Identification of Clostridium Difficile Toxin B Cardiotoxicity Using a Zebrafish Embryo Model of Intoxication

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

Elaine E. Hamm, Daniel E. Voth, and Jimmy D. Ballard*

Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104

Edited by Stanley Falkow, Stanford University, Stanford, CA, and approved July 31, 2006 (received for review June 6, 2006) Clostridium difficile toxin B (TcdB) has been studied extensively by life-threatening cases of CDAD, including cardiopulmonary using cell-free systems and tissue culture, but, like many bacterial arrest (18), acute respiratory distress syndrome (19), multiple toxins, the in vivo targets of TcdB are unknown and have been organ failure (20), renal failure (21), and liver damage (22). difficult to elucidate with traditional animal models. In the current Hence, identification of the cells targeted in vivo by TcdB is study, the transparent Danio rerio (zebrafish) embryo was used as needed to gain relevant insight into the disease-related activities a model for imaging of in vivo TcdB localization and organ-specific of this toxin and advance our understanding of CDAD. damage in real time. At 24 h after treatment, TcdB was found to Identification of systemic targets of bacterial toxins such as localize at the pericardial region, and zebrafish exhibited the first TcdB has been limited, because it is difficult to directly visualize signs of cardiovascular damage, including a 90% reduction in the impact of these proteins on major organs in real time. To systemic blood flow and a 20% reduction in heart rate. Within 72 h overcome this problem, zebrafish embryos were used herein to of exposure to TcdB, the ventricle chamber of the heart became characterize the systemic impact of TcdB in real time. Unlike deformed and was unable to contract or pump blood, and the fish other vertebrates, zebrafish embryos are transparent, and major exhibited extensive pericardial edema. In line with the observed organs can be visualized by standard light microscopy (23). Thus, defects in ventricle contraction, TcdB was found to directly disrupt zebrafish embryos provide a unique system for directly visual- coordinated contractility and rhythmicity in primary cardiomyo- izing the temporal and spatial effects of TcdB intoxication. By cytes. Furthermore, using a caspase-3 inhibitor, we were able to using the zebrafish embryo as a model, in the current work, we block TcdB-related cardiovascular damage and prevent zebrafish have found that TcdB functions as potent cardiotoxin, reducing death. These findings present an insight into the in vivo targets of blood flow and ventricle contraction. Furthermore, correspond- TcdB, as well as demonstrate the strength of the zebrafish embryo ing to TcdB’s known proapoptotic activity, a caspase-3 inhibitor as a tractable model for identification of in vivo targets of bacterial was found to alleviate the cardiotoxic effects of TcdB. These toxins and evaluation of novel candidate therapeutics. findings provide important insight into the in vivo activities of TcdB and present the zebrafish embryo as a model for deter- ͉ ͉ bacterial toxin Clostridium difficile-associated disease mining the systemic targets of bacterial toxins. large clostridial toxins Results rotein toxins are produced by bacterial pathogens during dis- Localizaton of TcdB in Zebrafish Embryos. To determine the local- Pease and have evolved different functions, ranging from pore ization of TcdB, zebrafish were treated with TcdBAlexa-546 and formation in plasma membranes to enzymatic activities that alter examined by fluorescence microscopy for sites of toxin tropism. intracellular signaling, cell cycle, apoptosis, and protein synthesis in As shown in Fig. 1, after a 24-h treatment with the toxin, targeted cells (1). Mechanisms of receptor binding, cell entry, TcdBAlexa-546 localized at the frontal ventral portion of the fish, membrane insertion, and enzymology are routinely determined by with specific foci formed within the pericardial region. Local- using a broad range of cell types in vitro, yet for many toxins, the cell ization of TcdBAlexa-546 was also observed in an anatomical types targeted during disease are unknown (2). region corresponding to the outflow chamber of the heart (see Clostridium difficile toxin B (TcdB) is an example of a bacterial arrow in Fig. 1A). Magnified views, as shown in Fig. 9 A and B, toxin studied extensively in vitro, but the in vivo activities remain which is published as supporting information on the PNAS web ϭ ͞ poorly understood (3). TcdB is a potent (LD50 200 ng kg) site, reveal intense localization near the cardiac region. In intracellular bacterial toxin; the protein enters cells by receptor- contrast, the negative control, BSAAlexa-546 did not show detect- mediated endocytosis; translocates to the cytosol; hydrolyzes able anatomical localization within the zebrafish (Fig. 9 C UDP-glucose; and transfers the liberated sugar to a reactive and D). threonine in the effector-binding loops of the small GTPases To further demonstrate specificity of TcdB localization, com- Rho, Rac, and Cdc42 (4–7). As a result, cultured cells treated petition experiments were performed by using the putative with TcdB exhibit changes in cell morphology and undergo receptor-binding domain (RBD) of TcdB. As shown in Fig. 1B, apoptosis, eventually leading to the death of the cell (8–10). cotreatment with a 30-fold molar excess of the TcdB RBD TcdB intoxicates numerous cell types in vitro, including fibro- reduced the detectable levels of labeled toxin to that observed blasts, neuronal cells, epithelial cells, endothelial cells, lympho- cytes, and hepatocytes (4, 11–15), yet whether any of these cell types are targeted during C. difficile-associated disease (CDAD) Author contributions: E.E.H. and J.D.B. designed research; E.E.H. and D.E.V. performed is unknown. research; E.E.H. and J.D.B. analyzed data; and E.E.H. and J.D.B. wrote the paper. In addition to TcdB, C. difficile also produces toxin A (TcdA), The authors declare no conflict of interest. which is known to function as an enterotoxin, causing gastroin- This paper was submitted directly (Track II) to the PNAS office. testinal damage (16, 17). Previous studies have shown that TcdB Abbreviations: TcdB, Clostridium difficile toxin B; CDAD, Clostridium difficile-associated is effective only when the intestinal mucosa is damaged (17), disease; PA, protective antigen; LFn, anthrax toxin lethal factor; RBD, receptor-binding suggesting that the intestinal effects of TcdA facilitate the entry domain; RCm, rat cardiomyocyte(s). of TcdB into the bloodstream. The TcdA-mediated release of *To whom correspondence should be addressed. E-mail: [email protected]. TcdB could explain the systemic complications observed in © 2006 by The National Academy of Sciences of the USA

