Multifaceted Interactions of Bacterial Toxins With the Gastrointestinal Mucosa

MR Popoff


Future Microbiol. 2011;6(7):763-797. 

In This Article

Toxins Crossing the Intestinal Barrier & Targeting a Remote Organ or Tissue

Bacterial toxins produced in the digestive tract, in addition to their effects on the gastrointestinal mucosa, may be transported through the blood circulation and may elicit disorders in other organs. For example, as described previously, Stx is responsible for renal and/or neurological complications. However, some bacterial toxins can transit through the digestive tract without inducing damage in enterocytes or gastrointestinal mucosa. Certain toxins can undergo a transcytotic passage through the intestinal barrier; others can use the paracellular route. They then diffuse in the underlying tissues where they interact with target cells other than enterocytes or can be disseminated by the general circulation to distant target organs or tissues. Toxins such as ɛ-toxin have the ability to interact with various organs and also to pass through the blood–brain barrier and to act on specific cells of the CNS, whereas other toxins (botulinum neurotoxins [BoNTs]) are highly specific of a cell type (motoneurons). Toxins that are absorbed from the intestine and diffuse through the blood circulation are responsible for multiorgan failure (C. perfringens ɛ-enterotoxemia), specific neurological diseases, such as botulism, which is characterized by a flaccid paralysis, or immunosuppression (listeriolysin), facilitating the progression of invasive bacteria (Listeria).

Clostridium Perfringens ɛ-toxin

ɛ-toxin is the major virulence factor of C. perfringens types B and D.[184] Fatal enterotoxemia, which is common in sheep and goat, and more rarely in cattle, is caused by C. perfringens type D. Overgrowth of C. perfringens type D in the intestine of susceptible animals, generally as a consequence of overeating of food containing a large proportion of starch or sugars, produces large amounts of ɛ-toxin. ɛ-toxin increases the intestinal barrier permeability mainly by opening tight junction by an unknown mechanism.[185] The toxin is then absorbed through the intestinal mucosa and spreads in the different organs by the blood circulation, causing blood pressure elevation, vascular permeability increase, lung edema and kidney alteration (pulpy kidney disease in lambs characterized by a post-mortem kidney softening).[186,187]

ɛ-toxin is synthesized as a protein containing a signal peptide (32 N-terminal amino acids). The secreted protein (32,981 Da) is poorly active and it is called prototoxin. The prototoxin is activated by proteases such as trypsin, α-chymotrypsin and C. perfringens λ-protease, which remove 11–13 N-terminal and 29 C-terminal amino acids.[188] ɛ-toxin retains an elongated form and contains three domains, which are mainly composed of β-sheets (Figure 2). The overall structure is significantly related to that of the pore-forming toxin aerolysin.[189]

Major pathological changes are observed in the brain: congestion and edema of the meninges, perivascular and intercellular edema, and necrotic foci of the nervous tissue. ɛ-toxin can pass through the blood–brain barrier and accumulates specifically in the brain.[190–192] The neurological disorders (retraction of the head, opithotonus, convulsions, agonal struggling and hazard roaming) seem to result from ɛ-toxin action on the hippocampus, leading to an excessive release of glutamate.[193]

Specific activity of ɛ-toxin is observed in cultured cells. Among the cell lines that have been tested, Madin Darby Canine Kidney (MDCK), murine renal cortical collecting duct principal cell line (mpkCCDcl4) and, to a lesser extent, the human leiomyoblastoma (G-402) cells are sensitive to ɛ-toxin. Marked swelling is observed in the first phase of intoxication, followed by blebbing and membrane disruption.[153,194,195]

ɛ-toxin binds to the MDCK cell surface, preferentially to the apical site, and recognizes a specific membrane receptor, which is not present in insensitive cells. Binding of the toxin to its receptor leads to the formation of large membrane heptamer complexes, which are very stable when the incubation is performed at 37°C.[194,196]

The cytotoxicity is associated with a rapid loss of intracellular K+, and an increase in Cl and Na+, whereas the increase in Ca2+ occurs later. In addition, the loss of viability also correlates with the entry of propidium iodide, indicating that the ɛ-toxin forms large pores in the cell membrane. Pore formation is evident in an artificial lipid bilayer. ɛ-toxin induces water-filled channels permeable to hydrophilic solutes up to a molecular mass of 1 kDa, which represent general diffusion pores slightly selective for anions.[197] In polarized MDCK cells, ɛ-toxin induces a rapid and dramatic increase in permeability. Pore formation in the cell membrane is likely responsible for the permeability change of cell monolayers. Actin cytoskeleton and organization of tight and adherens junctions are not altered, and the paracellular permeability to macromolecules is not significantly increased upon ɛ-toxin treatment.[198,199] ɛ-toxin causes rapid cell death by necrosis, characterized by a marked reduction in nucleus size without DNA fragmentation. Toxin-dependent cell signaling leading to cell necrosis is not yet fully understood but involves ATP depletion, AMP-activated protein kinase stimulation, mitochondrial membrane permeabilization and mitochondrial-nuclear translocation of apoptosis-inducing factor, which is a potent caspase-independent cell death factor.[199] Therefore, ɛ-toxin is a very potent toxin, which alters the permeability of cell monolayers such as epithelium and endothelium causing edema and cell death.

