Have you ever been a victim of diarrhea and suffered that awful churning stomach feeling? Regardless if you’ve experienced it or not, which you probably have in your lifetime, have you ever wondered what causes it? Diarrhea is a very common symptom that accompanies multiple diseases. It can also be produced by a plethora of different microbes, one of them being Clostridium difficile. This is a gram positive and spore forming bacterium that is known for producing toxins that can be transmitted through humans. It is most commonly known for causing diarrhea and inflammation of the colon, also known as Colitis, which can also be life threatening. Although this bacteria affects individuals who rely heavily on antibiotics and spend most of their time hospitalized, there has been a great increase of infections in the past few years. Studies have concluded that there were 453,000 cases of infection and 29,000 deaths in 2011 caused by C. difficile. I know what you're thinking, can 💩 cause these many deaths? It sure can!
As you can imagine, the human gastrointestinal (GI) tract is full of thousands of microbes that help keep us healthy, but it can also potentially be the home to bacteria that can lead to different types of infections. As mentioned earlier, C. difficile is a spore forming bacterium. It produces spores that are known to survive adverse conditions because they can thrive without oxygen. The GI tract, specifically the intestines, contain glycine and cholate, which are both acid derivatives that can lead to the germination of C. difficile’s spores. Normally, germination of this bacterium is suppressed by other bacteria that are able to process these derivatives. Patients who consume many antibiotics, like penicillins, are susceptible to C. difficile infection. This is because the consumption of antibiotics causes patients to lose all of the microflora in the intestines that normally process these derivatives. Without these derivatives, C. difficile is able to mass germinate and produce toxins that can potentially cause diarrhea and damages to the cytoskeleton of cells that often leads to Colitis.
C. difficile bacteria can be found in multiple places, such as air, water, soil, human and animal feces, and even in processed meats. The availability of this bacteria has led to the increase in infections. Unfortunately, as these C. difficile infections become more common, they have also become harder to treat due to resistance. Also, different strains of C. difficile have started to produce even higher levels of toxins that lead to quicker infection and rapid spread among communities. Therapies for infection have been created, but many more are needed due to the fact that this infection has become one of the top leading in the country. Studies have been conducted to try to find potential new therapies, especially ones using bacteriocins. Bacteriocins could potentially be consumed orally to treat bacterial infections such as the ones produced by C. difficile.
Bacteriocins are small proteins which are produced by a bacterium in an effort to kill strains of other bacteria within the same species. The production of these antimicrobial peptides (AMPs) are triggered by lack of nutrients or space in the surrounding environment -- they are a survival mechanism when the bacterium is pressed for resources. In the same way that royal families poisoned their own kin to ascend to the throne, bacteria produce these bacteriocins to eliminate other strains of the same bacteria. The strains that live are able to utilize the resources that would have otherwise been consumed by its “relatives”. Over 99% of bacteria make one or more bacteriocins! This is funny if you think about how hand sanitizers boast a 99% effectiveness rate at killing bacteria. Both antibiotics and bacteriocins kill bacteria, but antibiotics are molecules while bacteriocins are proteins. Therefore, bacteriocins can be broken down by enzymes called proteases, while antibiotics are unharmed by them.
Bacteriocins were first discovered by virologist Andre Gratia in 1925 during the early hunt for antibiotics to treat human disease. Later on, bacteriocins produced by gram-positive bacteria were categorized into one of three classes -- Class I, II, or III -- depending on their molecular weight and the modifications they undergo after protein synthesis.
In the context of our study of interest, we ought to specifically consider the Group A subgroup of the Class III bacteriocin. These include enzymes that have lytic activity, meaning that they kill the bacterium by targeting its cell wall. Any chinks in the cell wall “armor” will upset the pressure balance between the bacterial cytoplasm and its outside environment, and the cell will burst. This is known as bacteriolytic activity, and our interest in bacteriocins stems from the idea that we can harness this activity for human use.
One interesting application of bacteriocins includes one of our favorite things: food! The infamous probiotics are microorganisms that promote the health of the organism they reside in (found in “gut-healthy” foods like yogurt). The model organism Bacillus subtilis is one such bacterium that both resides in the gastrointestinal system and also produces bacteriocins. Another application is for biomedical purposes. Isolated bacteriocins can diminish the concentration of pathogenic bacterium in a system. They may also be utilized as anti-tumor drug, by targeting cancer cells -- or in the case of our paper, the C. difficile cells.
The researchers of this study sought to expand their knowledge of bacteriocins and their genetic location within C. difficile and the aerobic microbe, Bacillus subtilis (Gebhart et al, 2012). They had identified the same genetic location that encodes for bacteriocins like the ones in C. difficile in B. subtilis’s genome. By comparing both microbes, they hope to gain more insight as to how they could use gene therapy to produce bacteriocins in different bacterial strains and treat infectious diseases!
Researchers in this investigation mainly worked with two strains of C. difficile: CD4 and CD16. They are notorious for producing lethal bacteriocins known as diffocins. I guess you could say CD4 and CD16 have made bad reps for themselves! Through electron microscopy they were able to see what they described as “flower-like” appendages, which they believed might be the receptor-binding protein diffocins use to attach themselves to their victims and insert their harmful contents into the cell. Yikes! Who knew flowers and attachment could be so toxic!
The investigators ran experiments in which they tested for bactericidal activity of the diffocins in the presence of different C. difficile strains. Substances that can kill other bacteria are referred to as having bactericidal traits. In their experiment they found that certain strains were more sensitive to diffocins than others. They observed areas of clearing on their lawn spot assays which indicated bactericidal activity.
Because C. difficile has the largest locus that encodes for diffocins, investigators tried to locate the open reading frames (ORF) in the C. difficile genome. After identifying the ORF, the investigators were able to isolate, clone, and insert this portion of DNA in a gene of the aerobic, Bacillus subtilis cell. They wanted to see whether B. subtilis could generate active diffocins. Bactericidal activity was observed! I guess toxicity in life and bacterial cells can be contagious. B. subtilis strains that were known to be sensitive to diffocins showed bactericidal activity, however, diffocin production was a lot lower than that of C. difficile. So, it was back to the drawing board for these scientists!
They decided to continue their investigation but deleted B. subtilis gene that encodes for a phage that causes lysis of the cell. They believed this would increase diffocin production, which was a little contradicting because lysis is needed in order to release diffocins into the environment. After deleting the gene, they found even more diffocin production than that of the C. diff strains!
These experiments confirmed that diffocins are highly specific and only certain bacterial strains are responsive to the toxic nature of diffocins. Their experiments that involved cloning the genes showed that diffocins can be generated in different bacterial cell species, which leads to hope that diffocins can be generated in different disease causing agents. It would be interesting to continue studying the interaction between diffocins and other phages in the genomic map of C. diff to see how this could influence diffocin lethality. Do diffocins work their evil alone, or do they have other partners in crime? 😈 Bacteriocins are not perfect -- they can be destroyed by enzymes that unravel and destroy peptides. But it is important to study alternative ways of killing these bacteria because antibiotic resistance is increasing! Hopefully, bacteriocins can pave the way for such discoveries and halt these antibiotic resistant bacteria from flourishing and infecting further populations. Who knows? Perhaps the next explosion in human population growth will be attributed to bacteriocins, and antibiotics will be a thing of the past.
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