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CDT – a bacterial toxin that mediates its own delivery into cells

Clostridium difficile is totally harmless in healthy people. However, in combination with antibiotics it can cause severe diarrhoea and intestinal inflammation in elderly and debilitated people. But how does the spore-forming, rod-shaped bacterium deploy its power? And how does it enter the cell? Dr. Panagiotis Papatheodorou and his colleagues from the Institute of Experimental and Clinical Pharmacology and Toxicology (Director: Prof. Dr. Klaus Aktories) at the University of Freiburg are trying to find answers to these questions. The researchers have identified the molecular mechanism that enables the C. difficile toxin to enter the cells of the intestinal mucosa. This finding possibly provides new approaches for treating C. difficile infections.

Clostridium difficile is a so-called nosocomial pathogen and is one of the most common hospital pathogens. Although the bacterium is a rather inconspicuous inhabitant of the human intestines, under certain conditions it can cause potentially life-threatening diarrhoea. Normally, other bacteria in our intestinal flora prevent C. difficile from gaining the upper hand. However, long-term treatment with antibiotics kills off the intestinal bacteria that keep C. difficile in check. C. difficile, which is resistant to a variety of antibiotics, starts to produce toxins and overgrow the other bacteria. C. difficile infections often occur in hospitals where people are usually more vulnerable to disease. In the Western world, infections with so-called hypervirulent C. difficile strains are rapidly increasing. These strains are much more dangerous and more difficult to treat and can even affect young, healthy people who have not taken antibiotics.

Panagiotis Papatheodorou (centre) and his two doctoral students Sarah Hemmasi (left) and Bernd Czulkies (right) are investigating the internalisation of CTD. © Dr. Panagiotis Papatheodorou, University of Freiburg.

Hypervirulent Clostridium difficile bacteria

C. difficile produces two major types of toxin, i.e. enterotoxin A and cytotoxin B, which are very potent. They destroy intestinal cells and cause fluid loss in the intestine. This occurs as a result of damage to the cytoskeleton. "The toxins inactivate molecules that control the cytoskeleton, which leads to the loss of cell integrity," says Dr. Panagiotis Papatheodorou from the Institute of Experimental and Clinical Pharmacology and Toxicology at the University of Freiburg. This is why toxin A and B cause antibiotic-associated diarrhoea and in severe cases, life-threatening colitis, which can lead to bloating, resolution of the intestinal walls and sepsis. Increased virulence occurs in C. difficile strains that produce much higher than normal toxin quantities. One such hypervirulent strain, C. difficile ribotype 027, has become more common since 2001. In addition to producing higher than normal quantities of toxin A and B, it also produces the toxin CDT (Clostridium difficile transferase). CDT consists of two componenets, CDT-A and CDT-B, which together form a functional toxin. Inside the cell, CDT directly targets actin, which is the major constituent of the cytoskeleton. It transfers an ADP ribose to actin (ADP ribosylation). The attachment of the ribose leads to the inactivation of actin, resulting in the disintegration of the protein into monomers. Actin is then no longer able to form a coherent scaffold (depolymerisation). Prior to Papatheodorou's findings, it was not known how CDT enters cells.

 

After binding to LSR, the toxin-receptor complex on the cell surface enters the cells by way of vesicles. The CDT-B heptamer forms a pore in the vesicles, which then allows CDT-A to enter the cytosol. © Dr. Panagiotis Papatheodorou, University of Freiburg.

How toxins enter the cells

In 2011, Aktories and Papatheodorou identified the receptor that the CDT toxin uses to get into the cell. The receptor to which CDT binds is called LSR (lipolysis-stimulated lipoprotein receptor) and is found on the surface of intestinal mucosa cells. Papatheodorou and his team have now elucidated the molecular interactions between toxin and receptor. "We have identified which toxin region enables the toxin to interact with the receptor, as well as the receptor region that interacts with the toxin," says the scientist. The natural function of LSR has not yet been fully elucidated. One hypothesis is that it is required for the uptake of lipoproteins into cells; however, other receptors are known to be far more effective at doing this. Recent findings suggest that LSR is important for cell-cell contacts where three cells meet. Tricellular junctions connect three cells with each other at places where a good seal is needed to keep the cell epithelium absolutely tight. The study of the transfer of CDT into cells revealed that the toxin and receptor regions that come into contact with each other are smaller than previously thought. The toxin's CDT-B component is sufficient for receptor binding. The CDT-A component contains the enzymatically active and hence toxic domain that performs ADP ribosylation. The receptor-binding B domain forms oligomers and probably binds to LSR as a ring-shaped heptamer with seven individual molecules. Only then does the A domain bind to the complex and triggers the internalisation of a vesicle that contains both CDT components and the receptor in an elevator-like manner. Inside the vesicle, a membrane pore forms in the heptamer circle through which the enzymatic A component enters the cytosol where it binds to actin. "This is a very interesting mechanism," says Papatheodorou, "the toxin brings it own door with it, in a manner of speaking, so that it can leave the "elevator" when it has reached its destination."

CDT enables pathogen adhesion

The researchers have already observed another effect of CDT. Large quantities of actin are found in the cell cortex right below the membrane. They are there to support the membrane. Further towards the cell interior, where there is less cortical actin, there are larger numbers of microtubuli, which are also important for scaffold strength. CDT destabilises the cell membrane due to the depolymerisation of the actin polymers. "The microtubuli extrude, the membrane turns to the outside, and small cellular protrusions that look like hairs develop," says Papatheodorou. "When there is a large number of hairs, the bacteria get easily caught in them." This helps the pathogens to attach more effectively to the intestinal epithelium; adhesion and colonisation become easier. "A toxin is a poisonous substance that immediately causes considerable damage," says the toxicologist. "But in this case, the toxin appears to modify something in the cell to make it advantageous for the bacterium." Papatheodorou and his team are studying bacterial toxins, rather than the bacteria themselves. They introduce a CDT gene into harmless bacterial species such as Bacillus megaterium, and produce the A and B components separately from each other. They then isolate the components to study their effect in colon cancer cell lines. "CDT only becomes toxic when both components are present," says the researcher who wants to find out whether the binding of CDT to the receptor loosens the connections between cells. The researchers have already come up with ideas for potential C. difficile infection treatments. One possibility is antibodies and another is small peptides that block either the receptor binding domain or the LSR site where interaction takes place. This means that the toxin is captured before it can exert its effect. As many pathogens have become resistant to antibiotics, specifically targeting the toxin would be an effective solution. "The ideal solution would be to be able to attenuate the effect of the toxins by preventing them from being internalised in the cells," concludes Papatheodorou.

 

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