Toxic Little Molecules

There are various different ways that pathogenic bacteria can damage and kill human cells, but one of the most common is by the production of toxic molecules. These small molecules are made inside the bacterial cell, the protein chain built using the DNA template and then often modified within the cell before being secreted directly into, or in the general direction of, human cells. A recent paper in PLoS Pathogens took a look at some of these toxic molecules produced by C. difficile, a human pathogen that infects the human colon and is showing worrying levels of antibiotic resistance.

The rod shaped Clostridium difficile bacteria, image from the Centers for Disease Control and Prevention, part of the United States Department of Health and Human Services.

C. difficile produces two main toxic molecules, labelled TcdA and TcdB. These toxins lead to?fluid secretion, inflammation, and colonic tissue damage associated with the bacterial infection. Both of the toxins get into human cells and disrupt the internal structure, causing them to look round and blobby under a microscope. Both toxins are capable of activating apoptosis ? the internal pathway used by cells to kill themselves, however some studies have shown that when cells were exposed to high concentrations of TcdB they tended to just destroy themselves (necrosis) without using the apoptotic pathway.

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Normal cells on the left, cells treated with TcdB in the middle showing the rounded phenotype. Cells treated with high concentrations of TcdB on the right showing necrosis (cell death). Image from reference.

Before it is sent out of the bacterial cell to cause its damage, the TcdB molecule is processed inside the cell. This processing releases the catalytic effector part of the TcdB molecule which can then damage the host cell. By knocking out the genes responsible for this processing, the researchers were able to create TcdB mutants ? unprocessed forms of the TcdB. They then tested both the normal wild-type and the unprocessed TcdB to see what affect they had on human cells (using both standard HeLa cells, and cells derived from the human colon). Surprisingly, both forms of TcdB were able to kill the human cells. This suggests that the internal processing is not required for TcdB necrosis.

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Graph showing cell viability at increasing concentrations of TcdB for five different unprocessed mutants and wild type. Image from reference.

The researchers suggest that while low concentrations of TcdB have a ?cytopathic effect? i.e they damage the cells leading to apoptosis, high concentrations of TcdB lead to a ?cytotoxic effect? killing the cells off without causing the controlled cell death of apoptosis. Having shown the effects in culture the researchers then explored whether the same effect would be seen in actual tissues, using pig colon tissue. No significant difference was shown between the wild-type and unprocessed mutant; in the case of this toxin, internal processing is not required to cause cell damage.

Although the results might seem negative when put in those terms, it?s very important for pathology research to explore the effects of cell toxins, and to rule out areas where future research would not be useful for revealing anti-bacterial therapies. In this case, any antibiotic that targets the processing of TcdB would not be of use for therapy, although it would prevent the molecule being processed it would not stop the bacteria from causing damage.

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Reference:? Chumbler NM, Farrow MA, Lapierre LA, Franklin JL, Haslam D, et al. (2012)Clostridium difficile Toxin B Causes Epithelial Cell Necrosis through an Autoprocessing-Independent Mechanism. PLoS Pathog 8(12): e1003072. doi:10.1371/journal.ppat.1003072

Source: http://rss.sciam.com/click.phdo?i=1945cc374270c99c749dc773a444d9b6

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