14176–14181 ͉ PNAS ͉ September 19, 2006 ͉ vol. 103 ͉ no. 38 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0604725103 Downloaded by guest on September 25, 2021 Fig. 2. RBC perfusion rate and vein integrity of TcdB-treated zebrafish. RBC perfusion rate was used as a measurement of blood flow and calculated as the average number of RBC per 30 s in the intersegmental veins (n ϭ 27), with error bars representing standard error. Zebrafish treated with TcdB (solid bars) had a reduced RBC rate compared with the heat-inactivated TcdB control (hatched bars).

RBC per 30 s (P Ͻ 0.0001; see Fig. 2 and Movies 1 and 2, which are published as supporting information on the PNAS web site). Reduced blood flow was observed in the caudal and intersegmental veins and appeared to occur in the absence of detectable damage to the vascular endothelium. To confirm this, fli1::EGFP zebrafish, which express GFP in endothelial cells, were used to assess vein integrity after treatment with TcdB. As shown in Fig. 10B, which is published as supporting information on the PNAS web site, despite a loss in blood flow, the veins of toxin-treated zebrafish appeared to be intact. TcdB-treated zebrafish were examined for cardiac damage, as a possible explanation for the reduction in blood flow. Between 24 and 48 h after treatment, there was a decrease in ventricle Fig. 1. TcdB localization observed in zebrafish. Fluorescently labeled TcdB chamber contractility and a loss in heart looping (see Fig. 3; see was used to follow localization of the toxin to specific anatomical regions. (A) also Fig. 11 and Movies 3 and 4, which are published as Zebrafish treated with 37 nM TcdBAlexa-546 for 24 h. Arrows indicate toxin

