Tiny molecules in cells and mice stop bacteria from hijacking cells

Tiny molecules called nanobodies, which can be designed to mimic the structures and functions of antibodies, may be the key to blocking a tick-borne bacterial infection that remains beyond the reach of almost all antibiotics, new research suggests.

The infection is called human monocytic ehrlichiosis and is one of the most common and potentially fatal tick-borne diseases in the United States. The disease initially causes flu-like symptoms common to many illnesses and, in rare cases, can be fatal if left untreated.

Most antibiotics cannot build up to concentrations high enough to kill the bacteria responsible for the infection, Ehrlichia chaffeensis, because the microbes live and multiply inside human immune cells. Commonly known bacterial pathogens such as Streptococcus and E. coli cause their infectious damage outside of the host’s cells.

Researchers at Ohio State University have created nanobodies to target a protein that makes E. chaffeensis bacteria particularly infectious. A series of experiments in cell cultures and mice showed that a specific nanobody created in the lab could inhibit infection by blocking three ways the protein allows bacteria to hijack immune cells.

“If more than one mechanism is blocked, it’s better than just shutting down a function, and it gives us more confidence that these nanobodies will actually work,” said lead study author Yasuko Rikihisa, professor of veterinary biosciences. at the Ohio State.

The study provided support for the feasibility of treatment for ehrlichiosis using nanobodies, but much more research is needed before a treatment is available for humans. There is a certain urgency to offer an alternative to the antibiotic doxycycline, the only treatment available. The broad-spectrum antibiotic is dangerous for pregnant women and children, and it can cause serious side effects.

“With only one antibiotic available as a treatment for this infection, if antibiotic resistance were to develop in these bacteria, there would be no more treatment. It’s very scary,” said Rikihisa.

The research is published this week in Proceedings of the National Academy of Sciences.

The bacteria that cause ehrlichiosis are part of a family called obligate intracellular bacteria. E. chaffeensis not only requires internal access to a cell in order to live, but also blocks the ability of host cells to program their own death with a function called apoptosis – which would kill bacteria.

“Infected cells would normally kill themselves by apoptosis to kill the bacteria inside. But these bacteria block apoptosis and keep the cell alive so that they can multiply hundreds of times very quickly and then kill the host cell. “said Rikihisa.

A long-time specialist in the Rickettsial bacteria family to which E. chaffeensis belongs, Rikihisa developed the precise culture conditions that made it possible to cultivate these bacteria in the laboratory in the 1980s, which led to his dozens of discoveries explaining their operation. Among these findings was the identification of proteins that help E. chaffeensis block programmed cell death of immune cells.

Researchers synthesized one of these proteins, called Etf-1, to make a vaccine-like agent that they used to immunize a llama with the help of Jeffrey Lakritz, professor of veterinary preventive medicine at Ohio State. . Camels, llamas and alpacas are known to produce single chain antibodies that include a large antigen binding site on the tip.

The team cut out segments of this binding site to create a library of nanobodies that can function as antibodies that recognize and bind to the Etf-1 protein and stop infection with E. chaffeensis.

“They work the same as our own antibodies, but they are tiny and tiny nano-antibodies,” Rikihisa said. “Because they’re small, they get into nooks and crannies and recognize antigens much more efficiently.

“Large antibodies cannot enter a cell. And we don’t need to rely on nanobodies to block extracellular bacteria because they are on the outside and accessible to ordinary antibodies that attach to them.”

After selecting candidates for their efficacy, the researchers landed on a single nanobody that attached to Etf-1 in cell cultures and inhibited three of its functions. By making the nanobodies in the fluid inside the cells of E. Coli, Rikihisa said her lab could produce them on an industrial scale if needed – packing millions of them into a tiny drop.

She collaborated with co-author Dehua Pei, Ohio State Professor of Chemistry and Biochemistry, to combine the tiny molecules with a cell-penetrating peptide that allowed nanobodies to be delivered safely. to mouse cells.

Mice with weakened immune systems were inoculated with a highly virulent strain of E. Chaffeensis and treated with intracellular nanobodies one and two days after infection. Compared to mice that received control treatments, mice that received the most potent nanobodies showed significantly lower levels of bacteria two weeks after infection.

With this study providing proof of principle that nanobodies can inhibit E. chaffeensis infection by targeting a single protein, Rikihisa said that there are several additional targets that could provide even more protection with nanobodies administered alone or in combination. combination. She also said the concept is widely applicable to other intracellular diseases.

“Cancers and neurodegenerative diseases work in our cells, so if we want to block an abnormal process or an abnormal molecule, this approach can work,” she said.

This study was supported by the National Institutes of Health.

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