Clostridium perfringens Epsilon Toxin Binds to and Kills Primary Human Lymphocytes.

Clostridium perfringens epsilon toxin (ETX) is the third most lethal bacterial toxin and has been suggested to be an environmental trigger of multiple sclerosis, an immune-mediated disease of the human central nervous system. However, ETX cytotoxicity on primary human cells has not been investigated. In this article, we demonstrate that ETX preferentially binds to and kills human lymphocytes expressing increased levels of the myelin and lymphocyte protein MAL. Using flow cytometry, ETX binding was determined to be time and dose dependent and was highest for CD4+ cells, followed by CD8+ and then CD19+ cells. Similar results were seen with ETX-induced cytotoxicity. To determine if ETX preference for CD4+ cells was related to MAL expression, MAL gene expression was determined by RT-qPCR. CD4+ cells had the highest amount of Mal gene expression followed by CD8+ and CD19+ cells. These data indicate that primary human cells are susceptible to ETX and support the hypothesis that MAL is a main receptor for ETX. Interestingly, ETX bindings to human lymphocytes suggest that ETX may influence immune response in multiple sclerosis.

Epsilon toxin-producing Clostridium perfringens colonize the multiple sclerosis gut microbiome overcoming CNS immune privilege.

Multiple sclerosis (MS) is a complex disease of the CNS thought to require an environmental trigger. Gut dysbiosis is common in MS, but specific causative species are unknown. To address this knowledge gap, we used sensitive and quantitative PCR detection to show that people with MS were more likely to harbor and show a greater abundance of epsilon toxin-producing (ETX-producing) strains of C. perfringens within their gut microbiomes compared with individuals who are healthy controls (HCs). Isolates derived from patients with MS produced functional ETX and had a genetic architecture typical of highly conjugative plasmids. In the active immunization model of experimental autoimmune encephalomyelitis (EAE), where pertussis toxin (PTX) is used to overcome CNS immune privilege, ETX can substitute for PTX. In contrast to PTX-induced EAE, where inflammatory demyelination is largely restricted to the spinal cord, ETX-induced EAE caused demyelination in the corpus callosum, thalamus, cerebellum, brainstem, and spinal cord, more akin to the neuroanatomical lesion distribution seen in MS. CNS endothelial cell transcriptional profiles revealed ETX-induced genes that are known to play a role in overcoming CNS immune privilege. Together, these findings suggest that ETX-producing C. perfringens strains are biologically plausible pathogens in MS that trigger inflammatory demyelination in the context of circulating myelin autoreactive lymphocytes.

CNS endothelial derived extracellular vesicles are biomarkers of active disease in multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is a complex, heterogenous disease characterized by inflammation, demyelination, and blood-brain barrier (BBB) permeability. Currently, active disease is determined by physician confirmed relapse or detection of contrast enhancing lesions via MRI indicative of BBB permeability. However, clinical confirmation of active disease can be cumbersome. As such, disease monitoring in MS could benefit from identification of an easily accessible biomarker of active disease. We believe extracellular vesicles (EV) isolated from plasma are excellent candidates to fulfill this need. Because of the critical role BBB permeability plays in MS pathogenesis and identification of active disease, we sought to identify EV originating from central nervous system (CNS) endothelial as biomarkers of active MS. Because endothelial cells secrete more EV when stimulated or injured, we hypothesized that circulating concentrations of CNS endothelial derived EV will be increased in MS patients with active disease. METHODS: To test this, we developed a novel method to identify EV originating from CNS endothelial cells isolated from patient plasma using flow cytometry. Endothelial derived EV were identified by the absence of lymphocyte or platelet markers CD3 and CD41, respectively, and positive expression of pan-endothelial markers CD31, CD105, or CD144. To determine if endothelial derived EV originated from CNS endothelial cells, EV expressing CD31, CD105, or CD144 were evaluated for expression of the myelin and lymphocyte protein MAL, a protein specifically expressed by CNS endothelial cells compared to endothelial cells of peripheral organs. RESULTS: Quality control experiments indicate that EV detected using our flow cytometry method are 0.2 to 1 micron in size. Flow cytometry analysis of EV isolated from 20 healthy controls, 16 relapsing-remitting MS (RRMS) patients with active disease not receiving disease modifying therapy, 14 RRMS patients with stable disease not receiving disease modifying therapy, 17 relapsing-RRMS patients with stable disease receiving natalizumab, and 14 RRMS patients with stable disease receiving ocrelizumab revealed a significant increase in the plasma concentration of CNS endothelial derived EV in patients with active disease compared to all other groups (p = 0.001). CONCLUSIONS: For the first time, we have identified a method to identify CNS endothelial derived EV in circulation from human blood samples. Results from our pilot study indicate that increased levels of CNS endothelial derived EV may be a biomarker of BBB permeability and active disease in MS.

