It’s the world’s most poisonous substance; a pathogen able to wipe out the population of the United States with a few teaspoons - the entire Earth with a single kilo. It is manufactured in military installations and has been responsible for the deaths of thousands as a murder weapon and as a poison. Yet despite being so fatal, it is a ubiquitous substance in our daily lives. It lurks in our kitchens, in our water, in our food - even in our medicine as a treatment for migraines and muscle disorders. Astonishingly, some even pay thousands of dollars to have microscopic amounts injected into their faces. Many of us know it as Botox, however the bacterium Clostridium botulinum has been a part of our microbiome long before it was used to stop wrinkles.
Botulism bacteria, or Clostridium botulinum, frequently observed in poorly-preserved canned foods, is the world’s most lethal substance. Dr. Phil Luton/Science Photo Library/Corbis
Botulinum toxin (BoNT), often shortened to Botox, is an anaerobic spore forming bacterium first documented as early as the 18th century, when the consumption of meat and blood sausages gave rise to numerous deaths throughout the Württemberg kingdom of Southern Germany. Today, it is still transmitted through contaminated food and water sources resulting in severe and often fatal neuro-paralytic disease. A subspecies of BoNT, C. botulinum Group II is a particularly potent psychrotrophic saccharolytic bacterium that forms spores of moderate heat resistance, making it a particular hazard in minimally heated foods. A recent study by the BBSRC Institute Strategic Programme on Gut Health and Food Safety analyzing the genome of C. botulinum Group II was the first to identify its functional germination receptor, an orphan GR GerXAO essential for L-alanine stimulated spore development. Identification of this receptor has important implications for increasing food safety and limiting the growth of BoNT.
Receptors are regions in a cell that receive chemical signals which stimulate the growth of spores. In order to identify these receptors in botulism, researchers first studied the effect of two potential germinants, L-alanine and exogenous Ca2+-DPA, on the germination of C. botulinum strain Eklund 17B spores to determine which sent the most potent chemical signal. Analysis of more than 150 C. botulinum Group II genome sequences revealed the presence of a single gerA subunit, gerXAO, in each strain, indicating its importance to spore development. Each spore suspension was heat activated to mimic natural growing conditions before the addition of germinants. Following incubation, spore germination was assessed by measuring optical density (OD600) as a percentage of the initial OD600. To validate this measurement, the proportion of germinated spores was then visualized by observing 200 spores in at least ten fields using phase-contrast microscopy. A statistical analysis was performed using the two-tailed Student’s T-test with a significance level of 0.05. Results revealed that the addition at 50 mM of L-alanine initiated spore germination, as observed by a 50 percent drop of initial OD600, after approximately 30 min, while direct spore counts by phase contrast microscopy revealed a 50 percent drop in OD600 correlated to nearly 99 percent spore germination. Exogenous Ca2+-DPA, by contrast, failed to induce any spore germination in C. botulinum Eklund 17B. These findings are summarized in Figures 4 and 5.
After determining that L-alanine produces almost complete germination of Eklund 17B strain spores (as compared to exogenous Ca2+-DPA which appeared to inhibit germination), the researchers tested the effects of L-alanine on the Eklund 17B strain mutants (gerX3bA, gerX3bB, gerX3bC, and the orphan germinant receptor protein gerXAO) as compared to the wild type to identify the receptors responsible for germination. In the first 3 hours, gerX3bA and gerXAO showed the most promising results in inhibiting germination of spores after induction with L-alanine, while gerX3bB showed complete germination of spores (similar to the wild type) and gerX3bC originally showed inhibition but then slowly germinated after the first hour (Fig. 4a/4b). However, after 24 hours, gerXAO emerged as the only mutant able to inhibit spore germination, as all other mutants - including gerX3bA - showed some degree of partial germination (Fig. 4c). Figure 4d shows the rates of germination for each of the mutants of the Eklund 17B strain. These results provide important insight into the use of gerXAO in the prevention of spore germination in C. botulinum Group II species.
Figure 4: The effect of L-alanine (50 mM) on spore Germination of C. botulinum Eklund 17B gerX3bA−, gerX3bB−, gerX3bC−, gerXAO− and wild type.
Following this close comparison of spore development, researchers were able to identify a more complete germination model for C. botulinum Group II strains. They discovered that following heat activation, the nutrient germinant binds to the orphan GerXAO receptor, followed by Ca2+-DPA release via a specialized SpoVA protein channel. Lytic enzymes are then activated by the protein CspB, triggering hydrolysis of the cell cortex, degradation of the membrane and coat, the start of metabolism, and eventual cell growth. This is in contrast to the germination pathway of C. botulinum strain ATCC3502 (Group I). In Group I bacterium, nutrient germinants are recognized together by the GerX1a and GerX1d receptors, followed by Ca2+-DPA release through the proposed SpoVA channel, cortex hydrolysis, membrane and coat degradation, the start of metabolism and eventually cell growth. Unlike Group II species, there is no CspB activation, and the germinants are processed by two different germination receptors.
Figure 5: Comparison of the models proposed for the germination pathways of C. botulinum Group I and Group II.
So what? This study, although quite specific and narrowly focused, can have important implications down the line on the study of botulism and how to prevent the formation of the toxins. Since the spore-forming C. botulinum is only extremely hazardous when its dormant spores are germinated and allowed to grow into active multiplying bacterial cells that produce neurotoxins, the primary focus of research concerning how to prevent botulism is inhibiting the germination of these spores. And since this microbe is found seemingly everywhere, this research can make a huge difference. Group II strains are considered high risk, particularly in minimally heated foods, since they form spores that are moderately heat-resistant even at higher temperatures. Because of this, this study looked at ways to inhibit germination of these Group II strain spores by modifying the germinant receptor protein and testing mutants in an environment known to be favorable to spore germination (L-alanine). Because the orphan germinant receptor protein gerXAO emerged as being highly effective in preventing spore germination under these conditions, this provides a way for food scientists to possibly eventually prevent human botulism by treating food with this receptor protein. This research provides a deeper level of understanding into the “cause” of botulism - dormant spore germination - and exploration into a way to inhibit this process to stop neurotoxins from forming to be able to be ingested by humans. These results have important implications for microbiological food safety to hopefully prevent botulism deaths!
No comments:
Post a Comment