supporting information on the PNAS web site). As shown in Fig. MICROBIOLOGY accumulation around the pericardial sac as well as distinct foci on the yolk sac 3, in control fish, the ventricle exhibited a dynamic change in size Alexa-546 and upper cranial region. (B) Zebrafish treated with 37 nM TcdB and of 20% during contraction and expansion (see Movie 3). How- 370 nM TcdB (RBD). (C) Brightfield image of zebrafish treated with 37 nM TcdBAlexa-546 and 370 nM TcdB (RBD). Arrows 1, 2, and 3 denote the heart, yolk ever, treatment with TcdB substantially reduced the change in sac, and eye of zebrafish, respectively. ventricle size during beating (see Movie 4), indicating the heart was unable to contract and expand in a normal fashion. At Ϸ48 h after treatment, both the atrium and ventricle were deformed in the BSA control. Collectively, these observations suggested (Fig. 4). By 7 days after treatment, 100% of TcdB-treated fish TcdB exhibits specific tissue tropism in the zebrafish, with the exhibited pericardial edema, with 70% developing whole-body toxin primarily localizing to the yolk-sac, pericardial, and cardiac edema (Fig. 5). TcdB-treated fish survived between 7 and 10 regions of the zebrafish. days after initial exposure to TcdB, but by 11 days after treatment, 100% of the fish succumbed to the effects of the toxin. Treatment of Zebrafish Embryos with TcdB Results in Damage to the Similar cardiovascular defects were observed in fish treated with Cardiovascular System. Experiments were performed to deter- TcdB 24, 72, and 96 h after fertilization, indicating that toxin mine the effects of TcdB on zebrafish physiology. For initial effects did not depend upon treatment at a particular stage of analysis, zebrafish embryos were collected 24 h after fertilization and exposed to TcdB, heat-inactivated TcdB, or buffer alone. Treatment with doses ranging from 0.037 to 0.37 nM did not cause detectable damage to the zebrafish embryos (data not shown). However, exposure of the embryos to doses of toxin ranging from 3.7 to 37 nM resulted in distinct dose-dependent changes in zebrafish physiology and anatomy. The time course and specific changes in physiology are summarized in Table 2, which is published as supporting information on the PNAS web site. A decrease in heart rate was the first physiological change observed [72 Ϯ 6 beats per 30 s in the control compared with 57 Ϯ 3 beats per 30 s in TcdB-treated fish (P Ͻ 0.001)]. Corresponding to the reduced heart rate, a visible reduction in blood flow [calculated as the RBC perfusion rate] was also observed. In control fish, the RBC perfusion rate within Ϯ Fig. 3. Comparisons of contraction and expansion dynamics of the ventricle the intersegmental veins was 402 10.6 RBC per 30 s at 24 h in control- and TcdB-treated zebrafish embryos. Plot of relative changes in after treatment. In comparison, in TcdB-treated zebrafish, the ventricle size over a 10-s time period in zebrafish treated for 24 h with 37 nM RBC perfusion rate within intersegmental veins was 44 Ϯ 1.49 heat-inactivated TcdB (solid line) or 37 nM TcdB (dotted line).

Hamm et al. PNAS ͉ September 19, 2006 ͉ vol. 103 ͉ no. 38 ͉ 14177 Downloaded by guest on September 25, 2021 Fig. 4. Morphological changes in zebrafish heart after exposure to TcdB. (A) Zebrafish embryos treated with 37 nM heat-inactivated TcdB for 48 h. (B) Zebrafish embryos treated with 37 nM TcdB for 48 h. Fig. 6. Representative photographs of zebrafish after a 72-h exposure to PA plus LFn-TcdB1–556.(A) Zebrafish treated with 0.85 nM LFn-TcdB1–556 alone at 72 h after fertilization resembled untreated zebrafish. (B) Zebrafish treated development (data not shown). Collectively, these observed with 0.85 nM PA and LFn-TcdB1–556. Arrow indicates tissue damage (visualized defects indicated TcdB disrupts cardiac function. as tissue discoloration).