In vivo Blood-brain Barrier Permeability Assays Using Clostridium perfringens Epsilon Toxin.

In order for the brain to function properly, a carefully orchestrated homeostasis must be maintained. To help regulate this delicate balance, the brain has developed a highly selective blood-brain barrier (BBB). Under normal conditions, the BBB excludes harmful blood-borne material from the brain parenchyma. However, numerous neuropathological conditions can disrupt this barrier, causing BBB permeability and subsequent CNS dysfunction. Understanding the mechanisms involved in BBB permeability are essential to elucidating the pathology of various neurological disorders as well as identifying methods for drug delivery to the CNS. Here, we describe several in vivo methods to measure BBB permeability in mice using an array of diverse sized tracers including exogenous 376 Da fluorescein salt, 66.5 kDa bovine serum albumin, and 70 kDa dextran as well as endogenous 160 kDa mouse IgG. When administered intravenously, these substances are excluded from a healthy brain by the BBB. However, BBB dysfunction can allow entry of these tracers into the brain and this accumulation can be measured using spectrophotometry, fluorescent microscopy, and immunohistochemistry. We also describe a method to induce BBB permeability using Clostridium perfringens epsilon toxin. Finally, we include a short discussion about the advantages and disadvantages of each method and their appropriate downstream applications.

Clostridium perfringens epsilon toxin induces blood brain barrier permeability via caveolae-dependent transcytosis and requires expression of MAL.

Clostridium perfringens epsilon toxin (ETX) is responsible for causing the economically devastating disease, enterotoxaemia, in livestock. It is well accepted that ETX causes blood brain barrier (BBB) permeability, however the mechanisms involved in this process are not well understood. Using in vivo and in vitro methods, we determined that ETX causes BBB permeability in mice by increasing caveolae-dependent transcytosis in brain endothelial cells. When mice are intravenously injected with ETX, robust ETX binding is observed in the microvasculature of the central nervous system (CNS) with limited to no binding observed in the vasculature of peripheral organs, indicating that ETX specifically targets CNS endothelial cells. ETX binding to CNS microvasculature is dependent on MAL expression, as ETX binding to CNS microvasculature of MAL-deficient mice was not detected. ETX treatment also induces extravasation of molecular tracers including 376Da fluorescein salt, 60kDA serum albumin, 70kDa dextran, and 155kDA IgG. Importantly, ETX-induced BBB permeability requires expression of both MAL and caveolin-1, as mice deficient in MAL or caveolin-1 did not exhibit ETX-induced BBB permeability. Examination of primary murine brain endothelial cells revealed an increase in caveolae in ETX-treated cells, resulting in dynamin and lipid raft-dependent vacuolation without cell death. ETX-treatment also results in a rapid loss of EEA1 positive early endosomes and accumulation of large, RAB7-positive late endosomes and multivesicular bodies. Based on these results, we hypothesize that ETX binds to MAL on the apical surface of brain endothelial cells, causing recruitment of caveolin-1, triggering caveolae formation and internalization. Internalized caveolae fuse with early endosomes which traffic to late endosomes and multivesicular bodies. We believe that these multivesicular bodies fuse basally, releasing their contents into the brain parenchyma.

Clostridium perfringens Epsilon Toxin Compromises the Blood-Brain Barrier in a Humanized Zebrafish Model.

Clostridium perfringens epsilon toxin (ETX) is hypothesized to mediate blood-brain barrier (BBB) permeability by binding to the myelin and lymphocyte protein (MAL) on the luminal surface of endothelial cells (ECs). However, the kinetics of this interaction and a general understanding of ETX's behavior in a live organism have yet to be appreciated. Here we investigate ETX binding and BBB breakdown in living Danio rerio (zebrafish). Wild-type zebrafish ECs do not bind ETX. When zebrafish ECs are engineered to express human MAL (hMAL), proETX binding occurs in a time-dependent manner. Injection of activated toxin in hMAL zebrafish initiates BBB leakage, hMAL downregulation, blood vessel stenosis, perivascular edema, and blood stasis. We propose a kinetic model of MAL-dependent ETX binding and neurovascular pathology. By generating a humanized zebrafish BBB model, this study contributes to our understanding of ETX-induced BBB permeability and strengthens the proposal that MAL is the ETX receptor.

