Irregular spread of Listeria monocytogenes pioneer cells within the gut

By: Shuyang Lin '22, Maggie Anderson '21, and Halle Willeford '22

[Image source]

Have you ever wondered why your parents growing up would warn you to wash fruits and vegetables you’re about to eat or to be careful when storing raw meats in the fridge? The reason is bacteria, like Listeria monocytogenes are found in places like soil, water, and decaying organic matter that can at some point come into contact with your food products in the form of manure, the dirt it was grown in, or the water it was provided (plant or animal). The CDC reported around 1,600 people become infected by this bacteria overall through food or drink contamination each year with about 260 of the people infected dying as a result of the infection. This bacteria mostly impacts people over the age of 65, pregnant women, and immunocompromised people. These people usually become sick from the infection because listeria is able to cross the blood brain barrier in immunocompromised people and the placental barrier in expecting mothers.

Listeria has a gram positive cell wall, rod shaped appearance, and tumbling motility. However, what is most interesting and important about listeria is its ability to change its mode of transport depending on its location in relation to a cell. Outside of a cell or in the external environment L. monocytogenes uses tumbling motility but when inside the cell it uses polymerization of host cell actin to move within and across adjacent cells. This process works by the listeria bacteria first degrading the vacuole shell that covers it when it enters into the intestinal tract through either a goblet cell or apoptotic cell at the top of a villus with a toxin called LLO (listeriolysin O).

[Image source]

[Image source]

Once the bacteria is free inside the cell, it begins replicating and uses a protein called ActA on one end of itself to begin polymerizing the host cell’s actin to that end of the bacterium. This generates a force which then pushes the bacterium through the cell and potentially protrudes the bacterium through an adjacent cell membrane to then infect another host cell. The goal of the bacteria, regardless of which cell on a villus it infects, is to make it into the lamina propria or basement membrane of the GI tract before the entire apoptotic cell is extruded or the goblet cell extrudes its contents since it produces mucus secretions. Once the bacteria is in the lamina propria it can then easily access the bloodstream and spread even faster through the body. A few symptoms of L. monocytogenes infection include headache, confusion, loss of balance, muscle aches, and in the case of pregnant women potential complications or death of the fetus.

It has already been discovered that intracellular bacteria create protrusions that can be donated to a recipient cell. The donation can only be made if the recipient cell and donor cell are directly adjacent to each other. However, since L. monocytogenes are able to form long protrusions a recent study investigated if the bacterium can use their lengthy protrusions to reach non-adjacent host cells.

Ortega, Koslover, and Theriot tested this hypothesis by infecting merging Madin-Darby canine kidney (MDCK) epithelial cells with either the wildtype L. monocytogenes or the strain of L. monocytogenes that is known as glycine-rich repeat (GRR) mutant; cell to cell spread is typically impossible in mutant strain. MDCK epithelial cells were the cells of choice due to them being commonly used in studies with L. monocytogenes, and the fact that they form polarized and homogeneous monolayers in culture.

In order to understand how L. monocytogenes spread in an epithelial monolayer in culture, researchers use live microscopy to observe bacteria’s behaviors. Firstly, researchers determine the growth and spread of the infected focus and how the intracellular bacteria contributed. This was done by infecting the merging MDCK monolayers with a wild-type 10403 S L. monocytogenes strain that contains an mTagRFP open reading frame under the actA promoter. They observed and imaged the progression of the infection of L. monocytogenes in MDCK epithelial cells for up to 22 hours after infection. Figure 1A shows that bacteria (in red) spread intracellularly among host cells (in blue) at three different times. The green lines show the smallest boundary that encloses all bacteria, which is irregular. The shape of the L. monocytogenes spread tells researchers that bacteria spreads heterogeneously which can also be described as anisotropically. One possible reason for this anisotropic bacterial spread is that a small number of L. monocytogenes cells (denoted as white arrows in the figure) disperse farther than the rest in the infection focus. Researchers then defined these far-spreading cells that create irregular boundaries as “pioneers.” Figure 1B shows that heterogeneous spreading is common in L. monocytogenes as there are four more different infection foci boundaries of the bacteria. The green color gets darker as time passes.

Figure 1, “L. monocytogenes spreads anisotropically through a polarized, confluent MDCK cell monolayer”; panels A & B [image source, video 1]

What causes the anisotropic spreading? To figure out this question, researchers use models to simulate mobility of L. monocytogenes, which only includes random walks of the bacteria. Previous research studies gave them an equation (equation 1) to work on.