Delivery of the TcdB Enzymatic Domain with a Surrogate System domain with a system that targets multiple tissues, should reduce Results in Systemic Damage. Experiments were next performed to these effects, because the enzymatic domain would be distrib- determine whether the cardiotoxicity of TcdB was due to uted throughout the body. Alternatively, if cardiotoxicity were heightened sensitivity of this organ to the toxin’s enzymatic due to heightened sensitivity of cardiac tissue to the enzymatic activity, or whether this effect results from a preferential local- activity of TcdB, the heart should be preferentially impacted ization of the toxin to the heart. Arguably, if TcdB cardiotoxicity despite localization of the enzymatic domain to multiple tissues. were due to specific toxin tropism, then delivery of the enzymatic To address this issue, we took advantage of a previously described heterologous delivery system derived from the cell entry components of anthrax lethal toxin, which consists of Bacillus anthracis protective antigen (PA) and anthrax toxin lethal factor (LFn)TcdB1–556, a fusion protein consisting of the translocation active nontoxic 255 N-terminal residues of LFn and the enzymatic glucosylation region of TcdB (amino acids 1–556; refs. 24–26). Previous studies have shown that PA can target multiple tissues within the zebrafish (27, 28). As shown in Fig. 6, zebrafish embryos treated with PA and LFnTcdB1–556 exhibited wide- spread tissue damage, which differed substantially from that observed in TcdB-treated zebrafish. These findings suggest that TcdB-related cardiac damage may involve a specific tropism for cardiac tissue.

TcdB Modulates Cardiomyocyte Physiology. Results from the zebrafish treatments indicated that TcdB could influence cardiac function, perhaps by directly targeting functional cells of the heart. To further elucidate TcdB-cardiotoxic effects, experiments next sought to determine whether this toxin is capable of disrupting the physiology of cardiomyocytes. In these experiments, cultured cardiomyocytes were treated with TcdB and examined for changes in overall morphology, contraction, and viability. Within2hoftoxintreatment, the cardiomyocytes exhibited morphological changes. Phalloidin staining of actin in TcdB-treated cardiomyocytes revealed distinct changes in cel- lular structure when compared with cardiomyocytes treated with Fig. 5. Representative photographs of zebrafish after exposure to TcdB. (A) heat-inactivated TcdB (see Fig. 7 A and B). Control cells had Zebrafish treated with heat-inactivated 37 nM TcdB for 72 h. (B) Zebrafish numerous dense bodies of fibrils and bundled Z bands (Fig. 7A), treated with 37 nM TcdB for 72 h. Arrows indicate regions of massive edema whereas these structures were undetectable in cardiomyocytes observed after cardiac damage. treated with TcdB (Fig. 7B).By5haftertreatment, the

14178 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0604725103 Hamm et al. Downloaded by guest on September 25, 2021 Fig. 8. Caspase-3 inhibitor reduces the cardiotoxic effects of TcdB. After concomitant exposure to TcdB and caspase-3 inhibitor, zebrafish were examined for reduction of TcdB-related phenotype, specifically decrease in heart function. The RBC perfusion rate of treated zebrafish over time (C, heat- inactivated TcdB control; I, caspase-3 inhibitor IV; and T, TcdB). RBC perfusion rate is calculated as the average number of RBC per 30 s in the intersegmental veins (n ϭ 27), with error bars representing standard error. Heart rate (Inset) was determined 72 h after exposure to heat-inactivated TcdB, caspase-3 inhibitor IV, TcdB plus caspase-3 inhibitor IV, or TcdB. Heart rate was determined in triplicate for each condition and is reported as the mean and standard error of five experimental samples.

