Jimmy D. Ballard

Clostridium difficile Toxins: Mechanism of Action and Role in Disease

SUMMARY As the leading cause of hospital-acquired diarrhea, Clostridium difficile colonizes the large bowel of patients undergoing antibiotic therapy and produces two toxins, which cause notable disease pathologies. These two toxins, TcdA and TcdB, are encoded on a pathogenicity locus along with negative and positive regulators of their expression. Following expression and release from the bacterium, TcdA and TcdB translocate to the cytosol of target cells and inactivate small GTP-binding proteins, which include Rho, Rac, and Cdc42. Inactivation of these substrates occurs through monoglucosylation of a single reactive threonine, which lies within the effector-binding loop and coordinates a divalent cation critical to binding GTP. By glucosylating small GTPases, TcdA and TcdB cause actin condensation and cell rounding, which is followed by death of the cell. TcdA elicits effects primarily within the intestinal epithelium, while TcdB has a broader cell tropism. Important advances in the study of these toxins have been made in the past 15 years, and these are detailed in this review. The domains, subdomains, and residues of these toxins important for receptor binding and enzymatic activity have been elegantly studied and are highlighted herein. Furthermore, there have been major advances in defining the role of these toxins in modulating the inflammatory events involving the disruption of cell junctions, neuronal activation, cytokine production, and infiltration by polymorphonuclear cells. Collectively, the present review provides a comprehensive update on TcdA and TcdBs mechanism of action as well as the role of these toxins in disease.

pH-Induced Conformational Changes in Clostridium difficile Toxin B

ABSTRACT Toxin B from Clostridium difficile is a monoglucosylating toxin that targets substrates within the cytosol of mammalian cells. In this study, we investigated the impact of acidic pH on cytosolic entry and structural changes within toxin B. Bafilomycin A1 was used to block endosomal acidification and subsequent toxin B translocation. Cytopathic effects could be completely blocked by addition of bafilomycin A1 up to 20 min following toxin treatment. Furthermore, providing a low extracellular pH could circumvent the effect of bafilomycin A1 and other lysosomotropic agents. Acid pH-induced structural changes were monitored by using the fluorescent probe 2-(p-toluidinyl) naphthalene-6-sulfonic acid, sodium salt (TNS), inherent tryptophan fluorescence, and relative susceptibility to a specific protease. As the toxin was exposed to lower pH there was an increase in TNS fluorescence, suggesting the exposure of hydrophobic domains by toxin B. The change in hydrophobicity appeared to be reversible, since returning the pH to neutrality abrogated TNS fluorescence. Furthermore, tryptophan fluorescence was quenched at the acidic pH, indicating that domains may have been moving into more aqueous environments. Toxin B also demonstrated variable susceptibility to Staphylococcus aureus V8 protease at neutral and acidic pH, further suggesting pH-induced structural changes in this protein.

Variations in TcdB Activity and the Hypervirulence of Emerging Strains of Clostridium difficile

Hypervirulent strains of Clostridium difficile have emerged over the past decade, increasing the morbidity and mortality of patients infected by this opportunistic pathogen. Recent work suggested the major C. difficile virulence factor, TcdB, from hypervirulent strains (TcdBHV) was more cytotoxic in vitro than TcdB from historical strains (TcdBHIST). The current study investigated the in vivo impact of altered TcdB tropism, and the underlying mechanism responsible for the differences in activity between the two forms of this toxin. A combination of protein sequence analyses, in vivo studies using a Danio rerio model system, and cell entry combined with fluorescence assays were used to define the critical differences between TcdBHV and TcdBHIST. Sequence analysis found that TcdB was the most variable protein expressed from the pathogenicity locus of C. difficile. In line with these sequence differences, the in vivo effects of TcdBHV were found to be substantially broader and more pronounced than those caused by TcdBHIST. The increased toxicity of TcdBHV was related to the toxins ability to enter cells more rapidly and at an earlier stage in endocytosis than TcdBHIST. The underlying biochemical mechanism for more rapid cell entry was identified in experiments demonstrating that TcdBHV undergoes acid-induced conformational changes at a pH much higher than that of TcdBHIST. Such pH-related conformational changes are known to be the inciting step in membrane insertion and translocation for TcdB. These data provide insight into a critical change in TcdB activity that contributes to the emerging hypervirulence of C. difficile.