Equation 1 [source]: In equation 1, Φ represents the bacterial concentration as position and time, t stands for time, r is the position of the bacteria in polar coordinates, D stands for the effective diffusion coefficient, k represents exponential growth rate, and finally, the backwards looking six, ∂ represents a partial derivative.

Nevertheless, the simulation based on the equation yields circular spreading, which is significantly different from what they observed from Figure 1. To measure the circularity, the boundary that the infection focus created was used in the calculation. They calculated the smallest circle that fully enclosed the boundary, found the area and took the area of the boundary and divided it by the area of the circle (Figure 2D).

Figure 2D shows different shapes of simulation and observation; figure 2E shows how different they are, supported by statistical data. Therefore, simulation based on an equation is not enough.

Figure 2, “Stochastic simulations of cell-to-cell spread via random walks are inconsistent with observed shape of infection foci”; panels D & E [image source]

Then, researchers decided to take into account the effect of pioneer bacteria. But one question regarding pioneer bacteria is how do they arrive at a location so far from the infection foci? Based on observations (Figure 3A), researchers hypothesize that pioneers travel from a donor cell to a non-adjacent recipient directly through a long protrusion. Once landed, pioneers can spread and replicate. After a second simulation that takes pioneer cells as an account, researchers find the shape of the boundaries becomes more anisotropic (Figure 3B), which is more assembled to what they observed. However, not all cells have the potential to become pioneers, so researchers simulate the spreading many times with different probability of becoming pioneers, shown by Figure 3C and D. The result shows that when the pioneer probability is small enough, such as 0.001 and 0.01, the circularity of simulations are the most similar to what researchers observed. As a result, L. monocytogenes cell-to-cell spread is consistent with individual bacteria having a low but non-zero probability of becoming pioneers, while the majority of the bacteria spread locally.

Figure 3, “Allowing simulated bacteria to interconvert between pioneer and non-pioneer behavior recapitulates the non-circular phenotype of experimental foci”; panels A, B & C [image source,video 6]

Figure 3, “Allowing simulated bacteria to interconvert between pioneer and non-pioneer behavior recapitulates the non-circular phenotype of experimental foci”; panel D [image source]

Even though simulations of cell-to-cell spreading solidify that pioneer cells are critical to mobility of L. monocytogenes, models cannot support researchers’ hypothesis that pioneer cells reach a more distant recipient cell through protrusions (Figure 4A right). They propose that the spreading event takes place between two cells that do not form junctions directly with each other. To test their hypothesis, they use GRR mutants which have a lower frequency to enter protrusions than the frequency of the wild-type bacteria and GRR mutants form protrusions that are less straight. The assumption here is that if there is less protrusion, there should be less pioneer cells transferring to non-adjacent cells, which suggests that the probability of being a pioneer is really low. The consequence further leads to a more circular shape of boundary of GRR mutant than that of the wild-type. Figure on the left shows micrograph of phalloidin staining of MDCK cell monolayer; figure on the right is a cartoon expression of long protrusion of L. monocytogenes.

Figure 4, “Decreasing the rate of bacterial protrusion formation leads to more circular infection foci”; panel A [image source]

It turns out that researchers’ hypothesis is supported by their data. The sharp contrast between the mutant and the wild-type allows researchers to notice that the circularity of the GRR is significantly higher than that of the wild-type (Figure 4B, left), indicating that pioneer behavior is indeed critical for the complex, irregular shape of infection (Figure 4B, right). The orange and cyan data points correspond to the foci on the comparison graph. Green boundaries fully enclose all bacteria. Circular dashed lines represent the smallest circles that fully enclose green boundaries. Each black dot in Figure 4B symbolizes a spreading and every dot has different value in terms of circularity. The straight dash line at the top indicates simulations only with random walk, without pioneer cells. Therefore, from the decreasing circularity among three groups (simulated random walk, GRR mutant and wild-type), researchers gain further support for their result that pioneer cells spread through long extracellular protrusion is important for heterogeneous cell-to-cell spread in L. monocytogenes.

Figure 4, “Decreasing the rate of bacterial protrusion formation leads to more circular infection foci”; panel B [image source]

Even though researchers discovered novel spreading of L. monocytogenes some new questions arise. For example, how to differentiate between pioneer bacteria from typical bacterial cells? How would antibacterial treatments affect the pioneer cells, would it make the treatment easier? Future works could focus on solving these questions. Another direction of future research is to investigate where L. monocytogenes form protrusions, is it above, below, or between the host cell? By figuring out answers, we might decrease the chance of percisisant L. monocytogenes infections.