Fig. 7. Impact of TcdB on primary RCm. (A) Rhodamine phalloidin actin stain TcdB-treated zebrafish (see Fig. 12, which is published as of RCm treated with heat-inactivated TcdB (7.4 nM). (B) RCm treated with TcdB (7.4 nM) and stained for actin. (C) Plot of changes in the number of RCm supporting information on the PNAS web site). These results contractions over time. RCm cells were treated with heat-inactivated TcdB indicate the cardiotoxic effects of TcdB can be alleviated by a (solid line) or TcdB (dotted line). Contractions were measured 4 h after caspase-3 inhibitor. treatment as number of contractions per 60 s. Discussion Tissue damage and inactivation of specific cell types by bacterial cardiomyocyte contractions were less coordinated and arrhyth- toxins are an integral part of many infections and are important mic (Fig. 7C; see also Movies 5 and 6, which are published as for colonization, immune evasion, and progression of disease. supporting information on the PNAS web site). Finally, 48 h Thus, toxin immunogenicity and mechanisms of action have after treatment, there was a 60% decline in cardiomyocyte been studied for over a century, leading to new vaccines and an

viability (data not shown). understanding of these virulence factors at the molecular level. MICROBIOLOGY Yet, despite numerous advances in the study of bacterial toxins, Caspase-3 Inhibitor Reduces Cardiovascular Damage in TcdB-Treated little is known about cell types targeted in vivo (2). In particular, Zebrafish. TcdB is known to cause cytotoxicity by apoptosis, and and important to the current study, are the facts that, although blocking caspase activity in cell culture slows the rate of death TcdB is a potent cytotoxin, has a low LD50, and causes rapid after treatment with TcdB (10). Hence, experiments were de- death in animal models, the overall physiological systems im- signed to determine whether cardiotoxicity correlates with the pacted by this toxin have not been identified. ability of TcdB to induce apoptosis, and whether inhibition of Although in vitro studies have provided important insight into apoptosis could provide protection against the systemic effects TcdB’s mechanisms of cell entry, membrane translocation, and of this toxin. In these experiments, zebrafish embryos were enzymatic activity, it is difficult to apply this knowledge to the treated with TcdB and cotreated with Ac-DMQD-CHO, a in vivo setting. In vitro analysis can neither mimic toxin receptor water-soluble tetrapeptide inhibitor of caspase-3. TcdB-treated availability within the host nor reflect overall organ sensitivity to and control zebrafish were observed for changes in heart beat TcdB. Therefore, to characterize TcdB’s systemic effects, we and overall blood flow. As shown in Table 1, zebrafish treated sought an animal model that would allow direct in vivo visual- with TcdB exhibited a significant decrease in heartbeats per 30 s ization of events leading to death after exposure to the toxin. as compared with the control, yet zebrafish cotreated with Contemporary models, such as higher-order primates, rodents caspase-3 inhibitor presented heart rates similar to control (29), Drosophila melanogaster (30–32), and Caenorhabditis el- zebrafish along with a normal RBC perfusion rate (Fig. 8). The egans (33, 34), were considered for assessing the systemic effects events quantified in Fig. 8 can be viewed as a video (see Movie of TcdB, but these lacked many of the qualities needed for the 7, which is published as supporting information on the PNAS current study. It is difficult to directly visualize all of the major web site). Furthermore, the addition of caspase-3 inhibitor organs in higher-order models, and the fruit-fly and nematode decreased the frequency and severity of damage observed in the systems lack the organ complexity needed for a thorough study of systemic damage. In contrast, the zebrafish embryo provides several distinct advantages over these traditional models. In Table 1. Heart rate of treated zebraﬁsh addition to having many of the major organs found in humans, Heartbeat per 30 s, zebrafish embryos are transparent, which allows direct visual- Treatment 72 h after treatment ization of labeled toxin and toxin-induced changes in anatomy and physiology (23). Indeed, these same characteristics have Heat-inactivated TcdB 72 Ϯ 6 made the zebrafish a widely accepted model for the study of Caspase-3 inhibitor 76 Ϯ 7 embryonic development and genetics (35) and infectious dis- TcdB 57 Ϯ 3 eases (36–42). TcdB ϩ caspase-3 78 Ϯ 2 The phenotypes of TcdB-treated zebrafish support the notion inhibitor that intoxication with TcdB leads to cardiovascular damage.

