How can mankind use bacteria’s internal mechanism of fighting a virus to protect human cells from a bacterial infection? Mahavir Singh, Associate Professor, Molecular Biophysics Unit at the Indian Institute of Science, and his team are seeking to answer the question.

There is a constant struggle for survival between phages (eg viruses that attack bacteria) and bacteria, and both sides have evolved several mechanisms to defend themselves. One such strategy involves the use of the toxin-antitoxin (TA) systems.

Every bacterium hosts inside itself a combination of a toxin (T), usually a protein, and an antitoxin (A), which can be a protein or an RNA molecule. In its free-form, the toxin is poisonous to bacteria that contains it. Therefore the bacteria keep it in a complex form (toxin-antitoxin — TA), bound with the antitoxin. However, a question arises: why would any bacteria host a toxin that could potentially kill the bacteria itself? Because of this, the TA complexes are called ‘internal bombs’ because they serve as a defence mechanism against invading phages.

When a phage attacks bacteria, it takes over the host’s internal machinery to multiply its genetic material (DNA or RNA) inside, killing the bacteria in the process. In an act of altruism, as Prof Singh calls it, the bacteria breaks down the TA complexes to free up the toxins which activate to prevent the viral infection from spreading beyond its walls.

Singh and his team have set out to solve the question: How are the TA complexes assembled, and how can we use these systems to turn them against bacteria themselves? Of the eight types of TA systems identified so far, Singh’s lab is interested in the Type III TA system, where the antitoxin is not a protein but rather a stretch of ribonucleic acid (RNA). The toxin gene gets transcribed on to the RNA which then is used to produce the toxin protein, but the antitoxin gene only gets transcribed to RNA. In type III TA systems, says Singh, the toxin protein is an enzyme that “cuts up its own antitoxin RNA into precise bits that in turn bind to the toxin to form the inactive TA complex”.

Free the toxin!

Why is antitoxin as RNA important here? Being the RNA, it gets degraded faster than the protein toxin. Now, if there is a transcription shut-off (as happens in the case of a virus attack), no new antitoxin is being produced. As a result, more and more free toxin accumulates in the cell. This free toxin cleaves the phage RNA (along with the bacterial RNAs), effectively preventing phage propagation from infecting other bacteria.

Singh’s team has shown that at least five types (clusters) of such complexes can exist in different strains of E.coli bacteria. Further, he says, the team has also published material on a detailed crystal structure of an E. coli type III TA complex depicting the tightly bound toxin-RNA complex in the bacterium.

Simply put, Singh’s team has helped establish the structure and binding nature between the antitoxin RNA and the toxin protein. The next step, he says, is to find a molecule which can disrupt this complex arrangement and free the toxin. The free toxin will then destroy the bacteria thus preventing bacterial infection.

“The world of science can potentially design some peptide or molecule in a way that frees the toxin. To do this, you have to understand the structure of these TA systems first, which is what we have currently achieved,” he says.

“The hetero-hexameric closed complex structure helps bind the two tightly. The peptide/molecule should be able to bind with the antitoxin to set the toxin free.”

Now, the other question is, what if newly designed peptides or drugs harm human cells by inhibiting human proteins? “So far, no TA systems have been identified in human cells. They seem to be bacteria-specific. So, it is hoped that a peptide or the drug effective against bacteria will not act against the human cells,” says Singh.

His team is currently working on a small-scale project “to see whether they can dislodge this complex and free the toxin.”

How difficult is it to design a successful inhibitor? Singh says there are several unknowns. For example, “you design inhibitors for one TA complex; but, there may be another, slightly different antitoxin in bacteria which can bind and neutralise the toxin. So, there is potential for ‘crosstalk’ that makes designing an inhibitor challenging.”

“The other challenge is that the interaction of the toxin and antitoxin is very extensive, and they’re very tightly bound. The TA complex formation involves extensive surface involvement from both the toxin and the antitoxin. Finding inhibitors for such large binding surfaces is further challenging.”

If you want to find an inhibitor of the complex, you first need to understand how it is assembled before you can design an effective inhibitor, Singh points out.

Since the team has identified type III TA systems and characterised them for their function, assembly and structure, these can be “ used as antibiotic targets for designing novel antibiotics; that’s where scientists see potential during this era of emerging antibiotic resistance in bacteria,” says Singh.