Researchers in Hyderabad have successfully expressed and purified a novel antimicrobial protein from the milk of an egg-laying mammal, echidna, using a simple bacterial system. The protein displays antibacterial action against a wide spectrum of bacteria and could be useful in battling drug-resistant pathogens.
Egg-laying mammals have been studied extensively because of their unique position in the evolutionary tree and their unusual means of reproduction and milk secretion. In a recent study, researchers led by Satish Kumar at the Centre for Cellular and Molecular Biology (CCMB) have characterized an antimicrobial protein present in the milk of Echidna, an egg-laying mammal native to Australia and New Guinea.
Echidnas lay soft-shelled eggs and the hatchlings that emerge are significantly underdeveloped. For the first several months of their lives, these hatchlings are completely dependent on mother’s milk for survival. Since female echidnas lack nipples, the milk is secreted instead through skin pores on the body surface, which the hatchlings then lick off.
Echidnas reside and lay eggs in burrows in the ground. As a result, they are exposed to millions of soil bacteria, which the hatchlings ingest along with the milk. Some of these are potentially infectious, and the hatchlings’ first line of protection comes from antimicrobial compounds in the milk itself.
Kumar and colleagues decided to purify and characterize one such Echidna milk protein, EchAMP, using E. coli, a common bacterial system. The researchers artificially introduced the gene for EchAMP into E. coli, which acts as a tiny production factory for the protein. By altering the sequence of the gene slightly, they could purify a functional form of the protein in bulk.
The researchers found that the purified protein shows antibacterial activity against both major classes of bacteria: Gram-negative and Gram-positive, many of which are present in the soil. Alok Kumar, one of the lead authors of the study says, “The role that EchAMP plays in its native source, echidna, and its absence in other mammals, makes it most interesting”.
The team attempted to understand EchAMP’s mode of action and found that it kills bacteria by causing the cells to lyse. Following a thorough structural analysis, they found that the EchAMP belongs to a class called intrinsically disordered proteins (IDPs). Unlike most proteins, disordered proteins do not possess a concrete three-dimensional structure. Since protein functions are generally attributed to a defined structure, understanding how disordered proteins work is a difficult task.
There have been reports that a few antimicrobial proteins are natively disordered but acquire a shape after encountering the bacterial cell membrane. To test if EchAMP functions in the same manner, the researchers exposed the protein to molecules that mimic the bacterial cell membrane and found that indeed, the protein acquires a defined shape in the presence of these molecules. This led them to hypothesize that the otherwise disordered protein acquires a fixed structure within the cell membrane and forms pores which lead to cell lysis.
R Nagaraj, a principal scientist in CCMB, who was not involved in the study, says, “This is a great basic research finding. Its practical utility can be determined with further experiments like testing the protein for its action against clinical strains and multidrug-resistant bacteria.”
Given EchAMP’s wide spectrum of antimicrobial activity against many bacterial species and the low dose required for effective action, the researchers propose that it could potentially be used in topical applications like ointments or medicated creams for bacterial infections in livestock and humans.
This finding could be particularly important in the context of mastitis (an infection of breast tissue which causes swelling, pain and decreased milk production) in cattle caused by drug-resistant bacteria, which leads to severe economic losses for the dairy industry each year. With multi-drug-resistant infections on the rise, looking towards such unusual sources for antimicrobials could become an important strategy going forward.
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