Clostridium difficile Infection

Clostridium difficile is the leading cause of hospital-acquired diarrhea in Europe and North America and is a serious reemerging pathogen. Recent outbreaks have led to increasing morbidity and mortality and have been associated with a new strain (BI/NAP1/027) of C difficile that produces more toxin than historic strains. With the increasing incidence of C difficile infection, clinicians have also seen a change in the epidemiology with increased infections in previously low-risk populations. This chapter highlights the current knowledge on C difficile virulence, human disease, epidemic outbreaks and optimal treatment strategies.

Mapping dominant-negative mutations of anthrax protective antigen by scanning mutagenesis

The protective antigen (PA) moiety of anthrax toxin transports edema factor and lethal factor to the cytosol of mammalian cells by a mechanism that depends on its ability to oligomerize and form pores in the endosomal membrane. Previously, some mutated forms of PA, designated dominant negative (DN), were found to coassemble with wild-type PA and generate defective heptameric pore-precursors (prepores). Prepores containing DN–PA are impaired in pore formation and in translocating edema factor and lethal factor across the endosomal membrane. To create a more comprehensive map of sites within PA where a single amino acid replacement can give a DN phenotype, we used automated systems to generate a Cys-replacement mutation for each of the 568 residues of PA63, the active 63-kDa proteolytic fragment of PA. Thirty-three mutations that reduced PAs ability to mediate toxicity at least 100-fold were identified in all four domains of PA63. A majority (22) were in domain 2, the pore-forming domain. Seven of the domain-2 mutations, located in or adjacent to the 2β6 strand, the 2β7 strand, and the 2β10-2β11 loop, gave the DN phenotype. This study demonstrates the feasibility of high-throughput scanning mutagenesis of a moderate sized protein. The results show that DN mutations cluster in a single domain and implicate 2β6 and 2β7 strands and the 2β10–2β11 loop in the conformational rearrangement of the prepore to the pore. They also add to the repertoire of mutations available for structure–function studies and for designing new antitoxic agents for treatment of anthrax.

Activation and mechanism of Clostridium septicum alpha toxin.

Clostridium septicum produces a single lethal factor, alpha toxin (AT), which is a cytolytic protein with a molecular mass of approximately 48kDa. The 48kDa toxin was found to be an inactive protoxin (ATpro) which could be activated via a carboxy‐terminal cleavage with trypsin. The cleavage site was located approximately 4kDa from the carboxy‐terminus. Proteolytically activated ATpro had a specific activity of approximately 1.5 × 106 haemolytic units mg‐1. The trypsin‐activated toxin (ATact) was haemolytic, stimulated a prelytic release of potassium ions from erythrocytes which was followed by haemoglobin release, induced channel formation in planar membranes and aggregated into a complex of Mr >210000 on erythrocyte membranes. ATpro did not exhibit these properties. ATact formed pores with a diameter of at least 1.3‐1.6 nm. We suggest that pore formation on target cell membranes is responsible for the cytolytic activity of alpha toxin.

Identification of Clostridium difficile toxin B cardiotoxicity using a zebrafish embryo model of intoxication

Clostridium difficile toxin B (TcdB) has been studied extensively by using cell-free systems and tissue culture, but, like many bacterial toxins, the in vivo targets of TcdB are unknown and have been difficult to elucidate with traditional animal models. In the current study, the transparent Danio rerio (zebrafish) embryo was used as a model for imaging of in vivo TcdB localization and organ-specific damage in real time. At 24 h after treatment, TcdB was found to localize at the pericardial region, and zebrafish exhibited the first signs of cardiovascular damage, including a 90% reduction in systemic blood flow and a 20% reduction in heart rate. Within 72 h of exposure to TcdB, the ventricle chamber of the heart became deformed and was unable to contract or pump blood, and the fish exhibited extensive pericardial edema. In line with the observed defects in ventricle contraction, TcdB was found to directly disrupt coordinated contractility and rhythmicity in primary cardiomyocytes. Furthermore, using a caspase-3 inhibitor, we were able to block TcdB-related cardiovascular damage and prevent zebrafish death. These findings present an insight into the in vivo targets of TcdB, as well as demonstrate the strength of the zebrafish embryo as a tractable model for identification of in vivo targets of bacterial toxins and evaluation of novel candidate therapeutics.

