Bacterial enzyme uses vitamin C to neutralize immune defenses, study finds

Throughout evolution, pathogenic microorganisms, such as bacteria, viruses and fungi, have evolved sophisticated defense strategies to survive and multiply in the hostile environment of their hosts. These mechanisms increase their virulence and make infections more difficult to fight. One of the most effective strategies is to neutralize oxidants released by defense cells to eliminate invaders.

A research group led by Luis Eduardo Soares Netto, from the Institute of Biosciences of the University of São Paulo (IB-USP) and the Center for Redox Processes in Biomedicine (Redoxoma) describes how the protein LsfA (a 1-Cys type peroxiredoxin) protects the bacterium Pseudomonas aeruginosa against hydrogen peroxide produced during the immune response. LsfA catalyzes the removal of hydroperoxides using ascorbate (vitamin C) as a reducing agent, thereby strengthening the antioxidant defense of bacteria.

Unveiling the structure of a key bacterial enzyme

“A major contribution of our work is the structural determination of a protein involved in the virulence of a medically important bacteria. We also demonstrated that ascorbate can act as a reducing agent in a cellular system, which is novel. In technical terms, we are the first to use the HyPer7 probe in Pseudomonas,” said Rogério Luis Aleixo Silva, researcher at the Chan Medical School of the University of Massachusetts. Silva participated in the research as a doctoral student at IB-USP.

The unprecedented structural data obtained in the study could open new opportunities to develop specific inhibitors of the bacterial enzyme and advance new therapeutic strategies.

“This is the first study to characterize the biochemistry and structure of a bacterial Prx6. In the literature, among the three major domains of life – Eubacteria, Archaea and Eukarya – there are already many resolved structures of Prx6, mainly in Archaea and mammals, including humans. But this is the first structure of a bacterial Prx6. E. coli, which is a classic model bacteria, has not Prx6”, comments Netto.

The research results were published in a journal article Redox biology.

How Pseudomonas aeruginosa defends itself

Pseudomonas aeruginosa is an opportunistic bacteria that primarily causes infections in people with weakened immune systems. It causes various types of nosocomial infections, including pneumonia in patients with cystic fibrosis, urinary tract infections, and infections of burns and surgical wounds. It can also cause endocarditis and septicemia. Due to its resistance to antibiotics, it is one of the priority pathogenic bacteria for the development of new treatments listed by the World Health Organization (WHO).

When the body is infected by a pathogen, it reacts by mobilizing immune defenses such as phagocytes. These cells fight microorganisms by releasing reactive oxygen, nitrogen and chlorine species. Faced with this oxidative stress, bacteria such as P. aeruginosa activate protective mechanisms involving various antioxidant proteins, including peroxiredoxins (Prxs).

LsfA, a peroxiredoxin of the Prx6 subfamily, is present in P. aeruginosa and is associated with bacterial virulence. In the new study, researchers deepened their understanding of the molecular mechanisms involved in its protective function. They showed that although P. aeruginosa has an arsenal of antioxidant enzymes, LsfA stands out for its high efficiency in breaking down hydrogen peroxide.

One of the main findings of the study is the interaction between LsfA and ascorbate. In 2007, Netto’s group showed that this vitamin could reduce sulfenic acid, formed during the oxidation of 1-Cys peroxiredoxins. This finding challenges the prevailing view that these enzymes rely exclusively on thiol recycling. A thiol is an organic compound containing a sulfur atom bonded to a hydrogen atom, analogous to the sulfur in alcohols. Thiols play an important role as antioxidants, helping to protect cells.

Although the role of vitamin C as a Prx reducer remains unclear in biological systems, the study’s structural analyzes suggest that ascorbate interacts directly with the active site of LsfA. This interaction allows the enzyme to regenerate after oxidation and restore its antioxidant function.

Implications for new antibacterial therapies

Since bacterial LsfA has a human homolog, any potential inhibitor should only target the bacterial form without affecting the human form. The researchers demonstrated that, despite its structural similarity to other Prx6 proteins, bacterial LsfA exhibits unique electrostatic properties, resulting in distinct charges at its active sites. These differences influence how an inhibitor interacts with each version of the enzyme.

“The advantage of our study is that in addition to resolving the structure, we used in silico docking to show some of the interactions between LsfA and ascorbate that could perhaps be mimicked by an inhibitor,” says Aleixo-Silva.

Next steps, according to Netto, include investigating ascorbate metabolism in P. aeruginosa in more detail, as well as conducting studies using macrophage models to assess the effects of LsfA exclusion on bacterial defense and host inflammatory responses.