Regulating Productivity: A Trait Common to Both Students and Listeria monocytogenes

By: Kiera Sapp

Figure 1. Images of L. monocytogenes using electron microscopy and in stuffed animal form.

Environments often dictate changes in our level of productivity. Why is it that students are generally more productive in a library than at a raging party? Or that working at home often requires a designated office space? Additionally, even in a designated office space it is difficult to be productive without the right supplies (laptop, notebook. etc.), and you probably wouldn’t want to bring your laptop to that party. How do you coordinate what you want to bring with you depending on the environment you’re traveling to? This is a question that the bacterium Listeria monocytogenes faces every time it infects a host cell. L. monocytogenes, the causative agent of Listeriosis, is introduced to human tissue when it is consumed in contaminated food.

After eating something contaminated with L. monocytogenes, the bacteria waste no time growing and dividing in the human gut. Once infection spreads beyond the gut, the individual is diagnosed with invasive listeriosis and can experience symptoms such as fever and diarrhea, however the infection often escalates and requires hospital care. One in five people with invasive listeriosis die. Pregnant women, older adults, and individuals with weakened immunity are most susceptible to L. monocytogenes infection. Invasive listeriosis in pregnant women can cause miscarriage, stillbirth, or life-threatening infections of the newborn child. Invasive listeriosis in older adults or individuals with weakened immunity can cause confusion, loss of balance, or convulsion.

L. monocytogenes bacteria thrive in a wide variety of host environments, ranging from 4℃ (the temperature of the average household refrigerator) to 37℃ (body temperature). In 2014, a large collaboration project by Sureka et al. was published looking at a specific second messenger and its role in altering the metabolism of L. monocytogenes as it moves from one environment to another. When infecting a host cell, L. monocytogenes undergoes a major metabolic transition from obtaining its energy from glucose to utilizing alternative carbon sources. Glutamate, a negatively-charged amino acid, is crucial in this transition process, as it is used by L. monocytogenes to react to neutralize incoming positively-charged potassium as the pathogenic bacterium is stabilized osmotically. Students keep close watch on their laptops because they tend to dictate productivity, paying careful attention to when to bring them and when to leave them behind because a student can feel just as out of place without a laptop in a library as they can with a laptop at a party. Because glutamate plays such a crucial role in the regulation of metabolism/productivity in L. monocytogenes, it makes sense that it would be tightly regulated. Figure 2 shows the biosynthetic pathway to produce glutamate in L. monocytogenes.

Figure 2. The biosynthetic pathway to produce glutamate from glucose in L. monocytogenes.

Pyruvate carboxylase (PC) catalyzes the reaction to form oxaloacetate from pyruvate, and it acts early on in the glutamate biosynthetic pathway, before the commitment step. L. monocytogenes has its own type of PC, which will henceforth be referred to as LmPC. LmPC activity is a little bit like buying milk and eggs, products that can be used for a variety of baking projects, one of which is a special birthday cake for your sister. Sureka et al. showed that LmPC binds to a broadly conserved second messenger called cyclic di-adenosine monophosphate (c-di-AMP) using a chemical proteomics screen. This screen in itself is a fascinating topic to discuss, as it opens up an entire new area of research, but it is not the focus of this post. I have chosen to delve into this c-di-AMP second messenger and its role in regulating glutamate biosynthesis as L. monocytogenes transitions from one environment to another.

Second messengers are critical members of the cell environment, as they initiate signal transduction by binding to a protein and altering it to cause transcriptional, translational, and posttranslational changes within the cell. This is particularly important for immune detection within a cell, and, as a pathogenic bacterium, it aids L. monocytogenes during infection by responding to the particular environment the bacterium is in. As humans, our senses act as a second messenger of sorts to help us orient ourselves and distinguish one environment from another. Second messengers like c-di-AMP help regulate microbial growth and physiology through a signal transduction cascade in response to external stimuli.

