Pathogenic microorganisms such as bacteria, fungi, and protozoa rely on an arsenal of antioxidant enzymes to combat oxidative stress. That’s because animals and plants defend themselves from infections caused by these pathogens by generating oxidants derived from oxygen and nitrogen, including hydroperoxides, such as hydrogen peroxide, peroxynitrite, and organic hydroperoxides. Peroxiredoxins (Prxs), enzymes present in all living organisms, efficiently decompose these hydroperoxides, protecting cells against oxidative damage. In the case of infections, protecting invading organisms. Specific inhibitors for these microorganisms peroxiredoxins could, therefore, represent a new therapeutic approach to overcome the growing resistance of pathogens to antibiotics and antifungals.

In order to systematize knowledge about peroxiredoxins in pathogens, Professor Marcos Antonio de Oliveira, from the Universidade Estadual Paulista Júlio de Mesquita Filho (UNESP) and a member of the RIDC Redoxoma, and his group described, in a review article, several aspects of these enzymes, such as abundance, substrate diversity, and structural and functional peculiarities, showing that some classes are present only in pathogenic microorganisms, while others present structural peculiarities that differentiate them from the host’s isoforms. According to the authors, the intrinsic characteristics of these proteins can help in the development of new antimicrobial drugs. The article, written in collaboration with the group of Professor Luis Eduardo Soares Netto, from the Instituto de Biociências da USP and a member of the RIDC Redoxoma, was published in the journal Applied Microbiology and Biotechnology.

“What I find very interesting is that there was a gap – you have six classes of these enzymes and you didn’t have an approach to the role of each of them in the response to host oxidative/nitrosative defenses, cellular localization, reducing systems, in the virulence of microorganisms, antibiotics response, or even what is the natural substrate for each class. Our objective is to bring this information in a structured and organized way, to stimulate research in this area”, said the researcher.

A specialist in determining the crystallographic structure of antioxidant proteins, Oliveira has been investigating molecules with the potential to inhibit peroxiredoxins. Together with another research group, Oliveira and Netto have filed a patent application for the use of Adenantine, obtained from a natural compound of Chinese origin, as an inhibitor of bacterial peroxiredoxins. Adenanthin is three to thirty times more effective in bacterial Prxs than in human proteins and also enhances the effectiveness of existing antibiotics. Furthermore, “based on these results we established a research collaboration with the professors João Henrique Ghilardi Lago and Rodrigo Luiz Oliveira Rodrigues Cunha (CCNH – UFABC) and Marcos H. Toyama, UNESP (CLP), specialists in the purification of natural compounds and interaction between enzymes and inhibitors, and we have already identified two new compounds from the Brazilian biodiversity. We tested one of them and the inhibition rates are as high as those of the Chinese compound. It is able of inhibiting the enzyme and killing the bacteria.”

To assess the toxicity of the compounds, the researchers now intend to proceed with tests on human defense cells in a collaborative initiative with the groups of Flávia Meotti, from the IQ-USP and also a member of the RIDC Redoxoma, and Marcelo Brocchi from UNICAMP, which were delayed because of the pandemic. This research has also been supported by FAPESP.

Peroxiredoxins

Peroxiredoxins are versatile proteins, essential for cell homeostasis and redox signaling, with functions as antioxidants, peroxide sensors, and molecular chaperones. They are thiol-proteins classified into six different classes (Prx1-AhpC, BCP-PrxQ, Tpx, Prx5, Prx6, and AhpE), based on structural and biochemical characteristics. They efficiently catalyze the reduction of hydrogen peroxide, organic hydroperoxides, and peroxynitrite. The amino acid sequence of its catalytic triad containing a peroxidase cysteine (C_P), an arginine, and a threonine (or serine in some species) is highly conserved in all living things, from bacteria to humans.

They are widely distributed in several cell compartments, such as cytosol, nucleus, mitochondria, and membranes. According to Oliveira, in some microorganisms, Prxs are very abundant. “In Saccharomyces cerevisiae, a well-studied yeast, the amount of the best-known antioxidant enzymes (glutathione peroxidases and catalases) is roughly one-thirtieth the amount of the Prxs.” They can also be exported to the extracellular environment, and have already been detected in biofilms, which confer resistance to bacteria and fungi against host immune defenses and antimicrobial drugs. In addition to biofilms, Prx isoforms are also secreted into the extracellular environment and some are found on the cell surface of bacteria, fungi, and protozoa. This is an important aspect for the development of diagnostic tests and vaccines.

In bacteria, three to ten enzyme isoforms are expressed, representing some of the most abundant proteins in these organisms. The number of isoforms is also elevated in eukaryotic microorganisms, reaching up to six isoforms in fungi and three to five in protozoan.

Despite the abundance, Prx expression can be further increased in response to oxidative stress promoted by the host defense systems, by treatment with hydroperoxides, or by antibiotics. Consequently, some isoforms have been described as virulence factors. Oliveira explains that in the three classes of microorganisms studied – bacteria, fungi, and protozoa –, the deletion of genes encoding Prxs makes the pathogens more susceptible to death, either by the action of the host’s immune system or by microbicides, indicating the role of this proteins in virulence.

For all their diversity and the structural differences between isoforms found in pathogenic microorganisms and hosts (mammals and plants), Prxs are important targets for drugs. According to the researchers, the identity between the amino acid sequences of the microbial and host Prxs isoforms ranges from 35 to 60%.

“We do basic science to understand the mechanisms by which the enzyme works. It is essential to know the crystallographic structure, to study the interactions of ligands in the active site. If I have the structure, I can do some computer simulations to understand which would be the best ligand and then test in the laboratory”, explains Oliveira. However, the researcher concludes that many studies are still needed, with larger groups of microorganisms. “Comparative studies of inhibitors are lacking, mainly to discover what are the proteins natural substrates inside the cells, because maybe by mimicking these compounds, we can have more success with inhibitors.”

The article Relevance of peroxiredoxins in pathogenic microorganisms, by Marcos Antonio de Oliveira, Carlos A. Tairum, Luis Eduardo Soares Netto, Ana Laura Pires de Oliveira, Rogerio Luis Aleixo-Silva, Vitoria Isabela Montanhero Cabrera, Carlos A. Breyer, and Melina Cardoso dos Santos, can be accessed here