin Vaccine Immunol 2017 Aug 4;24(8). Epub 2017 Aug 4.

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

The host immune response affects pathogen virulence in infection (CDI). Thus, cytokine responses to CDI likely are associated with disease initiation and progression. Understanding the molecular drivers of inflammation and biochemical markers of disease severity is important for developing novel therapies and predicting disease prognosis. In this study, we investigated cytokine production in patients with CDI and evaluated the potential of cytokines to serve as biomarkers for CDI and predictors of disease severity. The systemic cytokine profiles of 36 CDI patients (20 with severe disease) and 8 healthy donors and the toxin-induced cytokine profiles of peripheral blood mononuclear cells (PBMC) were determined. Further, we evaluated glucosyltransferase (GT) activity in regulation of toxin-induced cytokine expression. We found upregulation of the majority of measured cytokines (11/20, 55%) in CDI patients. Interleukin-1β (IL-1β), IL-6, IL-8, IL-17A, and IL-16 were the most upregulated. High serum levels of IL-2 and IL-15 were associated with a poor prognosis in CDI patients, whereas high levels of IL-5 and gamma interferon (IFN-γ) were associated with less severe disease. Both TcdA and TcdB were potent inducers of cytokine responses, as demonstrated by stimulation of a greater number and amount of cytokines. In addition to confirming prior reports on the role of IL-8, IL-1β, and IL-6 in CDI, our data suggest that IL-16 and IL-17A, as well as the IL-1β/Th17 axis, play a key role in driving inflammatory responses in CDI. A functional GT domain of toxins was required for the induction of a majority of cytokines investigated.
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http://dx.doi.org/10.1128/CVI.00037-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5583471PMC
August 2017

Correction: Chen, S., et al. Identification of an Essential Region for Translocation of Clostridium difficile Toxin B. Toxins 2016, 8, 241.

Toxins (Basel) 2016 Dec 2;8(12). Epub 2016 Dec 2.

School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, China.

n/a.
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http://dx.doi.org/10.3390/toxins8120352DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5198547PMC
December 2016

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

Identification of an Essential Region for Translocation of Clostridium difficile Toxin B.

Toxins (Basel) 2016 08 15;8(8). Epub 2016 Aug 15.

School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, China.

Clostridium difficile toxin A (TcdA) and toxin B (TcdB) are the major virulence factors involved in C. difficile-associated diarrhea and pseudomembranous colitis. TcdA and TcdB both contain at least four distinct domains: the glucosyltransferase domain, cysteine protease domain, receptor binding domain, and translocation domain. Few studies have investigated the translocation domain and its mechanism of action. Recently, it was demonstrated that a segment of 97 amino acids (AA 1756-1852, designated D97) within the translocation domain of TcdB is essential for the in vitro and in vivo toxicity of TcdB. However, the mechanism by which D97 regulates the action of TcdB in host cells and the important amino acids within this region are unknown. In this study, we discovered that a smaller fragment, amino acids 1756-1780, located in the N-terminus of the D97 fragment, is essential for translocation of the effector glucosyltransferase domain into the host cytosol. A sequence of 25AA within D97 is predicted to form an alpha helical structure and is the critical part of D97. The deletion mutant TcdB∆1756-1780 showed similar glucosyltransferase and cysteine protease activity, cellular binding, and pore formation to wild type TcdB, but it failed to induce the glucosylation of Rho GTPase Rac1 of host cells. Moreover, we found that TcdB∆1756-1780 was rapidly degraded in the endosome of target cells, and therefore its intact glucosyltransferase domain was unable to translocate efficiently into host cytosol. Our finding provides an insight into the molecular mechanisms of action of TcdB in the intoxication of host cells.
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http://dx.doi.org/10.3390/toxins8080241DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4999857PMC
August 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

Pilin Vaccination Stimulates Weak Antibody Responses and Provides No Protection in a C57Bl/6 Murine Model of Acute Infection.

J Vaccines Vaccin 2016;7(3). Epub 2016 May 27.

