In stressful situations, when bacteria compete for nutrients, they produce toxins that kill other bacteria. These molecules, called bacteriocins, can be a therapeutic alternative for the treatment of infections caused by multi-drug-resistant (MDR) microorganisms - those that do not respond to available antibiotics. Pyocins are toxins produced by the bacterium Pseudomonas aeruginosa. Among them, pyocin S8 has potent bactericidal activity against multi-resistant strains. To understand the mechanism by which this molecule causes cell death, researchers led by Professor Luis E. S. Netto, from the Instituto de Biociências at Universidade de São Paulo (USP) and a member of the RIDC Redoxoma, performed the biochemical, microbicidal and structural characterization of pyocin S8.

“The WHO (World Health Organization) has released a list of the families of bacteria that pose the greatest threat to human health, and strains of Pseudomonas aeruginosa resistant to carbapenems are at the critical level of priority for the development of new antibiotics. The pyocin S8 can be an alternative to treat infections caused by antibiotic-resistant bacteria”, said postdoctoral fellow Helena Turano, first author of the article, published in the Journal of Bacteriology.

Pseudomonas aeruginosa is an opportunistic bacterium capable of colonizing a wide variety of hosts. In humans, this pathogen is commonly found in burn wounds, urinary tract infections, and obstructive pulmonary diseases, common in patients with cystic fibrosis. It is easily found and disseminated in the hospital environment, and is a serious problem for immunocompromised patients.

Unlike antibiotics, pyocins are proteins and have a specific spectrum of action, that is, they have activity against strains phylogenetically related to that of the producing cell. “The pyocin produced by P. aeruginosa can kill another strain of P. aeruginosa and not an E. coli, for instance. This means that the use of pyocins also has the advantage of not interfering with the host’s microbiota, improving the patient’s health, and increasing the treatment effectiveness”, said the researcher, who started studying these toxins during her doctorate, at the Instituto de Ciências Biológicas at USP, under the orientation of Professor Nilton Lincopan, one of the co-authors of the work. She identified a pyocin that had potent activity against multi-drug-resistant strains of P. aeruginosa and, when sequencing the genome, discovered that it was the S8. “This pyocin had only been described in silico, that is, by computer simulation. It was the first time that it was tested in vivo”.

Structure and Mechanism

There are three types of pyocins, classified according to their structure as R, F, and S. The first two are high-molecular-weight complexes, and the S-type, which were the focus of this research, are molecules of low molecular weight, consisting of only two protein subunits. The larger component is the one that kills the target cells and the smaller one gives immunity to the cell that produces the pyocin by binding to the larger component and inhibiting its activity, preventing the bacteria that produces the toxin from being eliminated by it.

The researchers described the structure of pyocin S8 by X-ray crystallography, with a resolution of 1.38 Å. They also did the biochemical characterization of the molecule, which involved assessing the DNase activity of its cytotoxic domain. “This DNase activity is non-specific, that is, when entering the target cell, the enzyme will cleave the DNA in several places, as it is a DNase that doesn’t have sequence specificity. This will result in cell death, as the cell cannot handle these countless lesions in the DNA molecule”, explains Fernando Gomes, who is a postdoctoral fellow at the same laboratory and co-author of the article.

The DNase activity depends on an enzymatic cofactor, in this case, a metal, a divalent cation. The metal binds to the enzyme by four highly conserved histidine residues, which make up the H-N-H motif of the molecule. To characterize the enzyme binding affinity with different metals, the researchers used the Isothermal titration calorimetry (ITC) technique, in partnership with the group of professor Cristiano L. P. Oliveira, from the Instituto de Física at USP. They concluded that the enzyme binds with a high affinity for nickel and zinc, the first inducing DNase activity and the second inhibiting it.

An important finding of the study was that the amino acid glutamate, which is a highly conserved residue of the H-N-H motif, is fundamental for catalytic activity. “Previous studies with the colicins (the toxins produced by Escherichia coli) have shown that the glutamate residue forms an interaction with an arginine. It has been shown that this interaction is important to distort the DNA molecule and bring the phosphodiester skeleton closer to the enzyme active site, allowing cleavage. However, so far, there is no crystallographic structure of pyocins in complex with DNA, and therefore we are working on it”, Gomes said.

The researchers point out that the motif that binds the metal in these enzymes, H-N-H, is widely distributed in nature. Several DNA metabolism enzymes, such as those that act in homologous recombination and DNA repair have this H-N-H motif. “Understanding how this motif works will also provide a basis for understanding other super important enzymes in biology. This is a very important contribution to this work”.

However, according to the researchers, the great potential of these molecules can be reduced if the bacteria develop resistance to pyocin. Hence the importance of understanding the molecular mechanisms by which pyocins kill target cells. “One of our goals now is to resolve the structure of the molecule complexed with DNA”, the researchers said.

In collaboration with other researchers, who provided a collection of strains of multi-resistant bacteria, Helena intends to conduct preclinical trials to find out what is the minimum concentration of pyocin S8 necessary to kill the bacteria. The next step will be to perform animal tests. “Unfortunately, with the pandemic, we stopped, but the idea is that when we resume normality, it can be done,” concludes the researcher.

The article Molecular structure and functional analysis of pyocin S8 from Pseudomonas aeruginosa reveals the essential requirement of a glutamate residue in the HNH motif for DNase activity, by Helena Turano, Fernando Gomes, Renato M. Domingos, Maximilia FS Degenhardt, Cristiano LP Oliveira, Richard C. Garratt, Nilton Lincopan and Luis ES Netto, can be accessed here.