Hamm et al. PNAS ͉ September 19, 2006 ͉ vol. 103 ͉ no. 38 ͉ 14179 Downloaded by guest on September 25, 2021 Indeed, many of the changes observed in the TcdB-treated (24–26). The 2,165 nucleotides from the 3Ј end of the tcdb embryos have been reported in mutant lines of zebrafish defec- gene, which encode for the putative RBD of TcdB, were cloned tive in genes necessary for a functional heart. For example, dead in-frame into the pET15b plasmid (Novagen, San Diego, CA). beat (ded; refs. 43 and 44) and heartstrings (hst; ref. 45) are All recombinant proteins were expressed as a His6 fusion in documented to possess cardiac defects that result in poor Escherichia coli͞BL-21 DE3 and isolated by using Ni2ϩ affinity contractility and pericardial edema (ded) or stretching and loss chromatography according to the manufacturer’s protocol of function of cardiac chambers as well as a loss in circulation (Novagen). (hst). These phenotypes were observed in the TcdB-treated embryos, further suggesting that the toxin impacts cardiac Fluorescent Labeling of Protein. TcdB and BSA were labeled with function. Additionally, results from toxin localization experi- a reactive fluorescent dye Alexa-Fluor-546, according to the ments using TcdB labeled with a trackable marker and the manufacturer’s instructions [Molecular Probes͞Invitrogen delivery of the TcdB enzymatic domain with PA and LFn (Carlsbad, CA)]. The relative activity of labeled TcdB to unla- support the idea that the toxin preferentially localizes to the beled TcdB was determined by using a standard cytotoxicity heart. A specific in vivo affinity for cardiac tissue may explain assay. Labeling of TcdB did not reduce the effective cytotoxic why TcdB can damage many cell types in vitro but primarily dose of the toxin by Ͼ20%. impacts the heart in the zebrafish. In zebrafish embryo studies, loss in chamber contractility was Zebrafish Maintenance and Care. Wild-type zebrafish were ob- observed, allowing for the identification of a relevant candidate tained from Aquatic Eco-System (Apopka, FL), and mutant cell type, cardiomyocytes, for further studies in cell culture. fli1::EGFP fish were obtained from the Zebrafish Information Whether cardiomyocytes are the only cardiac cells impacted by Network (ZFIN, University of Oregon, Eugene, OR). Ze- TcdB is not known; however, loss of cardiomyocytes will have the brafish were maintained at 28.5°C on a 14-h light͞10-h dark most dramatic impact on the heart and may provide an expla- cycle in a Z plex unit (Aquatic Habitats, Apopka, FL) and nation for TcdB-related defects in contractility. These cells are matings, embryo collection, and preparation were performed central to heart contraction and the movement of blood; thus, as described (23). even subtle intoxication events could have dramatic effects. Furthermore, unlike cardiac fibroblasts and endothelial cells, In Vivo Toxin Localization Studies Using Fluorescently Labeled TcdB. cardiomyocytes are not renewable, and loss of these cells can Zebrafish embryos were placed into a 96-well plate (five result in chronic heart problems (46, 47). It seems reasonable to embryos per well) 24 h after fertilization and allowed to predict that if other cells are impacted by TcdB within the heart, incubate with TcdBAlexa-546 (3.7–100 nM) or TcdBAlexa-546 death of cardiomyocytes would have a more severe and sustained (3.7–100 nM) and a 30-fold molar excess of TcdB RBD for 24 h. effect on the host. Control zebrafish were incubated with 100 nM BSAAlexa-546. In cardiac diseases that involve activation of apoptosis in Subsequently, zebrafish were rinsed 10 times in embryo water cardiomyocytes, caspase inhibitors have been promoted as prom- for 20 min and visualized by using an Olympus (Melville, NY) ising treatments (48–50). Moreover, prior work has shown that BX81 epifluorescent microscope. Images were captured inhibitors of apoptosis, caspase inhibitors in particular, reduce and processed by using the Nikon (Florham Park, NJ) Spot the cytotoxic effects of TcdB (10). Thus, it was hypothesized that Software. inhibition of caspase-3 would alleviate the cardiac damage