Identification of epsilon toxin-producing Clostridium perfringens strains in American retail food.

Food samples (n = 216) from New York city were tested for the presence of C. perfringens via PCR for specific toxin genes. Thirty-four (16%) samples were positive for C. perfringens. Of these 34, 31 (91.2%) were type A or E, one (2.9%) was type B, and two (5.9%) were type D.

A Novel Panel of Rabbit Monoclonal Antibodies and Their Diverse Applications Including Inhibition of Clostridium perfringens Epsilon Toxin Oligomerization.

The pore-forming epsilon toxin (ETX) produced by Clostridium perfringens is among the most lethal bacterial toxins known. Sensitive antibody-based reagents are needed to detect toxin, distinguish mechanisms of cell death, and prevent ETX toxicity. Using B-cell immuno-panning and cloning techniques, seven ETX-specific monoclonal antibodies were generated from immunized rabbits. ETX specificity and sensitivity were evaluated via western blot, ELISA, immunocytochemistry (ICC), and flow cytometry. ETX-neutralizing function was evaluated both in vitro and in vivo. All antibodies recognized both purified ETX and epsilon protoxin via western blot with two capable of detecting the ETX-oligomer complex. Four antibodies detected ETX via ELISA and three detected ETX bound to cells via ICC or flow cytometry. Several antibodies prevented ETX-induced cell death by either preventing ETX binding or by blocking ETX oligomerization. Antibodies that blocked ETX oligomerization inhibited ETX endocytosis and cellular vacuolation. Importantly, one of the oligomerization-blocking antibodies was able to protect against ETX-induced death post-ETX exposure in vitro and in vivo. Here we describe the production of a panel of rabbit monoclonal anti-ETX antibodies and their use in various biological assays. Antibodies possessing differential specificity to ETX in particular conformations will aid in the mechanistic studies of ETX cytotoxicity, while those with ETX-neutralizing function may be useful in preventing ETX-mediated mortality.

Clostridium perfringens Epsilon Toxin Causes Selective Death of Mature Oligodendrocytes and Central Nervous System Demyelination.

UNLABELLED: Clostridium perfringens epsilon toxin (ε-toxin) is responsible for a devastating multifocal central nervous system (CNS) white matter disease in ruminant animals. The mechanism by which ε-toxin causes white matter damage is poorly understood. In this study, we sought to determine the molecular and cellular mechanisms by which ε-toxin causes pathological changes to white matter. In primary CNS cultures, ε-toxin binds to and kills oligodendrocytes but not astrocytes, microglia, or neurons. In cerebellar organotypic culture, ε-toxin induces demyelination, which occurs in a time- and dose-dependent manner, while preserving neurons, astrocytes, and microglia. ε-Toxin specificity for oligodendrocytes was confirmed using enriched glial culture. Sensitivity to ε-toxin is developmentally regulated, as only mature oligodendrocytes are susceptible to ε-toxin; oligodendrocyte progenitor cells are not. ε-Toxin sensitivity is also dependent on oligodendrocyte expression of the proteolipid myelin and lymphocyte protein (MAL), as MAL-deficient oligodendrocytes are insensitive to ε-toxin. In addition, ε-toxin binding to white matter follows the spatial and temporal pattern of MAL expression. A neutralizing antibody against ε-toxin inhibits oligodendrocyte death and demyelination. This study provides several novel insights into the action of ε-toxin in the CNS. (i) ε-Toxin causes selective oligodendrocyte death while preserving all other neural elements. (ii) ε-Toxin-mediated oligodendrocyte death is a cell autonomous effect. (iii) The effects of ε-toxin on the oligodendrocyte lineage are restricted to mature oligodendrocytes. (iv) Expression of the developmentally regulated proteolipid MAL is required for the cytotoxic effects. (v) The cytotoxic effects of ε-toxin can be abrogated by an ε-toxin neutralizing antibody. IMPORTANCE: Our intestinal tract is host to trillions of microorganisms that play an essential role in health and homeostasis. Disruption of this symbiotic relationship has been implicated in influencing or causing disease in distant organ systems such as the brain. Epsilon toxin (ε-toxin)-carrying Clostridium perfringens strains are responsible for a devastating white matter disease in ruminant animals that shares similar features with human multiple sclerosis. In this report, we define the mechanism by which ε-toxin causes white matter disease. We find that ε-toxin specifically targets the myelin-forming cells of the central nervous system (CNS), oligodendrocytes, leading to cell death. The selectivity of ε-toxin for oligodendrocytes is remarkable, as other cells of the CNS are unaffected. Importantly, ε-toxin-induced oligodendrocyte death results in demyelination and is dependent on expression of myelin and lymphocyte protein (MAL). These results help complete the mechanistic pathway from bacteria to brain by explaining the specific cellular target of ε-toxin within the CNS.