Intravenous injection of ɛ-toxin in experimental animals leads to damage and permeability increase of the blood–brain barrier, accumulation of the toxin in the brain and formation of perivascular edema.[200,201] Endothelial cells are possibly the primary target cells, but ɛ-toxin also alters the blood–brain barrier and binds specifically to the brain.[191,192,201,202] ɛ-toxin directly interacts with a subset of neurons, such as granule cells from the cerebellum and, possibly, with hippocampus neurons, leading to an excessive release of glutamate, which is probably responsible for the nervous symptoms of excitation.[192,203] The precise target cell in the host and mechanism of ɛ-toxin action on neuronal cells remain to be elucidated.

Botulinum Neurotoxins

Clostridium botulinum secretes very potent neurotoxins, which are responsible for neurological disorders in humans and animals, botulism, characterized by a flaccid paralysis. Botulism is most often acquired through the digestive tract.

Botulinum neurotoxins are synthesized as precursor proteins (150 kDa), which are inactive or weakly active. The precursor, which does not contain a signal peptide, is released from the bacteria by an as yet undetermined mechanism. BoNTs are proteolytically cleaved in their light chain, which contains the enzymatic site, and heavy chain, which harbors the receptor binding domain and translocation domain, both chains remain linked by a disulfide bridge. BoNTs are associated with nontoxic proteins (ANTPs) to form large complexes. ANTPs encompass a nontoxic and nonhemagglutinin component (NTNH) and several hemagglutinin components (HA34, HA17 and HA70 in C. botulinum A).[204]

Botulinum neurotoxins enter the body orally or are produced directly in the intestine subsequent to C. botulinum intestinal colonization, and then undergo receptor-mediated transcytosis across the digestive mucosa.[205–210] However, the mechanism of BoNT passage through the intestinal barrier is not fully understood. The hemagglutinin component (HA34), which targets E-cadherin, might also facilitate BoNT transport through disassembled baso–lateral junctions between enterocytes.[211,212] After diffusion into the extracellular fluid and blood stream circulation, BoNTs target motorneuron endings.

Each type of BoNT recognizes specific receptors on demyelinated terminal nerve endings, which consist of two parts, a ganglioside of the G1b series (GD1b, GT1b), associated with a membrane protein. It has been found that synaptotagmin II associated with ganglioside GT1b is involved in the binding of BoNT/B and the synaptotagmin isoforms I and II mediated the binding of BoNT/G.[213–216] BoNT/A interacts with another membrane protein of synaptic vesicles called SV2, preferentially isoform C whereas BoNT/E recognizes SV2A and SV2B as a receptor in conjunction with gangliosides.[217–219] Neurotoxin bound to its receptor is internalized by receptor-mediated endocytosis. The light chains of all clostridial neurotoxins are zinc metalloproteases that cleave one of the three members of the SNARE proteins. BoNT/B, D, F and G cut synaptobrevin (or vesicular associated membrane protein), BoNT/A and E cut SNAP25, and BoNT/C1 cut both SNAP25 and syntaxin. The cleavage sites are different for each neurotoxin. When an individual SNARE protein is cleaved by a clostridial neurotoxin, SNARE complex formation is not inhibited but its stability is reduced and the release of neurotransmitter is blocked resulting in a flaccid paralysis.[204]


Listeriolysin (LLO) is a pore-forming toxin from the cholesterol-dependent cytolysin family, which is produced by Listeria monocytogenes and related pathogenic species. LLO (58 kDa) is not an enterotoxin, but has multiple roles in the pathogenesis of L. monocytogenes, a facultative intracellular pathogen responsible for food-borne diseases including gastroenteritis, meningoencephalitis and materno–fetal infections. It is well documented that LLO, which has an optimal lytic activity at acidic pH, is involved in the lysis of phagosome membrane permitting L. monocytogenes to escape the endocytic vacuole and to multiply in the cytosol. In addition, extracellularly released LLO modulates the host immune response. At sublytic concentration, LLO forms pores on plasma membranes causing an efflux of Ca2+ and activation of subsequent intracellular signaling such as MAPK and NF-κB pathways leading to upregulation of adhesion molecules, release of proinflammatory cytokines and other cell effects. LLO targets various cell types including endothelial cells and lymphocytes. Thereby, LLO participates in the progression of L. monocytogenes infection by inducing an immunosuppressive response (for example, by induction of protective cytotoxic T lymphocytes, lymphocyte apoptosis and anti-LLO antibodies) and mobilization of more cells available for bacterial replication.[220,221]