Small DNA-based inhibitor blocks entry of TB bacteria into host cells

Jaya Sivaswami Tyagi-Optimized

HupB is an essential protein of TB bacteria and so an attractive drug target,” say Jaya Sivaswami Tyagi (left), Priya Kalra and Tarun Kumar Sharma (right).

A team of researchers from three institutions in India has used aptamer-based inhibitor that binds to the HupB protein found on TB bacteria cell surface to significantly inhibit the entry of the bacteria into host cells. The researchers are now trying to deliver the aptamer into TB bacterial cell. If successful, it will make the bacteria vulnerable to stresses inside host cells leading to their death.

By using a small single-stranded DNA molecule (DNA aptamer) that specifically binds to a single protein (HupB) present in TB bacteria, researchers have been able to achieve 40-55% reduction in the bacteria’s ability to enter into human cells and infect them. Besides facilitating entry into host cells, HupB also helps the TB bacteria survive various stresses encountered inside host cells.

The HupB protein was discovered in late 1990s by Prof. H. Krishna Prasad, formerly with AIIMS, while looking at specific TB bacterial antigens that induced immune response in humans. He found the protein was associated with the DNA of the bacteria (Tubercle and Lung Disease journal). “Since it is associated with the DNA, we didn’t expect it to be present on the surface of the bacteria. But to our surprise, it was seen on the surface of the TB bacteria too,” recalls Prof. Prasad. Further studies showed that the HupB protein was able to interact and bind to proteins found on the surface of host cells, which will facilitate the entry into host cells.

“HupB is an essential protein of TB bacteria and so an attractive drug target,” says Prof. Jaya Sivaswami Tyagi from the Department of Biotechnology at AIIMS and one of the corresponding authors of a paper published a few days ago in the journal Molecular Therapy – Nucleic Acid.

The cell-surface associated HupB protein facilitates the entry into host cells, while  the protein that binds to the TB bacteria’s DNA protects the bacteria from multiple stresses inside the host cell.

Despite being an attractive drug target, designing inhibitors against HupB has been largely unsuccessful as the full length crystal structure of the protein is not known due to the disordered nature of the end portion of the protein (C-terminal). As a result, the classical drug discovery route, which relies on the structure, cannot be used for designing inhibitors against HupB protein.
And this is where aptamers come in handy; there is no need to know the structure of the protein when aptamers are used. “Since aptamers are selected based on their affinity to bind to the molecule being targeted, we turned our attention to search for aptamer-based inhibitors,” says Prof. Tyagi.

Two aptamers chosen for detailed study

From a collection of two types of DNA libraries, the researchers selected 23 aptamers. Of the 23, only two aptamers (4T and 13T) were chosen based on high binding affinity and specificity to the HupB protein. Both the aptamers remained stable when exposed to serum, an essential requirement for an inhibitor. The aptamers were found to bind at different positions of the protein. Also, the binding was four-fold higher when the aptamers bound to the full-length protein than when bound to only the N-terminal portion of the truncated protein, suggesting the importance of C-terminal for efficient binding.

While the cell-surface associated HupB protein facilitates the entry into host cells, the protein that binds to the TB bacteria’s DNA protects the bacteria from multiple stresses inside the host cell.

The binding of the aptamers to the cell-surface associated HupB protein was first confirmed using live, disease-causing TB bacteria. The researchers then treated the disease-causing TB bacteria with the aptamers to test the ability of the bacteria to enter human cells.

Inhibiting entry into cells

“Compared with controls, the aptamer-treated bacteria showed reduced ability to enter the host cells. At 55%, the HupB-13T aptamer had greater ability to inhibit TB bacteria entry than the HupB-4T (42%) aptamer,” says Dr. Tarun Kumar Sharma from Centre for Biodesign and Diagnostics, Translational Health Science and Technology Institute (THSTI), Faridabad and another corresponding author of the paper.

“The TB bacteria use a number of proteins to enter host cells. But their entry into host cells is inhibited 40-55% when the HupB protein alone is inhibited. This shows how vital the HupB protein is in modulating the bacterial entry into host cells,” says Dr. Priya Kalra from the Department of Biotechnology at AIIMS and first author of the paper.

But will the inhibition to enter host cells be greater if both the aptamers are used simultaneously? “Ideally, both the aptamers can be used. But no study was done to determine this. We speculate that the cocktail will have a complementary effect. But it is hard to say by how much the invasiveness will be reduced when the aptamers are used together,” says Dr. Kalra.

Killing the bacteria

Using the HupB protein alone, the researchers tested the ability of the aptamers to inhibit DNA binding. At 75%, the HupB-4T aptamer showed greater DNA binding inhibition than the HupB-13T aptamer (25%). Inhibition of DNA binding will make the bacteria vulnerable to stress inside the host cells, leading to death.

“We are now trying to deliver the aptamer into the TB bacterial cell to inhibit the DNA-binding function of HupB. But it is a huge challenge as there are three barriers to cross before getting inside the bacteria membrane. The infective TB bacteria present inside a human cell are enclosed by a phagosome vesicle. So the aptamer has to first cross the human cell wall, then the vesicle and finally the TB bacteria cell membrane, which is particularly thick,” says Prof. Tyagi.

“With increasing drug resistance and the difficulty to come up with safe and efficacious drugs, the aptamer-based approach is ideal alternative as HupB protein is vital for survival of the bacteria,” says Dr. Sharma. “So by targeting the HupB protein using an aptamer-based inhibitor can effectively block TB infection and will be effective against both drug-sensitive and drug-resistant bacteria.”

Published in The Hindu on September 8, 2018