Mycobacterium tuberculosis is the causal agent of tuberculosis (TB), which is transmitted through air droplets from person to person and causes severe lung defects. Many great strides – including the development of antibiotics – have been taken to reduce the spread of TB, however it remains a global health concern, still impacting millions of people today.

This is because M. tuberculosis is rapidly acquiring new tools in its arsenal to become resistant to antibiotics, such as thickening its cell wall so drugs can’t enter the cytoplasm. Finding novel targets to treat TB is therefore an urgent priority.

Researchers are therefore trying to uncover clues in the way that M. tuberculosis organises its genome to see whether this allows the bacterium to defend itself.

Last month, Maurizio et al. published a study in Nature Communications investigating the role of DNA looping structures called G-quadruplexes in M. tuberculosis. These structures may help it acquire resistance to oxidative stress (more on this later).

What are G-quadruplexes (G4s)?

G4s were mentioned once fleetingly during my undergraduate course on a slide at the very end of a lecture, so I had pretty much forgotten they existed until I saw this paper! So, let’s break down what they are.

When four guanine (G) bases in DNA form weak bonds with each other, this forms a G-tetrad. These tetrads then stack on top of each other with a central metal (often potassium or sodium) ion holding it all together.

You can think of G4s as scaffolding layers on a building, with the potassium ion being the building that ensures the scaffolding doesn’t fall apart. G4s have diverse roles in cells, including regulating DNA replication and maintaining telomere length.

There’s a first time for everything

The authors’ first step was to profile the locations of G4s in the M. tuberculosis genome with an antibody-based epigenomic method called CUT&Tag.

I should say that CUT&Tag has never been used in bacteria until this paper (it’s been used in virtually everything else you can think of). This is really quite something, as the authors would have had to spend many months optimising the method to suit bacterial chromatin.

CUT&Tag identified novel G4-containing regions, which the standard method of chromatin immunoprecipitation (ChIP) was not able to identify.

Moreover, it revealed that G4s in M. tuberculosis were found in gene bodies, in contrast to eukaryotic cells (e.g. animal cells) where they are found in the promoter regions of genes and are usually involved in upregulating transcription. This suggests that G4s may have a different, perhaps opposite function in bacteria compared to in our cells.

But the mystery was that G4s in M. tuberculosis didn’t appear to have anything to do with gene expression… under normal conditions.¹

Next step: change the conditions

The authors hypothesised that G4s may confer a survival advantage to the bacteria, and started investigating whether they had anything to do with oxidative stress protection.

Oxidative stress involves highly reactive molecules called radical oxygen species, which cause all sorts of intracellular damage, including breaking DNA strands.

But why did they investigate oxidative stress specifically, and not something like heat? By treating M. tuberculosis bacteria with hydrogen peroxide (the same chemical used to bleach hair!), this allowed the authors to mimic what occurs when the immune system, specifically macrophages, launches a response against them.

Intriguingly, the number of G4s jumped up under oxidative stress. At the same time, genes harbouring G4 loops saw a drop in expression² (remember, they did NOT under normal conditions), suggesting that G4s may act as genomic sensors.

Using Gene Ontology analysis, they were able to match these downregulated genes to specific functions. Downregulated genes harbouring G4s included those involved in cell wall synthesis and metabolic function, potentially allowing M. tuberculosis to lower its rate of cell division and conserve energy in order to survive during stress.

The authors therefore concluded that G4s may therefore act to protect the cell from environmental stress. Theoretically, therefore, targeting or blocking G4 formation may prevent the cell from sensing oxidative stress, ultimately leading to cell death – indeed, G4-targeting molecules have been used before to limit bacterial growth.

“Yeah, and?”

Since the 1980s, we have not discovered any new classes of antibiotics. Targeting G4s may open a new therapeutic avenue to treat bacterial diseases like TB. However, the authors did mention that not all G4s are identical in structure, and this may vary the effectiveness of any drugs.

Additionally – and this was not mentioned by the authors – but releasing oxygen radicals into the M. tuberculosis cells also occurs during rifampin treatment, a first-line antibiotic used for TB patients. Maurizio and colleagues’ research may therefore also be useful for understanding resistance to this drug too.

Finally, this is the first time CUT&Tag has been used in bacteria, which is remarkable. More epigenetic studies can now be conducted on the bacterial genome to allow us to better understand things like infectiousness and resistance to drugs.

---

Notes

1. For those who are curious, the authors determined this by overlapping the locations of G4s with RNA-seq data, which shows you which genes across the entire genome are being actively transcribed/expressed. Whether a gene harboured a G4 or not didn’t make any difference to the level of transcription. ↩︎
2. This may also suggest why the G4s were in gene bodies instead of promoters, like in eukaryotes: opposite location, opposite effect on expression (under stress). ↩︎

---

Discussion point

What other chromatin features could we investigate as a target for new TB treatments?

---