Hanping Feng

Expression of recombinant Clostridium difficile toxin A and B in Bacillus megaterium.

BackgroundMajor Clostridium difficile virulence factors are the exotoxins TcdA and TcdB. Due to the large size and poor stability of the proteins, the active recombinant TcdA and TcdB have been difficult to produce.ResultsThe toxin genes tcdA and tcdB were amplified by PCR using chromosomal DNA from a toxigenic strain as a template, and cloned into a shuttle vector pHis1522. The sequences of both tcdA and tcdB genes in the vector have been verified by DNA sequencing. The constructs were transformed into B. megaterium protoplasts and the protein expression was controlled under a xylose promoter. The recombinant toxins (rTcdA and rTcdB) were purified from bacterial crude extracts. Approximately 5 – 10 mg of highly purified recombinant toxins were obtained from one liter of bacterial culture. The resulting rTcdA and rTcdB had similar molecular masses to the native toxins, and their biological activities were found to be similar to their native counterparts after an extensive examination.ConclusionWe have generated the full length and active recombinant TcdA and TcdB in Bacillus megaterium.

Tumor-derived chaperone-rich cell lysates are effective therapeutic vaccines against a variety of cancers

Abstract. With the clinical use of purified, tumor-derived chaperone proteins as anti-cancer vaccines already in clinical trial stages, we have focused our attention on the utility of chaperone-rich cell lysates (CRCL) in cancer immunotherapy. CRCL, as prepared from tumor lysates via a free solution-isoelectric focusing (FS-IEF) technique, is a high-yield vaccine enriched for numerous chaperone proteins. We have compared the efficacy of CRCL vaccines to that of individual chaperone protein vaccines in in vivo settings, including ELISPOT assays, tumor-growth assays and survival assays. In all experiments, CRCL vaccines were at least as effective, and in some settings perhaps even more effective, than either of the two most heavily studied components of CRCL, HSP70 and GRP94/gp96, in reduction in tumor growth and prolongation of survival in both prophylactic and pre-existing tumor settings against tumors of diverse origin and genetic background. Combining CRCL preparations with dendritic cells ex vivo resulted in a cellular vaccine that could eradicate pre-existing tumors in a high percentage of cases. The high yields of CRCL vaccines from small quantities of starting materials, the relative ease of its procurement and the functional data presented here suggest that CRCL vaccines are worthy of evaluation in pilot clinical trial cancer immunotherapy protocols.

Mouse Relapse Model of Clostridium difficile Infection

ABSTRACT 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 diseases pathogenesis and permits critical testing of new therapeutics targeting recurrent disease.

A Direct Role for C1 Inhibitor in Regulation of Leukocyte Adhesion

Plasma C1 inhibitor (C1INH) is a natural inhibitor of complement and contact system proteases. Heterozygosity for C1INH deficiency results in hereditary angioedema, which is mediated by bradykinin. Treatment with plasma C1INH is effective not only in patients with hereditary angioedema, but also in a variety of other disease models, in which such therapy is accompanied by diminished neutrophil infiltration. The underlying mechanism has been explained primarily as a result of the inhibition of the complement and contact systems. We have shown that C1INH expresses the sialyl-Lewisx tetrasaccharide on its N-linked glycan, via which it binds to E- and P-selectins and interferes with leukocyte-endothelial adhesion in vitro. Here we show that both native C1INH and reactive center cleaved C1INH significantly inhibit selectin-mediated leukocyte adhesion in several in vitro and in vivo models, whereas N-deglycosylated C1INH loses such activities. The data support the hypothesis that C1INH plays a direct role in leukocyte-endothelial cell adhesion, that the activity is mediated by carbohydrate, and that it is independent of protease inhibitory activity. Direct involvement of C1INH in modulation of selectin-mediated cell adhesion may be an important mechanism in the physiologic suppression of inflammation, and may partially explain its utility in therapy of inflammatory diseases.