The Myelin and Lymphocyte Protein MAL Is Required for Binding and Activity of Clostridium perfringens ε-Toxin.

Clostridium perfringens ε-toxin (ETX) is a potent pore-forming toxin responsible for a central nervous system (CNS) disease in ruminant animals with characteristics of blood-brain barrier (BBB) dysfunction and white matter injury. ETX has been proposed as a potential causative agent for Multiple Sclerosis (MS), a human disease that begins with BBB breakdown and injury to myelin forming cells of the CNS. The receptor for ETX is unknown. Here we show that both binding of ETX to mammalian cells and cytotoxicity requires the tetraspan proteolipid Myelin and Lymphocyte protein (MAL). While native Chinese Hamster Ovary (CHO) cells are resistant to ETX, exogenous expression of MAL in CHO cells confers both ETX binding and susceptibility to ETX-mediated cell death. Cells expressing rat MAL are ~100 times more sensitive to ETX than cells expressing similar levels of human MAL. Insertion of the FLAG sequence into the second extracellular loop of MAL abolishes ETX binding and cytotoxicity. ETX is known to bind specifically and with high affinity to intestinal epithelium, renal tubules, brain endothelial cells and myelin. We identify specific binding of ETX to these structures and additionally show binding to retinal microvasculature and the squamous epithelial cells of the sclera in wild-type mice. In contrast, there is a complete absence of ETX binding to tissues from MAL knockout (MAL-/-) mice. Furthermore, MAL-/- mice exhibit complete resistance to ETX at doses in excess of 1000 times the symptomatic dose for wild-type mice. We conclude that MAL is required for both ETX binding and cytotoxicity.

Galectin-3 plays an important role in protection against disseminated candidiasis.

Recent in vitro studies have implicated galectin-3 as an important receptor in host recognition and response to specific Candida species; however, its role in protection against disseminated candidiasis in vivo has not been evaluated. This study investigated the importance of galectin-3 in host defense against systemic infection with the highly virulent species Candida albicans, and the less virulent species, C. parapsilosis. Mice deficient in galectin-3 (gal3-/-) were more susceptible to infection than wild-type (WT) mice. When infected with C. albicans, gal3-/- mice died significantly faster and exhibited a trend towards increased fungal burden and increased abscess formation in infected brains compared to WT mice. When infected with C. parapsilosis, gal3-/- mice had significantly higher renal fungal burdens and abscess formation compared to WT mice. To evaluate whether galectin-3 may contribute to susceptibility to candidiasis in human infants, galectin-3 levels in sera of newborn infants, a patient population uniquely susceptible to infections with both C. albicans and C. parapsilosis, were compared to serum galectin-3 levels of adults. Galectin-3 levels were significantly lower in newborn infant sera compared to adult sera. These data indicate that galectin-3 plays an important role in a murine model of disseminated candidiasis and suggest a potential mechanism of neonatal susceptibility to these infections.

The role of galectin-3 in phagocytosis of Candida albicans and Candida parapsilosis by human neutrophils.