Clostridium difficile toxin B activates dual caspase‐dependent and caspase‐independent apoptosis in intoxicated cells

Clostridium difficile toxin B (TcdB) inactivates the small GTPases Rho, Rac and Cdc42 during intoxi‐cation of mammalian cells. In the current work, we show that TcdB has the potential to stimulate caspase‐dependent and caspase‐independent apoptosis. The apoptotic pathways became evident when caspase‐3‐processed‐vimentin was detected in TcdB‐treated HeLa cells. Caspase‐3 activation was subsequently confirmed in TcdB‐intoxicated HeLa cells. Interestingly, caspase inhibitor delayed TcdB‐induced cell death, but did not alter the time‐course of cytopathic effects. A similar effect was also observed in MCF‐7 cells, which are deficient in caspase‐3 activity. The time‐course to cell death was almost identical between cells treated with TcdB plus caspase inhibitor and cells intoxicated with the TcdB enzymatic domain (TcdB1–556). Unlike TcdB treated cells, intoxication with TcdB1–556 or expression of TcdB1–556 in a transfected cell line, did not stimulate caspase‐3 activation yet cells exhibited cytopathic effects and cell death. Although TcdB1–556 treated cells did not demonstrate caspase‐3 activation these cells were apoptotic as determined by differential annexin‐V/propidium iodide staining and nucleosomal DNA fragmentation. These data indicate TcdB triggers caspase‐independent apoptosis as a result of substrate inactivation and may evoke caspase‐dependent apoptosis due to a second, yet undefined, activity of TcdB. This is the first example of a bacterial virulence factor with the potential to stimulate multiple apoptotic pathways in host cells.

Inhibition of Bacillus anthracis Spore Outgrowth by Nisin

ABSTRACT The lantibiotic nisin has previously been reported to inhibit the outgrowth of spores from several Bacillus species. However, the mode of action of nisin responsible for outgrowth inhibition is poorly understood. By using B. anthracis Sterne 7702 as a model, nisin acted against spores with a 50% inhibitory concentration (IC50) and an IC90 of 0.57 μM and 0.90 μM, respectively. Viable B. anthracis organisms were not recoverable from cultures containing concentrations of nisin greater than the IC90. These studies demonstrated that spores lose heat resistance and become hydrated in the presence of nisin, thereby ruling out a possible mechanism of inhibition in which nisin acts to block germination initiation. Rather, germination initiation is requisite for the action of nisin. This study also revealed that nisin rapidly and irreversibly inhibits growth by preventing the establishment of oxidative metabolism and the membrane potential in germinating spores. On the other hand, nisin had no detectable effects on the typical changes associated with the dissolution of the outer spore structures (e.g., the spore coats, cortex, and exosporium). Thus, the action of nisin results in the uncoupling of two critical sequences of events necessary for the outgrowth of spores: the establishment of metabolism and the shedding of the external spore structures.

Effects of Endogenous d-Alanine Synthesis and Autoinhibition of Bacillus anthracis Germination on In Vitro and In Vivo Infections

ABSTRACT Bacillus anthracis transitions from a dormant spore to a vegetative bacillus through a series of structural and biochemical changes collectively referred to as germination. The timing of germination is important during early steps in infection and may determine if B. anthracis survives or succumbs to responsive macrophages. In the current study experiments determined the contribution of endogenous d-alanine production to the efficiency and timing of B. anthracis spore germination under in vitro and in vivo conditions. Racemase-mediated production of endogenous d-alanine by B. anthracis altered the kinetics for initiation of germination over a range of spore densities and exhibited a threshold effect wherein small changes in spore number resulted in major changes in germination efficiency. This threshold effect correlated with d-alanine production, was prevented by an alanine racemase inhibitor, and required l-alanine. Interestingly, endogenous production of inhibitory levels of d-alanine was detected under experimental conditions that did not support germination and in a germination-deficient mutant of B. anthracis. Racemase-dependent production of d-alanine enhanced survival of B. anthracis during interaction with murine macrophages, suggesting a role for inhibition of germination during interaction with these cells. Finally, in vivo experiments revealed an approximately twofold decrease in the 50% lethal dose of B. anthracis spores administered in the presence of d-alanine, indicating that rates of germination may be directly influenced by the levels of this amino acid during early stages of disease.