Host S-nitrosylation inhibits clostridial small molecule-activated glucosylating toxins

The global prevalence of severe Clostridium difficile infection highlights the profound clinical significance of clostridial glucosylating toxins. Virulence is dependent on the autoactivation of a toxin cysteine protease, which is promoted by the allosteric cofactor inositol hexakisphosphate (InsP6). Host mechanisms that protect against such exotoxins are poorly understood. It is increasingly appreciated that the pleiotropic functions attributed to nitric oxide (NO), including host immunity, are in large part mediated by S-nitrosylation of proteins. Here we show that C. difficile toxins are S-nitrosylated by the infected host and that S-nitrosylation attenuates virulence by inhibiting toxin self-cleavage and cell entry. Notably, InsP6- and inositol pyrophosphate (InsP7)-induced conformational changes in the toxin enabled host S-nitrosothiols to transnitrosylate the toxin catalytic cysteine, which forms part of a structurally conserved nitrosylation motif. Moreover, treatment with exogenous InsP6 enhanced the therapeutic actions of oral S-nitrosothiols in mouse models of C. difficile infection. Allostery in bacterial proteins has thus been successfully exploited in the evolutionary development of nitrosothiol-based innate immunity and may provide an avenue to new therapeutic approaches.

Piglet Models of Acute or Chronic Clostridium difficile Illness

We examined the piglet model of Clostridium difficile illness (CDI) in humans, because swine are naturally susceptible to C. difficile. The piglet is a reproducible model of acute or chronic CDI with characteristic pseudomembranous colitis. Germ-free piglets were consistently and extensively colonized after oral challenge with the human strain 027/BI/NAP1, establishing an infectious dose-age relationship. This allowed a demarcation between acute fatal and chronic models. The clinical manifestations of disease inclusive of gastrointestinal and systemic symptoms and characteristic mucosal lesions of the large bowel (including pseudomembranous colitis) are described. Additionally, we demonstrate the presence of toxins in feces, body fluids, and serum and a significant elevation in interleukin 8 levels in animals with severe disease. We conclude that piglets infected with C. difficile mimic many of the key characteristics observed in humans with CDI and are suitable animals in which to investigate the role played by virulence attributes, drug efficacy, and vaccine candidates.

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

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

Listeria-Infected Myeloid Dendritic Cells Produce IFN-β, Priming T Cell Activation

The intracellular bacterium Listeria monocytogenes infects dendritic cells (DC) and other APCs and induces potent cell-mediated protective immunity. However, heat-killed bacteria fail to do so. This study explored whether DC differentially respond to live and killed Listeria and how this affects T cell activation. To control for bacterial number, a replication-deficient strain, Lmdd, defective in d-alanine biosynthesis, was used. We found that DC internalize both live and heat-killed Lmdd and similarly up-regulate the expression of costimulatory molecules, a necessary step for T cell activation. However, only live Lmdd-infected DC stimulate T cells to express the early activation marker CD69 and enhance T cell activation upon TCR engagement. Infection with live, but not heat-killed, Lmdd induces myeloid DC to secrete copious amounts of IFN-β, which requires bacterial cytosolic invasion. Exposure to high concentrations of IFN-β sensitizes naive T cells for Ag-dependent activation.

The Enterotoxicity of Clostridium difficile Toxins

The major virulence factors of Clostridium difficile infection (CDI) are two large exotoxins A (TcdA) and B (TcdB). However, our understanding of the specific roles of these toxins in CDI is still evolving. It is now accepted that both toxins are enterotoxic and proinflammatory in the human intestine. Both purified TcdA and TcdB are capable of inducing the pathophysiology of CDI, although most studies have focused on TcdA. C. difficile toxins exert a wide array of biological activities by acting directly on intestinal epithelial cells. Alternatively, the toxins may target immune cells and neurons once the intestinal epithelial barrier is disrupted. The toxins may also act indirectly by stimulating cells to produce chemokines, proinflammatory cytokines, neuropeptides and other neuroimmune signals. This review considers the mechanisms of TcdA- and TcdB-induced enterotoxicity, and recent developments in this field.

Granzyme B Binds to Target Cells Mostly by Charge and Must Be Added at the Same Time as Perforin to Trigger Apoptosis

Perforin (PFN) delivery of granzymes (Gzm) into the target cell at the immunological synapse is the major pathway for inducing apoptosis of virus-infected cells and tumors. A validated model for how PFN delivers Gzm into the cytosol is still lacking. PFN was originally thought to work by forming pores in the target cell plasma membrane that allow Gzm entry. This model was questioned when it was shown that GzmB is endocytosed without PFN. Moreover, apoptosis could be triggered by adding PFN to washed cells that have previously endocytosed GzmB. In this study, we show that GzmB binds to the plasma membrane mostly via nonspecific charge interactions. Washing in saline does not remove bound Gzm. However, if externally bound GzmB is completely removed, subsequent addition of PFN does not release previously endocytosed GzmB and does not trigger apoptosis. Therefore, PFN must be coendocytosed with GzmB to deliver it into the cytosol.