Department of Medicine, Division of Infectious Disease, University of Maryland School of Medicine, Baltimore, Maryland, USA.

is the leading cause of nosocomial infections in the United States, adding billions of dollars per year to health care costs. A vaccine targeted against the bacterium would be extremely beneficial in decreasing the morbidity and mortality caused by -associated disease; a vaccine directed against a colonization factor would hinder the spread of the bacterium as well as prevent disease. Type IV pili (T4Ps) are extracellular appendages composed of protein monomers called pilins. They are involved in adhesion and colonization in a wide variety of bacteria and archaea, and are putative colonization factors in . We hypothesized that vaccinating mice with pilins would lead to generation of anti-pilin antibodies, and would protect against challenge. We found that immunizing C57Bl/6 mice with various pilins, whether combined or as individual proteins, led to low anti-pilin antibody titers and no protection upon challenge. Passive transfer of anti-pilin antibodies led to high serum anti-pilin IgG titers, but to undetectable fecal anti-pilin IgG titers and did not protect against challenge. The low antibody titers observed in these experiments may be due to the particular strain of mice used. Further experiments, possibly with a different animal model of infection, are needed to determine if an anti-T4P vaccine would be protective against infection.
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http://dx.doi.org/10.4172/2157-7560.1000321DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4927082PMC
May 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

The Brucella melitensis M5-90 phosphoglucomutase (PGM) mutant is attenuated and confers protection against wild-type challenge in BALB/c mice.

World J Microbiol Biotechnol 2016 Apr 29;32(4):58. Epub 2016 Feb 29.

College of Animal Science and Technology, Shihezi University, Shihezi, 832000, Xinjiang Province, People's Republic of China.

Brucellae are Gram-negative intracellular bacterial pathogens that infect humans and animals, bringing great economic burdens to developing countries. Live attenuated Brucella vaccines (strain M5-90 or others) are the most efficient means for prevention and control of animal brucellosis. However, these vaccines have several drawbacks, including residual virulence in animals, and difficulties in differentiating natural infection from vaccine immunization, which limit their application. A vaccine that can differentiate infection from immunization will have extensive applications. A Brucella melitensis (B. melitensis) strain M5-90 pgm mutant (M5-90Δpgm) was constructed to overcome these drawbacks. M5-90Δpgm showed significantly reduced survival in embryonic trophoblast cells and in mice, and induced high protective immunity in BALB/c mice. Moreover, M5-90Δpgm elicited an anti-Brucella-specific immunoglobulin G response and induced the secretion of gamma interferon (IFN-γ) and interleukin-2 (IL-2). In addition, M5-90Δpgm induced the secretion of IFN-γ in immunized sheep. Serum samples from sheep inoculated with M5-90Δpgm were negative by the Rose Bengal Plate Test (RBPT) and Standard Tube Agglutination Test (STAT). Furthermore, the PGM antigen allowed serological differentiation between infected and vaccinated animals. These results suggest that M5-90Δpgm is an ideal live attenuated vaccine candidate against B. melitensis 16 M and deserves further evaluation for vaccine development.
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http://dx.doi.org/10.1007/s11274-016-2015-6DOI Listing
April 2016

The Complete Genome of Brucella Suis 019 Provides Insights on Cross-Species Infection.

Genes (Basel) 2016 Jan 26;7(2). Epub 2016 Jan 26.

College of Life Sciences, Nankai University, Tianjin 300071, China.

Brucella species are the most important zoonotic pathogens worldwide and cause considerable harm to humans and animals. In this study, we presented the complete genome of B. suis 019 isolated from sheep (ovine) with epididymitis. B. suis 019 has a rough phenotype and can infect sheep, rhesus monkeys and possibly humans. The comparative genome analysis demonstrated that B. suis 019 is closest to the vaccine strain B. suis bv. 1 str. S2. Further analysis associated the rsh gene to the pathogenicity of B. suis 019, and the WbkA gene to the rough phenotype of B. suis 019. The 019 complete genome data was deposited in the GenBank database with ID PRJNA308608.
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http://dx.doi.org/10.3390/genes7020007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4773751PMC
January 2016

Defective mutations within the translocation domain of Clostridium difficile toxin B impair disease pathogenesis.

Pathog Dis 2016 Feb 26;74(1):ftv098. Epub 2015 Oct 26.

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

The Clostridium difficile toxin B is one of the main virulence factors and plays an important role in the pathogenesis of C. difficile infection (CDI). We recently revealed crucial residues in the translocation domain of TcdB for the pore formation and toxin translocation. In this study, we investigated the effects of mutating a critical site involved in pore formation, Leu-1106, to residues that differ in size and polarity (Phe, Ala, Cys, Asp). We observed a broad range of effects on TcdB function in vitro consistent with the role of this site in pore formation and translocation. We show that mice challenged systemically with a lethal dose (LD100) of the most defective mutant (L1106K) showed no symptoms of disease highlighting the importance of this residue and the translocation domain in disease pathogenesis. These findings offer insights into the structure function of the toxin translocation pore, and inform novel therapeutic strategies against CDI.
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http://dx.doi.org/10.1093/femspd/ftv098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4882082PMC
February 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

Identification of toxemia in patients with Clostridium difficile infection.