Candida albicans causes the majority of invasive candidiasis in immunocompromised adults while Candida parapsilosis is a leading cause of neonatal candidiasis. While much work has focused on how the immune system recognizes and responds to C. albicans, less is known about host interaction with C. parapsilosis. This study investigates the human neutrophil phagocytic response to these species. Neutrophils underwent phagocytosis of C. parapsilosis yeast and C. albicans hyphae much more efficiently than C. albicans yeast. Treatment of neutrophils with a galectin-3 (gal3) blocking antibody inhibited phagocytosis of C. parapsilosis yeast and C. albicans hyphae, but not C. albicans yeast. The majority of neutrophil gal3 was expressed intracellularly and was secreted from neutrophils after treatment with C. parapsilosis mannan. When neutrophils were treated with exogenous gal3, phagocytosis of both C. albicans and C. parapsilosis yeast increased. Exposure of neutrophils to C. parapsilosis yeast increased phagocytosis of C. albicans yeast and was inhibited by gal3 blocking antibody. Taken together, these data indicate that gal3 secreted from neutrophils may act as a pro-inflammatory autocrine/paracrine signal in neutrophil phagocytosis and suggest that gal3 has a unique role in neutrophil response to C. parapsilosis yeast and C. albicans hyphae distinct from C. albicans yeast.

Candida parapsilosis and the neonate: epidemiology, virulence and host defense in a unique patient setting.

Invasive candidiasis is a common problem in premature infants that leads to high morbidity and mortality. Although Candida albicans has historically been the most prominent species involved in these infections and has therefore been the subject of the most study, Candida parapsilosis is increasing in frequency, and neonates are disproportionately affected. This article reviews unique aspects of the epidemiology of this organism as well as strategies for prophylaxis against invasive candidiasis in general. Additionally, important differences between C. parapsilosis and C. albicans are coming to light related to virulence determinants and interactions with components of host immunity. These developments are reviewed while highlighting the significant gaps in our understanding that remain to be elucidated.

High efficiency opsonin-independent phagocytosis of Candida parapsilosis by human neutrophils.

Candida species are associated with invasive fungal infections, and C. parapsilosis has become increasingly prevalent. As key antifungal effector cells, the function of human neutrophils confronting C. parapsilosis was investigated. We hypothesized that interaction between neutrophils and Candida species may not be uniform. Opsonins were omitted from these studies to understand the antifungal mechanisms at their most basic level. Human neutrophils underwent phagocytosis of C. parapsilosis with much higher efficiency than with C. albicans. Immunofluorescence assays with ss-glucan specific antibody detected more surface exposed ss-glucan on C. parapsilosis than C. albicans. However, blockade of the ss-glucan receptor Dectin-1, reduced phagocytosis of C. albicans but not C. parapsilosis. Inclusion of excess beta-glucan, mannan, or chitin also had no effect on phagocytosis of C. parapsilosis. Consistent with the differences noted in phagocytosis, neutrophils mediated damage to C. parapsilosis but not C. albicans in assays of residual metabolic activity. C. parapsilosis was more sensitive to oxidative stress, and inclusion of antioxidant in toxicity assays decreased neutrophil mediated damage, suggesting that generation of reactive oxygen species contributes to the toxicity mechanism. These data suggest that the interaction between neutrophils and Candida species is not uniform and may partially account for differences observed in the epidemiology and natural history of infections caused by these species.

Oxidative burst and phagocytosis of neonatal neutrophils confronting Candida albicans and Candida parapsilosis.

BACKGROUND: Candida albicans and Candida parapsilosis are important causes of sepsis among premature neonates. The neutrophil is a key element in the control of Candida infections, yet specific neutrophil mechanisms that may contribute to the susceptibility of the premature neonate to candidiasis are not well understood. AIMS: The hypothesis for this study is that neonatal neutrophils have a developmental deficiency in their capacity to generate an oxidative burst in response to Candida species. STUDY DESIGN: Neutrophils were isolated from cord blood of term and preterm infants and from peripheral blood of adult volunteers. Neutrophils were exposed to Candida species, and assays of oxidative burst and phagocytosis were conducted. RESULTS: Oxidative burst of neutrophils from term and preterm (22-29 weeks) neonates exposed to C. albicans hyphae was similar to adult neutrophils. No detectable burst was induced in either group by exposure to C. parapsilosis yeast, and was attenuated by exposure to C. albicans yeast. Because no deficiency in oxidative burst was seen, phagocytosis was also studied. Phagocytosis of unopsonized C. albicans yeast was low in both adult and neonatal neutrophils (10-12%), but was more efficient with C. parapsilosis as target (76-88%). Neutrophils from both term and preterm infants were capable of phagocytosis equivalent to adults. CONCLUSION: A deficiency in generation of an oxidative burst or phagocytosis may not contribute to the increased susceptibility of preterm neonates to infections with Candida.