Clostridioides difficile (C. difficile)
Clostridioides difficile, abbreviated as C. diff, is a bacteria that causes a lot of trouble for hospitals around the world everyday. Once you’ve smelled it, there’s no forgetting its uniquely horrible scent. Patients taking certain antibiotics have had their natural gut flora wiped out leaving little defense against C. diff which primarily causes diarrhea but can escalate to more severe symptoms. While diarrhea is unpleasant it is survivable, but the dehydration it causes can adversely affect a patient in an already fragile state or who is older. Not only are hospital patients at risk for this infection, but elders particularly those in nursing homes are susceptible. In 2015, the CDC reported that 29,000 people died within 30 days of being diagnosed with C. diff, with 15,000 of those deaths estimated to be caused by C. diff. Of those deaths more than 80 percent were aged 65 or older.
C. diff is a gram positive rod shaped bacteria that prefers anaerobic environments. While the active form dislikes oxygen, the microbe can survive exposed to oxygen in a vegetative state as well as leaving spores that can linger for two years on surfaces. But wait, there’s more! This little microbe is highly motile as well, evenly surrounding the cell are flagella that allow a tumbling motion for travel. C. diff is a major cause of hospital acquired diarrhea but also poses a serious threat to the vulnerable populations like nursing homes. Due to the link to antibiotics and depleted microbiota of the patient it was thought that the body was mostly defenseless to the bacteria in this state, but John Tam and his team have found evidence that suggests otherwise.
Gram positive C. diff
Without normal gut bacteria, the key players of the body in the fight against C. diff are bile acids. Bile acids are synthesized in the liver and are known to play roles in metabolic regulation and cholesterol regulation. There are two types of bile acids, primary and secondary. All bile acids are primary when made in the liver, and secondary bile acids are made when they are modified in the intestines by gut bacteria. They aid in digestion by helping to absorb lipids into the body among other important functions elsewhere. Bile acids have been found to play a role in the endocrine system by activating diverse signaling pathways, they can regulate their own cholesterol, energy, and glucose homeostasis among many other things. But because of this ability they could be the next drug treatment for diseases like type II diabetes. Recently, bile acids have been found to play a larger role in functions of the gut. Research has shown that bile acids help deter C. diff from infecting the gut in the absence of protective gut microbiota. The way that bile acids prevent C. diff from infecting as quickly is that these acids actually change the shape of the protein toxin that causes the symptoms of a C. diff infection. By changing the shape of the toxin C. diff can no longer bind to cell receptors efficiently, slowing the rate of infection and upon further studying, bile acids are even bound to and inhibit the toxin.
C. diff can produce multiple protein toxins that cause the symptoms of an infection, the Tam et al. study focused on the toxin TcdB. This toxin is the most likely to determine if someone has the disease and shows symptoms, so it is important to see how it can be slowed down or inhibited. The study shows two main results: that both primary and secondary bile acids balled up the shape of the toxin TcdB leading to decreased cell receptor binding and that small molecule scaffolds bind and inhibit TcdB through an acid-like mechanism.
The scientists hypothesized that bile acids could induce a conformational change that affected the function of the toxin. To test this, they exposed the toxin to three bile acids. Two of the bile acids were inhibitory bile acids (mCA and Taurochenodeoxycholate) while the third (dehydrochlorate) was nonbinding. The dehydrochlorate serves as a control that will tell us if the bile acids are in fact changing the structure by binding to the toxin.
TcdB toxin balling up in the presence of bile acids
In the study, TcdB was exposed to Methyl cholate (mCA), Taurochenodeoxycholate, and dehydrochlorate. These are all bile acids that show promise in having an effect on the shape of the TcdB toxin. On the far left of the figure is a picture taken with an electron scanning microscope that shows the normal shape of the TcdB toxin with a cartoon of the shape above the photo. Moving to the right the line of pictures on the top shows the concentration of bile acid increasing five times with the highest concentration on the far right. As the pictures display more concentrated solutions, the shape of the toxin is becoming rounder and balled up in appearance. The cartoon on the far right displays the shape of the toxin after coming in contact with the bile acid. The middle row shows TcdB coming in contact with Taurochenodeoxycholate and undergoing conformational changes. As a control we see the dehydrochlorate does not affect the shape meaning that if it cannot bind to the toxin, the toxin’s shape cannot be altered. In these two rows the final concentration is equivalent to 1000 µM, when converted to the same units as µM.
We know that the structure was changed by the binding bile acids, but is it permanent? It doesn’t seem to be, when diluted the bile acids can no longer alter the toxin. To test this, the scientists evaluated the melting point of the toxin in the presence of Taurochenodeoxycholate, where they were bound together and the melting point of the toxin and bile acid diluted to a point where they were only partially bound. In the mixture of toxin and bile acid that are fully bound, the melting point increased from 3 degrees celsius to 4 while the diluted sample’s melting point decreased showing that the change is reversible.
Conditions to test reversibility of conformational changes in TcdB
Change in the melting points of the standard conditions (blue) and the dilution (red)
Current treatments for C. diff infections include the drugs vancomycin or fidaxomicin which are antibiotics that stop C. diff growth. Unfortunately up to 20% of people treated for C. diff are reinfected with another strain or the initial infection hasn’t been completely cleared. However, antibiotic resistance is the major reason that finding alternative treatments is so important. This research can help scientists immensely in trying to create a drug to combat C. diff by providing data that will help in the development of anti-toxins. Bile acids alone are not enough to be used in therapies as the effects of overloading the body with them could harm a patient more than help them. However, this research shows promise that an anti-toxin treatment could be developed and used widely in place of antibiotics in the future.
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