IISc’s potent molecules show promise for TB therapy

SEM photo of Mycobacterium tuberculosis bacteria. - Photo NIAD-Optimized

The two molecules were able to prevent biofilm formation and even disrupt biofilms that had formed.

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.

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Inhibiting (p)ppGpp synthesis would target the survival of the bacteria, says Dr. Kirtimaan Syal.

“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.

Inhibiting biofilm

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.

Published in The Hindu on April 15, 2017

Indian researchers find a new bacterial target for drug development

Anshika Andaleeb Richa-Optimized

(From left) The study by Anshika Singhal, Andaleeb Sajid and Richa Misra helped understand how bacteria form biofilm.

Indian researchers have found a new target that can potentially be used for developing new antibiotics that will be effective against many bacteria. The new target is made of two proteins — which form a complex that is responsible for the formation of biofilm — that perform very important functions and are critical for bacterial ability to successfully infect humans. The results were published in the journal Biofilms and Microbiomes.

Bacteria form biofilms, a kind of matrix, during infection in plants and animals. Biofilm shields the bacteria from antibiotics and help bacteria to survive harsh conditions such as extreme temperature or stress. Now a study by Indian researchers has found the molecular signaling events that play a crucial role in biofilm formation in Bacillus anthracis, the causative agent of anthrax.

Till now, all attention has been on developing antibiotics that target disease-causing bacteria and not the biofilm itself.
One of the basic questions that scientists have been trying to answer is how and when bacteria decide to form biofilm. “One possibility is that bacteria has sensors on the surface which senses some signal and helps in biofilm formation,” says Andaleeb Sajid from the Institute of Genomics and Integrative Biology (IGIB), Delhi and one of the authors of the paper.

“It was serendipity. Our lab was working on signaling in bacteria and we were studying PrkC and similar proteins. When PrkC protein is deleted, Bacillus bacteria are unable to form biofilm. So we started studying the mechanism by which PrkC protein controls biofilm formation,” she says.

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Gunjan Arora says the GroEL-PrkC complex could be a target for developing new drugs.

“Our hypothesis is that PrkC senses some signal and transmits it from outside to inside the cell. This signal goes to other proteins like GroEL. PrkC adds phosphate group (phosphorylate) to different proteins. The mystery to biofilm formation lies with one chaperone protein called GroEL. The addition of phosphate to this tiny machine initiates a course of events within bacterial cells leading to complex biofilm formation,” Dr. Sajid says.

The team found several proteins receive signals from PrkC protein. Using cutting edge genetics, molecular biology and proteomics techniques, they confirmed that GroEL was regulated by PrkC.

“From other unrelated bacteria we already had a clue that GroEL has a role in biofilm formation. We looked at the molecular level and found six amino acid residues where phosphate was getting added to the GroEL protein. Through a series of steps, we ascertained how important phosphorylation was for proper functioning of GroEL,” says Gunjan Arora from IGIB and the first author of the paper.

“We wanted to know if the bacteria has any other compensation mechanism to form biofilm in the absence of PrkC. So we made PrkC mutant bacteria to produce more of GroEL. The bacteria were able to form biofilm even in the absence of PrkC. This experiment helped us understand that PrkC is the influencer and GroEL is key to biofilm formation,” Dr. Arora says.

Both PrkC and GroEL perform very important functions and are critical for bacterial ability to successfully infect humans. “We think GroEL-PrkC complex could be a target for developing new antibiotic that will be effective against many bacterial pathogens such as the ones that cause MRSA, TB and pneumonia. One strategy to tackle drug resistant bacteria will be to develop multi-drug regimen that combines traditional antibiotics with candidate drugs that can block bacterial signaling and prevent biofilm formation,” Dr. Arora says.

Published in The Hindu on March 26, 2017