How Mtb safeguards itself from foreign DNA

Researchers at the Indian Institute of Science (IISc), with collaborators from the Institute of Mathematical Sciences (IMSc), have discovered how a key protein in the tuberculosis bacterium helps protect it from the influence of foreign DNA inserted into its genome. Understanding how this protein – called Lsr2 – functions could help develop drugs that target it, thereby aiding in the fight against TB.

“This protein is interesting to study as it controls a large set of genes in the bacteria,” says Mahipal Ganji, Assistant Professor at the Department of Biochemistry, IISc, and corresponding author of the study.

Scientists have earlier shown that Lsr2 is crucial for Mycobacterium tuberculosis (Mtb) to infect other organisms.

In the current study, the team found that this protein silences regions of Mtb DNA which contain genetic material that foreign organisms such as viruses may have inserted into its genome. When a cell produces proteins encoded by foreign DNA, it may interfere with its own functioning. So, it is crucial for the cell to identify and stop these genes from being expressed.

Proteins typically regulate DNA expression by binding to specific sequences of bases (adenine, guanine, thymine or cytosine) on the genome, and stopping the cell’s machinery from “reading” the DNA in order to produce the protein.

The team found that Lsr2 binds to large regions of the DNA that are rich in the bases adenine (A) and thymine (T). Intriguingly, if there are enough Lsr2 proteins close to each other, they start sticking together. This clumping or condensation prevents the DNA regions from being read and transcribed. This is a unique mechanism that Lsr2 uses to block the production of proteins that may harm the bacteria.

“We never expected the protein to form condensates,” says Ganji. “We were initially doing single molecule experiments and we saw hints that maybe it was forming these condensates, and then we investigated further.”

The team used a combination of single-molecule imaging, computer simulations, and microscopy to visualise individual Mtb cells in order to piece together the exact mechanism of this protein’s operation. They created several long strands of DNA with different concentrations of bases to observe how the protein behaves. One of the methods used in the study allows the imaging of single strands of DNA by stretching it over a piece of glass. This technique was developed by Ganji during his work as a post-doctoral researcher at TU Delft in The Netherlands. “Very few labs around the world currently use this method,” he explains.

Using these techniques, they identified which parts of the Lsr2 protein specifically come into play to form these condensates. They found that one region of the protein is crucial for forming links with other protein molecules, while another region allows it to attach itself to the DNA segment.

Prakshi Gaur, PhD student in Ganji’s lab and one of the lead authors, says, “The Mtb genome is highly GC (guanine and cytosine) rich. The foreign gene … is high in AT (adenine and thymine) content, which is why this protein silences these sections of the DNA.”

“If we can design some proteins that target these regions, we could stop Lsr2 from forming these condensates,” adds Thejas Satheesh, former Master’s student and another lead author. This can be a possible approach to develop interventions to stop this bacterium from infecting its hosts.

The team is now studying the mechanism by which these condensates stop the transcription of the genome in the bacterium, and how other proteins in Mtb interact with and regulate Lsr2 and its functioning.