Kingdoms collide as bacteria and cells form captivating connections

In biology textbooks, the endoplasmic reticulum is often depicted as a compact, distinct organelle near the nucleus, which is known to be responsible for protein transport and secretion. In fact, the ER region is vast and dynamic, spread throughout the cell and able to establish contact with and between other organelles. These membrane communications regulate diverse processes such as lipid metabolism, sugar metabolism, and immune responses.

Exploring how pathogens manipulate and hijack essential processes to promote their own life cycles can reveal much about basic cellular functions and provide insight into viable treatment options for pathogens that have not yet been studied.

New research conducted recently by the Lamason Lab in the Department of Biology at MIT published in Journal of Cell Biology He showed that Rickettsia parkeri, a bacterial pathogen that lives freely in the cytosol, can interact in a broad and stable manner with the rough endoplasmic reticulum, forming previously unseen connections with the organelle.

It is the first known example of a direct in vivo contact site between intracellular bacterial pathogens and the eukaryotic membrane.

Lamason’s laboratory studies R. parkeri as a model for more virulent rickettsial infections. R. rickettsii, which is carried and transmitted by ticks, causes Rocky Mountain spotted fever. If the infection is left untreated, it can cause serious symptoms such as organ failure and death.

Rickettsia is difficult to study because it is an obligate pathogen, meaning it can only live and reproduce inside living cells, much like a virus. Researchers must get creative to analyze fundamental questions and molecular players in the R. parkeri life cycle, and much remains unclear about how R. parkeri spreads.

Turn into the intersection

First author Yamilex Acevedo-Sánchez, a graduate of the BSG-MSRP-Bio program and a graduate student at the time, found ER and R interactions. parkeri while trying to monitor the arrival of rickettsiae at the cell junction.

The current model of Rickettsia infection involves spreading Rickettsia parkeri from cell to cell by traveling to specialized contact sites between cells and being engulfed by the adjacent cell for dissemination. The bacterium Listeria monocytogenes, which Lamason’s lab also studies, uses actin tails to force itself into a neighboring cell. In contrast, R. parkeri can form an actin tail, but loses it before reaching the cell junction. Somehow, R. parkeri is still able to spread to neighboring cells.

After an MIT symposium on the lesser-known functions of the endoplasmic reticulum, Acevedo-Sanchez developed a cell line to observe whether rickettsiae might spread to neighboring cells by hitchhiking to the cell junction.

Instead, she saw an unexpectedly high percentage of R. parkeri bacteria surrounded and coated by the ER network, at a distance of about 55 nanometers. This distance is important because membrane contacts for communication between organelles in eukaryotic cells form connections 10–80 nm wide. The researchers ruled out that what they saw was not an immune response, and that the emergency room departments reacting to the R. parkeri bacteria were still connected to the broader network of the emergency network.

“I’m convinced that if you want to learn new biology, just look at cells,” says Acevedo Sanchez. “Manipulating an organelle that establishes contact with other organelles could be a great way for pathogens to take control during infection.”

Stable communications were unexpected because the emergency room constantly interrupted and patched communications, lasting for seconds or minutes. It was surprising to see the emergency unit so consistently threaded around the bacteria. As a cytosolic pathogen that exists freely in the cytosol of the cells it infects, one would also not expect to see R. parkeri surrounded by a membrane at all.

Small margins

Acevedo-Sanchez collaborated with the Center for Nanosystems at Harvard University to view her preliminary observations at higher resolution using an ion beam scanning electron microscope. FIB-SEM involves taking a sample of cells and blasting them with a focused ion beam in order to shave off part of the cell mass. With each layer, a high-resolution image is captured. The result of this process is a stack of images.

From there, Acevedo-Sanchez identified the different regions of the images — such as mitochondria, rickettsia, or the emergency room — and a program called ORS Dragonfly, a machine learning program, sorted through the thousands or so images to identify those categories. This information was then used to create 3D models of the samples.

Acevedo-Sanchez noted that less than 5% of R. parkeri bacteria formed contacts with the emergency network, but small amounts of certain characteristics are known to be critical in R. parkeri infection. R. may be present. Parkiri are in two states: motile, with the actin tail, and immotile, without it. In mutants that are unable to form actin tails, R. parkery from progressing to neighboring cells, but in non-mutants, R. Barkerii have tails of about 2 percent in early infestation and never exceed 15 percent when they are high.

ER interacts only with non-motile R. parkeri, and these interactions were increased 25-fold in mutants that cannot form tails.

Create connections

Co-authors Acevedo Sanchez, Patrick Weyda, and Carolyn Anderson also investigated potential ways in which emergency room communications are mediated. VAP proteins, which mediate ER interactions with other organelles, are known to be co-opted by other pathogens during infection.

During R. parkeri infection, VAP proteins are recruited to the bacteria; When VAP proteins were inactivated, the frequency of interactions between R. parkeri and the ER was reduced, suggesting that R. parkeri may take advantage of these cellular mechanisms for its own purposes during infection.

Although Acevedo Sanchez now works as a senior scientist at AbbVie, Lamason’s lab continues to work to explore the molecular factors that may be involved, how these interactions are mediated, and whether the connections affect the life cycle of the host or bacteria.

These potential interactions are particularly interesting because bacteria and mitochondria are thought to have evolved from a common ancestor, noted Rebecca Lamason, senior author and associate professor of biology. Lamason’s lab has been exploring whether R. parkeri can form the same membrane connections that mitochondria do, although they have not yet proven this. To date, R. parkeri is the only cytoplasmic pathogen observed to behave in this way.

“It’s not just that the bacteria accidentally hit the emergency network,” Lamason says. “These interactions are very stable. The emergency room is clearly wrapped extensively around the bacteria, and is still connected to the emergency network.” “It appears to have a purpose, and what that purpose is remains a mystery.”