In times of stress, different people react in different ways. Some buckle down to tackle the source of the stress, a good portion lean on those around them for support, and a few respond by completely shutting down. Just like humans, microbes respond to stressful conditions in various manners, depending on what type of bacteria they are and what environments they are exposed to. One microbe that takes on interesting characteristics during times of stress is a bacteria known as Mycobacterium smegmatis.
Scanning Electron Microscopy image of M. smegmatis. Source
Found most often in soil, water, and plants, M. smegmatis is an acid-fast gram positive bacteria that readily forms aggregates known as biofilms and moves around by sliding across surfaces. These characteristics of the bacteria arise in part because of a molecule covering the surface of M. smegmatis, known as glycopeptidolipids (GPL), which is a mouthful. If we break it down, these molecules are composed of a sugar (glyco), a peptide chain (peptido) and a fatty acid chain (lipid), seen below in pink, blue, and green respectively. As they make up the outer surface of the bacteria, GPLs play a role in how the bacteria interacts with other organisms, and how it reacts to its environment.
Structure of one type of M. smegmatis glycopeptidolipid, known as glycopeptidolipid-3. The three components of the molecule are marked in green (lipid), blue (peptide) and pink (carbohydrate). Source
The name Mycobacterium might sound familiar if you’ve heard of Mycobacterium tuberculosis (Mtb), the bacteria that causes the disease tuberculosis. Responsible for 1.5 million deaths annually, Mtb is the world’s top infectious killer. Understanding the bacteria’s behavior and how it causes disease is a game changer to be able to both prevent and treat tuberculosis. Unfortunately, Mtb is extremely hard to study, as it grows incredibly slowly, and the risk of infection while performing research on the bacteria is immensely high. To avoid these setbacks, scientists have had to look for other avenues to study the bacteria, and this is where we turn back to Mycobacterium smegmatis. A member of the same genus as Mtb, M. smegmatis offers a perfect alternative as: (1) it’s genome is extremely similar to that of Mtb, (2) it doesn’t cause disease in humans, and (3) it grows at a much faster rate than Mtb, making it much easier to study. M. smegmatis is therefore the ideal model organism for Mtb, and learning more about its many characteristics has become a top priority in microbiology.
A feature of Mtb that makes it such a dangerous pathogen is its ability to resist antibiotics. Part of this resistance arises because of the bacteria’s capacity to shut down and enter a dormant state when exposed to the stressful conditions of the host immune cell it infects, the macrophage (seen below). Whether or not M. smegmatis similarly enters this dormant state when exposed to the same stressors remains unknown. If it does, studying M. smegmatis would be a fantastic avenue in order to learn more about drug resistance in Mtb and how the dormant state of Mycobacterium contributes to this feature.
Diagram of Mtb dormancy establishment. The bacteria infects an immune cell known as the macrophage, and upon exposure to the harsh conditions of the cell, goes into a dormant non-replicating state that allows it to avoid clearance by antibiotics. Created with BioRender
Fortunately, Sushanta Ratna and Jaiyanth Daniel recently published an article that explored how M. smegmatis responds when exposed to stress. More specifically, how the characteristics of the bacteria change when exposed to the exact stress conditions that are present within macrophages: low oxygen (hypoxia), low pH (acidity), and reduced access to nutrients (nutrient starvation).
To address this question, Ratna and Daniel grew M. smegmatis in an environment that combined these three conditions, and as time passed, they measured how the number of colonies of the bacteria changed. What they found was that, immediately after they started the experiment, the number of live bacteria began to drop drastically. However, after three days of the bacteria’s numbers slowly dwindling, suddenly, the amount of M. smegmatis held constant. The number of colonies didn’t decrease, didn’t increase, and instead stayed the exact same until the experiment concluded, noticeable in the figure below as a straight line across days 3, 5 and 7. For Ratna and Daniel, these results were quite clear: when exposed to this extreme stress, a small portion of M. smegmatis are able to survive by shutting down. As a result, they can’t grow or replicate, and instead are fixed in a dormant state.
Viable number of M. smegmatis colonies (CFUs/mL) on different days throughout the experiment after exposure to a hypoxic, acidic, nutrient depleted environment. Source.