Sureka et al. show in their paper that c-di-AMP functions as an allosteric inhibitor of LmPC, thus decreasing glutamate synthesis. They showed that this decrease in glutamate production is a result of LmPC activity using a strain of L. monocytogenes with inducible expression of LmPC and a gene called dacA. The gene dacA encodes a protein that limits the amount of c-di-AMP produced in the cell. Adding the drug theophylline to the cells would result in expression of LmPC, and adding IPTG to the cells would induce dacA expression and ultimately reduce the amount of c-di-AMP being produced. Figure 3 shows that when they induced expression of LmPC at increasing concentrations of theophylline in an presence of c-di-AMP, they saw an increase in the amount of glutamate. Interestingly, they did not notice a change in the amount of alanine or aspartate, even though aspartate biosynthesis occurs downstream of LmPC activity. The function of LmPC and c-di-AMP is somehow specific for the glutamate pathway, and it is not limiting activity for aspartate biosynthesis.

Figure 3. Glutamate enrichment in L. monocytogenes strains with inducible dacA and LmPC as a result of increasing theophylline concentrations.

Why would LmPC specifically affect glutamate biosynthesis? How does the fact that c-di-AMP allosterically inhibits LmPC function play a role in this process? Sureka et al. hypothesized that the metabolic imbalance resulting from differences in LmPC function is responsible for changes in bacterial growth and host colonization in low levels of c-di-AMP. They decided to infect macrophages with the same strain of L. monocytogenes with inducible LmPC and dacA expression at various concentrations of theophylline. They saw a positive correlation between cell growth and theophylline concentration to a certain point, after which the growth was similar to normal LmPC expression. This means that the cells grow more when more LmPC is produced at constant levels of the second messenger c-di-AMP, but at some point a stable growth is reached and excess LmPC returns growth to levels similar as in the absence of LmPC. Figure 4 shows that, in an excess of theophylline, the cell produces an excess of glutamate. In limited amounts of c-di-AMP in the presence of excess LmPC, cell growth is reduced because of the hypothesized need for balanced metabolic activity.

Figure 4. The changes in L. monocytogenes growth in strains with inducible dacA and LmPC expression at increasing concentrations of theophylline.

Sureka et al. transitioned their focus to citZ, which encodes an enzyme that functions as the first committed step to glutamate biosynthesis. Using a listeria mouse model, Sureka et al. were able to show that a mutation in citZ rescues growth in the L. monocytogenes strain with a mutated dacA, which has an excess of c-di-AMP. When citZ is not disrupted in the mutant dacA strain, Figure 5 shows that there was limited infection in both spleen and liver mouse tissue. When they introduced the mutation in citZ in the same strain of dacA-mutated L. monocytogenes, the growth was almost rescued to wild-type levels in both the spleen and the liver. This has interesting implications when you consider the changing environment for L. monocytogenes and the resulting differences in accessible glucose.

Figure 5. Changes in bacterial growth in the spleen and the liver of mice in wild-type, mutant dacA, and mutant dacA and citZ strains of L. monocytogenes.

By showing that L. monocytogenes growth depends on the levels of LmPC and c-di-AMP second messengers that are produced, Sureka et al. were able to demonstrate that the bacteria can regulate their metabolism in accordance with their environment. A balanced metabolism is certainly important for regulating cell growth and infection of human tissue. Little is known about how exactly L. monocytogenes regulates glutamate biosynthesis, but this project is a first step in the process of understanding how it changes its metabolism in different environments. It would be interesting to do more studies to determine why changes in LmPC concentration do not alter alanine or aspartate biosynthesis. Additionally, it would be interesting to include information about other bacteria with similar (or different) responses to their environments. Just like students can be more productive with a laptop in the library than at a party, L. monocytogenes changes its metabolism and biosynthetic pathways based on the environment it is in.

More Information: Sureka et al. (2014) The cyclic dinucleotide c-di-AMP is an allosteric regulator of metabolic enzyme function. Cell 158: 1389. https://doi.org/10.1016/j.cell.2014.07.046

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Meet the author: Kiera Sapp

Kiera is a senior at Mount Holyoke College, about to graduate with a major in biochemistry and a minor in anthropology. She is a member of Professor Amy Camp’s 2017 Microbiology course, and she has tremendously enjoyed learning about the secret lives of bacteria this semester. Kiera hopes to continue studying microbiology during her graduate studies.