caused by this toxin. The results of our study show that TcdB’s 1–556 damaging and fatal cardiotoxic effects could be prevented Treatment of Zebrafish Embryos with TcdB and LFnTcdB . For through the use of a caspase-3 inhibitor. This is an example in TcdB studies, zebrafish embryos were placed (five embryos per well) into a 96-well plate and treated with 37 nM TcdB, which a caspase inhibitor blocked in vivo effects of a bacterial ⅐ exotoxin, which has not been previously described. Moreover, heat-inactivated TcdB, or 20 mM Tris HCl buffer in replicates of 10 (50 embryo per experimental condition). Similarly, these results suggest these compounds and other modulators of 1–556 apoptosis could be promising therapeutics for treating advanced LFnTcdB -treated zebrafish were placed (five embryos per well) into a 96-well plate and treated with 0.42–0.85 nM PA and CDAD. 1–556 It is important to consider the current findings in the context LFnTcdB in replicates of 10 (50 embryos per treatment). 1–556 of CDAD in humans. Cardiotoxicity could explain many of the Controls included 0.85 nM PA or LFnTcdB and 20 mM ⅐ observed clinical signs of serious CDAD. Patients with advanced Tris HCl. The embryos were observed for 7 days after treatment CDAD experience multiorgan failure, and decreases in cardiac for morphological changes by using a SZX-7 microscope with a function could be among the factors contributing to this event DP70 camera (Nikon). All still and video images were captured (20). Death of CDAD patients has also been directly associated and processed by using DP controller and DP manager software with cardiac arrest (18, 51–53). Collectively, these data suggest (Nikon). To calculate blood circulation, the RBC perfusion rate that inactivation of Rho, Rac, and Cdc42 by TcdB leads to was measured by using the SZX-7 Nikon microscope with a altered cardiac activity and cell death in the heart of CDAD DP70 video camera and is recorded as the number of blood cells patients. Ongoing studies assessing cardiac damage in infectious detected within the intersegmental veins over a 30-s time period. models of CDAD will help in directly addressing this hypothesis. RBC perfusion rate was measured in replicate countings in three In summary, results from the current study provide insight into separate veins in three separate fish and is reported as number a possible mechanism by which C. difficile causes severe systemic of RBC per 30 s. damage during CDAD. By functioning as a cardiotoxin, TcdB may directly or indirectly cause much of the systemic damage Treatment of Rat Cardiomyocytes with TcdB. Rat cardiomyocyte observed in CDAD patients. These findings also indicate that the (RCm) cells (Cell Applications, San Diego, CA) were seeded zebrafish embryo is a valuable model for identifying systemic into 96-well plates, treated in triplicate with 7.4 nM TcdB or targets of bacterial virulence factors, and that this model is useful heat-inactivated TcdB for 5 h, and subsequently stained with in the in vivo assessment of toxin therapeutics. rhodamine phalloidin according to the manufacturer’s instructions (Molecular Probes͞Invitrogen). Images were acquired by Materials and Methods using a TCS NT confocal microscope (Leica, Deerfield, IL) Protein Isolation. TcdB was isolated as described (54). Bacillus and processed by using Confocal Software (Leica). RCm cell anthracis PA and LFnTcdB1–556 were isolated as described viability after TcdB treatment was quantified across a 72-h

14180 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0604725103 Hamm et al. Downloaded by guest on September 25, 2021 time period by using the Cell Counting Kit-8 (CCK-8; Dojindo, rate and RBC perfusion rate, as well as a phenotype using an Gaithersburg, MD). SZX-7 Nikon microscope with a DP70 camera. All still and video images were captured and processed by using the DP Caspase-3 Inhibitor Assays. Zebrafish embryos were placed into a controller and manager software. 96-well plate (five embryos per well) in sterile embryo water, treated concomitantly with TcdB (37 nM) and caspase-3 inhib- Statistical Analysis of Data. All statistical data were calculated by itor IV (Calbiochem, 500 ␮M), and observed up to 1 wk after using Student’s two-tailed t test. treatment. Controls for this experiment included 500 ␮M caspase-3 inhibitor IV, TcdB (37 nM), and heat-inactivated This work was supported in part by Public Health Service, National TcdB (37 nM). The embryos were examined for changes in heart Institute of Child Health and Human Development Grant HD044861.

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