PLoS One 2015 17;10(4):e0124235. Epub 2015 Apr 17.

Department of Microbial Pathogenesis, University of Maryland Dental School, Baltimore, Maryland, United States of America.

Toxemia can develop in Clostridium difficile-infected animals, and correlates with severe and fulminant disease outcomes. Circumstantial evidence suggests that toxemia may occur in patients with C. difficile infection (CDI), but positive diagnosis is extremely rare. We analyzed the potential for C. difficile toxemia in patients, determined its characteristics, and assessed challenges. C. difficile toxins in serum from patients were tested using an ultrasensitive cell-based assay and further confirmed by Rac1 glucosylation assay. The factors that hinder a diagnosis of toxemia were assessed, including investigation of toxin stability, the level of toxins-specific neutralizing antibodies in sera and its effect on diagnosis limits. CDI patients develop detectable toxemia in some cases (2.3%). Toxins were relatively stable in stored sera. Neutralizing anti-toxin antibodies were present during infection and positively correlated with the diagnosis limits. Thus, the masking effect of toxin-specific neutralizing antibodies is the major obstacle in diagnosing C. difficile toxemia using cell-based bioassays.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0124235PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4401762PMC
April 2016

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

Pathog Dis 2015 Apr 27;73(3). Epub 2015 Jan 27.

Department of Microbial Pathogenesis, University of Maryland Dental School, Baltimore, MD 21021, USA

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.
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http://dx.doi.org/10.1093/femspd/ftv008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4435672PMC
April 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

Chondroitin sulfate proteoglycan 4 functions as the cellular receptor for Clostridium difficile toxin B.

Cell Res 2015 Feb 30;25(2):157-68. Epub 2014 Dec 30.

Biodynamic Optical Imaging Center (BIOPIC), State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing 100871, China.

As a gram-positive, spore-forming anaerobic bacillus, Clostridium difficile (C. difficile) is responsible for severe and fatal pseudomembranous colitis, and poses the most urgent antibiotic resistance threat worldwide. Epidemic C. difficile is the leading cause of antibiotic-associated diarrhoea globally, especially diarrhoea due to the emergence of hypervirulent strains associated with high mortality and morbidity. TcdB, one of the key virulence factors secreted by this bacterium, enters host cells through a poorly understood mechanism to elicit its pathogenic effect. Here we report the first identification of the TcdB cellular receptor, chondroitin sulfate proteoglycan 4 (CSPG4). CSPG4 was initially isolated from a whole-genome human shRNAmir library screening, and its role was confirmed by both TALEN- and CRISPR/Cas9-mediated gene knockout in human cells. CSPG4 is critical for TcdB binding to the cell surface, inducing cytoskeleton disruption and cell death. A direct interaction between the N-terminus of CSPG4 and the C-terminus of TcdB was confirmed, and the soluble peptide of the toxin-binding domain of CSPG4 could protect cells from the action of TcdB. Notably, the complete loss of CSPG4/NG2 decreased TcdB-triggered interleukin-8 induction in mice without significantly affecting animal mortality. Based on both the in vitro and in vivo studies, we propose a dual-receptor model for TcdB endocytosis. The discovery of the first TcdB receptor reveals a previously unsuspected role for CSPG4 and provides a new therapeutic target for the treatment of C. difficile infection.
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http://dx.doi.org/10.1038/cr.2014.169DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4650570PMC
February 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

Single domain antibody coated gold nanoparticles as enhancer for Clostridium difficile toxin detection by electrochemical impedance immunosensors.

Bioelectrochemistry 2015 Feb;101:153-8

This work presents a sandwich-type electrochemical impedance immunosensor for detecting Clostridium difficile toxin A (TcdA) and toxin B (TcdB). Single domain antibody conjugated gold nanoparticles were applied to amplify the detection signal. Gold nanoparticles (Au NPs) were characterized by transmission electron microscopy and UV–vis spectra. The electron transfer resistance (Ret) of the working electrode surface was used as a parameter in the measurement of the biosensor. With the increase of the concentration of toxins from 1 pg/mL to 100 pg/mL, a linear relationship was observed between the relative electron transfer resistance and toxin concentration. In addition, the detection signal was enhanced due to the amplification effect. The limit of detection for TcdA and TcdB was found to be 0.61 pg/mL and 0.60 pg/mL respectively at a signal-to-noise ratio of 3 (S/N = 3). This method is simple, fast and ultrasensitive, thus possesses a great potential for clinical applications in the future.
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http://dx.doi.org/10.1016/j.bioelechem.2014.10.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4706752PMC
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