Scientists at the Indian Institute of Science (IISc) Bengaluru have developed two new, potent molecules that can severely impact the survival of mycobacteria, including Mycobacterium tuberculosis that causes TB. The results were published in the journal Antimicrobial Agents and Chemotherapy.
Unlike most antibiotics that target the bacterial metabolism by aiming at the cellular components, the novel molecules inhibit the stress response pathway of mycobacteria. The stress response pathway is crucial for bacteria to survive during hostile conditions such as lack of nutrients and the presence of antibiotics, to name a few. So any inhibition of this pathway will lead to its death.
The master regulator of stress pathway in the case of mycobacteria is (p)ppGpp (Guanosine pentaphospahte or Guanosine tetraphosphate). Though a molecule that inhibits the (p)ppGpp formation has already been synthesised, the efficacy is not much. “Very high concentration of Relacin molecule is needed to inhibit the pathway and, therefore, the efficacy is low. So we synthesised two new molecules — acetylated compound (AC compound) and acetylated benzoylated compound (AB compound) — by bringing about a modification in the base of the Relacin molecule,” says Prof. Dipankar Chatterji from the Division of Biological Sciences at IISc and the corresponding author of the paper.
“We found both the molecules to be very good inhibitors of stress response. The two compounds affected the rate of synthesis of (p)ppGpp and also reduced the cell survival,” he says. Laboratory studies showed that the two molecules were not toxic to human cells and were able to penetrate the human lung epithelial cells.
“We found our compounds were targeting the Rel gene. The Rel gene makes Rel protein, which in turn synthesises (p)ppGpp. When the Rel gene is knocked out, the long-term survival of Mycobacterium smegmatis decreases,” says Prof. Chatterji.
“The Alarmone molecule “(p)ppgpp”, a modified nucleotide, is ubiquitous in bacteria and absent in humans. Inhibiting (p)ppgpp synthesis would specifically target the survival of bacteria without having any effects on humans,” says Dr. Kirtimaan Syal from the Division of Biological Sciences, IISc and the first author of the paper.
Earlier studies have shown that when the rel gene is deleted, the long-term survival ability under stress was lost; the M. tuberculosis bacteria was unable to persist in mice and unable to form tubercle lesions in guinea pigs.
“The major reason for prolonged treatment of TB is the bacterium’s ability to persist in dormant form, which is tolerant to most antibiotics used in the treatment regimen. So inhibition of (p)ppGpp-mediated persistence could help in shortening the treatment regime, dealing with the emergence of multiple drug resistance and treatment of chronic infections, Dr. Syal says.
Under hostile conditions, bacteria tend to form biofilms, which protect the bacteria from stress and induce tolerance to antibiotics. Recent studies have shown that tuberculosis bacteria that cannot form a biofilm cannot survive inside the host. Evidences have shown that at the time of infection, the M. tuberculosis display a biofilm-like phenotype and this helps the bacteria to survive inside the host.
Studies carried out by the researchers showed that both the molecules were able to inhibit biofilm formation by M. tuberculosis and M. smegmatis and also disrupt the already formed biofilm. “The biofilm formed by TB bacteria is very dangerous. The ability of the molecules to destroy the biofilm and even prevent its formation is a very important achievement,” says Prof. Chatterji.
Since there are very few antibiotics that target the stress response pathway of the bacteria, the two molecules offer great promise. “The next step is to test the molecules on animals. We have not thought about it. It will also be interesting to see if the bacteria develop resistance against these molecules,” Prof. Chatterji says.