Bacteria have an arsenal of molecular weaponry that they use to wage wars for survival. One of these weapons is Nisin — a bacterial toxin or bacteriocin made of 34 amino acids known to punch holes into the membranes of other bacteria to kill them. Although Nisin is a weapon of bacterial origins, humans use it as a food preservative. However, since drug resistance can evolve rapidly in bacteria, we are constantly trying to create better antibiotics to preserve food and cure diseases.

And to create better antibiotics, one needs to understand how antibiotics work. 

Nisin is known to kill bacteria by perforating their membranes and interrupting cell-wall biosynthesis by docking onto a fat molecule known as lipid II. Now, new research from Mohammed Saleem’s and Yusuf Akhter’s groups from the National Institute of Technology, Rourkela, and the Central University of Himachal Pradesh, Dharamsala, hints at another mechanism used by Nisin to kill bacteria. The scientists have shown that at high concentrations, Nisin interacts with phospholipids on bacterial membranes to deform them, eventually killing the bacteria.

“Although the specific lipid II — Nisin interaction has been found to result in pores that destabilise bacterial cell integrity, the amount of lipid-II present in bacterial membranes varies and is many folds less in number compared to other phospholipids,” says Saleem. ​“We therefore wondered if Nisin could interact non-specifically with membrane phospholipids on the bacterial cell membrane,” he adds.

Using a mix of approaches that included molecular dynamics simulations (MDS), microbiology and in vitro biophysical experiments, the researchers have shown that Nisin can interact with membrane phospholipids at high concentrations. The MDSs indicated that when Nisin forms five-molecule complexes, or pentamers, it can insert itself into lipid bilayers to destabilise membranes. Through its interactions with phospholipids, Nisin forms ​“water cavities” that warp these membranes. Furthermore, the researchers have used simulations to confirm that this effect of Nisin was independent of its lipid II-based mechanism of action. 

The group then tested their predictions in a biological system — two types of bacteria with vastly differing levels of lipid II on their surfaces. In micromolar concentrations, Nisin was capable of killing both the lipid II-rich Bacillus subtilis and the lipid II-poor Escherechia coli bacteria. Surprisingly, Saleem’s team also found that at these concentrations, Nisin was able to efficiently inhibit E. coli growth. This counters the commonly held belief that this antibiotic can target gram-negative bacteria only in the presence of heat shock or the chelating agent EDTA (Ethylene-diamine-tertaacetic-acid).

Further investigations via electron microscopy revealed that high surface concentrations of Nisin distorted bacterial membranes by causing roughness and blebbing. Experiments on Nisin treatment of bacteria at different stages of growth have also brought to light some interesting observations. 

“Most previous work only considered growth kinetics spanning 8 hours,” says Saleem. ​“Surprisingly, we found that at low concentrations, Nisin induces an extended lag phase, from which bacteria are able to recover. This suggests that the lag phase of bacterial growth may sometimes be mistaken for bacterial cell death. We need to be more careful about interpretations of the minimal inhibitory concentrations of various peptide antibiotics, especially in terms of bacterial growth dynamics,” he adds.

Based on their results which have been published in a paper in the journal Scientific Reports, a generalised mechanism of how Nisin affects bacterial membranes emerges. Depending on the concentration of membrane-bound Nisin, pores in the bacterial membrane form, causing distortion and loss of membrane potential. As the concentration of Nisin molecules increase, pores last longer and grow bigger. As lipid molecules in the membrane move to close pores, surface tension in the membrane forces lipids out of the membrane. This eventually leads to the reduced surface area and blebbing deformities seen in Nisin-treated bacteria. 

“Undoubtedly, specific biochemical interactions are critical to any antimicrobial mechanism,” says Saleem. ​“However, as bacteria can sense physical forces acting on or within their membrane surfaces, it becomes important to explore the possibility of non-specific modes of action in antibiotics,” he says summing up the importance of the collaborative work the teams have carried out.