This finding was huge, as it meant that just like Mtb, M. smegmatis enters a non-replicating, dormant state when exposed to the same harsh conditions of the macrophage. Having answered their big picture question, Ratna and Daniel decided to probe a bit further to see what they could learn about the characteristics of M. smegmatis when it enters this period of dormancy. In particular, they were interested in seeing if the production of the bacteria’s glycopeptidolipids, which are so critical for how M. smegmatis interacts with its surroundings, changed under these same stress conditions.
As previously mentioned, glycopeptidolipids (GPLs) are made up of a combination of sugars, peptides, and fatty acids. Two known fatty acids that make up the lipid portion of the molecule are called palmitic acid and oleic acid. During the synthesis of new GPLs, these two fatty acids are incorporated into the growing GPL molecule. This means that measuring how much palmitic acid and oleic acid are present on the surface of the bacteria would be an effective way of measuring how much GPL has been synthesized.
This is the approach that Ratna and Daniel decided to take. They again grew M. smegmatis under the same conditions of extreme stress, exposing them to an environment with low oxygen, low pH, and low nutrients. This time though, they added something in with the bacteria: radiolabelled palmitic acid and oleic acid. Radiolabelling is a process in which you attach a radioactive molecule to your fatty acids of interest. By monitoring the radioactivity of the bacteria, you could then identify where the two fatty acids ended up, and see whether or not they were used to synthesize glycopeptidolipids.
Amount of Palmitic acid (top) and Oleic acid (bottom) incorporated into glycopeptidolipids of M. smegmatis upon exposure to many different combinations of stress conditions. Log phase indicates normal conditions, pH 5 indicates acidic conditions, 10% Dubos indicates nutrient depletion, and 5-day hypoxia indicates low oxygen. Source.
Ratna and Daniels’ results were striking. When M. smegmatis was exposed to the triple stress conditions (10% Dubos pH 5 in the figures above), the amount of palmitic acid and oleic acid incorporated into the bacteria increased dramatically in comparison to when the bacteria was growing under normal conditions and was in its abundant, or “log” phase of growth. What exactly does this imply? It suggests that when M. smegmatis is subject to harsh conditions and is forced to enter a dormant state, the bacteria also responds by increasing the synthesis of its glycopeptidolipids. On top of that, because the two fatty acids were not produced by the bacteria, but instead were added in by the researchers, M. smegmatis appears to manufacture these extra GPLs by taking in fatty acids from its surrounding environment instead of using ones that the bacteria itself had made.
These observations were incredibly exciting, and have huge implications. Being able to identify how Mycobacterium respond to stress is critical in order to gain a stronger understanding of how Mtb establishes and maintains the dormant state that makes it so tricky to treat. Learning about how M. smegmatis, the ideal model organism for Mtb, behaves in the triple stress macrophage environment is the first step to tackle this issue, and thus Ratna and Daniel’s work fills a huge gap in our knowledge of this topic. To take this work even further, they intend to try and identify what genes, regions in the DNA that often encode for proteins, are involved in this ability of the bacteria to incorporate fatty acids from outside of the bacteria into their glycopeptidolipids.
However, reading this paper, one of the immediate questions that arises is how do these results translate to other pathogenic Mycobacterium? Mtb is often at the forefront of discussions regarding antibiotic resistance and the harmful nature of Mycobacterium. However, other bacteria in this genus such as Mycobacterium leprae, the causative agent of leprosy, similarly take on a pathogenic nature that is heightened by its ability to enter a period of dormancy. The difference between Mtb and M. leprae lies in what types of cells the bacteria infect, with M. leprae attacking and establishing dormancy in a particular group of cells of the nervous system known as Schwann cells. Using M. smegmatis to study dormancy under a wider range of conditions which reflects the intracellular environment of different cell types would broaden the implications and importance of these results, and allow us to better understand how a range of Mycobacterium are capable of causing disease.
Although the results of this experiment are huge for the scientific field, I found them particular interesting for an additional reason: In our day to day lives, each individual encounters stress, even bacteria, and just like most people, it appears that M. smegmatis responds to this stress by shutting down, and eating some ice cream.
About the Author:
Lucie Berclaz ‘25 is a Biochemistry major from Valais Switzerland. She is deeply interested in studying sexual and reproductive health, and found this passion during her time in the Lijek lab at Mount Holyoke College. In her free time she enjoys playing volleyball, watching movies with her friends, and spending time with her family and her dog.
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