Possible ‘Trojan Horse’ found for treating stubborn bacterial infections

Bacteria can be tricked into sending death signals to stop the growth of their slimy, protective homes that lead to deadly infections, a new study demonstrates.

Black and white microscope image of a bacterial cell with an extracellular vesicle attached.

Transmission electron microscope (TEM) image of the bacterial cell with an extracellular vesicle attached.

The discovery by Washington State University researchers could someday be harnessed as an alternative to antibiotics for treating difficult infections. Reporting in the journal, Biofilm, the researchers used the messengers, which they named death extracellular vesicles (D-EVs), to reduce growth of the bacterial communities by up to 99.99% in laboratory experiments.

“Adding the death extracellular vesicles to the bacterial environment, we are kind of cheating the bacteria cells,” said Mawra Gamal Saad, first author on the paper and a graduate student in WSU’s Gene and Linda Voiland School of Chemical Engineering and Bioengineering. “The cells don’t know which type of EVs they are, but they take them up because they are used to taking them from their environment, and with that, the physiological signals inside the cells change from growth to death.”

Bacterial resistance is a growing problem around the world. In the U.S., at least 2 million infections and 23,000 deaths are attributable to antibiotic-resistant bacteria each year, according to the U.S. Centers for Disease Control. When doctors use antibiotics to treat a bacterial infection, some of the bacteria can hide within their tough-to-penetrate, slimy home called a biofilm. These subpopulations of resistor cells can survive treatment and are able to grow and multiply, resulting in chronic infections.

“They are resistant because they have a very advanced and well-organized adaptive system,” said Saad. “Once there is a change in the environment, they can adapt their intracellular pathways very quickly and change it to resist the antibiotics.”

In their new study, the researchers discovered that the extracellular vesicles are key to managing the growth of the protective biofilm. The vesicles, tiny bubbles from 30 to 50 nanometers or about 2,000 times smaller than a strand of hair, shuttle molecules from cells, entering and then re-programming neighboring cells and acting as a cell-to-cell communications system.

TEM and confocal laser scanning microscopy (CLSM) of the interactions
of the G-EVs/D-EVs and P. aeruginosa PAO1 bacterial cells

A: TEM image of the bacterial cell after incubation with G-EVs added to the cell culture. After 24 h, the cells were negatively stained with 2% uranyl acetate before TEM images were taken (magnification power = 60000X and scale bar = 200 nm). Blue arrows indicate either an individual EV or an EV that is interacting with the P. aeruginosa cell. B: CLSM images of the bacterial cells that were incubated with DAPI-dye-labeled G-EVs. The images were taken with an excitation/emission of 359 nm/461 nm. Under this UV excitation, the recipient cells of DAPI-labeled G-EVs emit green fluorescence. The image on the left shows an overlay of the brightfield and fluorescent images, while the image on the right shows the fluorescent image only. The green fluorescence indicates the presence of the DAPI-labeled G-EVs. C: TEM image of bacterial cells in the log phase after incubation with D-EVs. The cells were negatively stained with 2% uranyl acetate before TEM images were taken (magnification power = 40000X and scale bar = 100 nm). The image on the left shows a cell without D-EV treatment; double membrane layers are visible. The image on the right shows two cells treated with D-EVs. The membranes and cell walls of the cells have become damaged. The average sizes of P. aeruginosa PAO1 bacterial cells observed in images A and C are between 0.6 and 1.2 um, consistent with the literature report. D: CLSM images of the bacterial cells that were positively stained with LIVE/DEAD BacLight Bacterial Viability Kits (excitation/emission: 480/500 nm for SYTO 9 and 490/635 nm for propidium iodide). The image on the left shows the green fluorescent live cells treated with PBS buffer; the image on the right shows the red dead cells (scale = 75 μm) after incubation with D-EVs. 

As part of this study, the researchers extracted the vesicles from one type of bacteria that causes pneumonia and other serious infections. They determined that the bacteria initially secrete vesicles, called growth EVs, with instructions to grow its biofilm, and then later, depending on available nutrients, oxygen availability and other factors, send EVs with new instructions to stop growing the biofilm.

The researchers were able to harness the vesicles with the instructions to stop growth and use them to fool the bacteria to kill off the biofilm at all stages of its growth. Even when the biofilms were healthy and rapidly growing, they followed the new instructions from the death EVs and died. The death EVs can easily penetrate the biofilm because they are natural products secreted by the bacteria, and they have the same cell wall structure, so the cells don’t recognize them as a foreign enemy.

“By cheating the bacteria with these death EVs, we can control their behavior without giving them the chance to develop resistance,” said Saad. “The behavior of the biofilm just changed from growth to death.”

WSU Professor and corresponding author Wen-Ji Dong, who has been studying the vesicles for several years initially thought that all of the bacterial-secreted vesicles would promote cell growth. The researchers were surprised when they found that older biofilms provided instructions on shutting themselves down.

“So now we’re paying attention to the extracellular vesicles secreted by older biofilms because they have therapeutic potential,” he said.

The researchers are applying for research funding from the National Institutes of Health to continue investigating exactly how the messengers work and how well the process works with other bacterial types or fungi. They are working with WSU’s Office of Commercialization and have applied for a provisional patent.

SourceWashington State University

Saad MG, Beyenal H, Dong WJ. (2024) Dual roles of the conditional extracellular vesicles derived from Pseudomonas aeruginosa biofilms: Promoting and inhibiting bacterial biofilm growth. Biofilm 7:100183. [article]

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