Involvement of a 1-Cys Peroxiredoxin in Bacterial Virulence

Abstract

The killing of bacterial pathogens by macrophages occurs via the oxidative burst and bacteria have evolved to overcome this challenge and survive, using several virulence and defense strategies, including antioxidant mechanisms. We show here that the 1-Cys peroxiredoxin LsfA from the opportunistic pathogen Pseudomonas aeruginosa is endowed with thiol-dependent peroxidase activity that protects the bacteria from H ₂O ₂ and that this protein is implicated in pathogenicity. LsfA belongs to the poorly studied Prx6 subfamily of peroxiredoxins. The function of these peroxiredoxins has not been characterized in bacteria, and their contribution to host-pathogen interactions remains unknown. Infection of macrophages with the lsfA mutant strains resulted in higher levels of the cytokine TNF-α production due to the activation of the NF-kB and MAPK pathways, that are partially inhibited by the wild-type P. aeruginosa strain. A redox fluorescent probe was more oxidized in the lsfA mutant-infected macrophages than it was in the macrophages infected with the wild-type strain, suggesting that the oxidative burst was overstimulated in the absence of LsfA. Although no differences in the phagocytosis rates were observed when macrophages were infected with wild-type and mutant bacteria in a gentamicin exclusion assay, a higher number of wild-type bacterial cells was found in the supernatant. This difference was not observed when macrophages were pre-treated with a NADPH oxidase inhibitor, confirming the role of LsfA in the bacterial resistance to ROS generated via NADPH oxidase. In an acute pneumonia model, mice infected with the mutant strains presented higher cytokine release in the lungs and increased activated neutrophil recruitment, with reduced bacterial burden and improved survival rates compared to mice infected with the wild-type bacteria. LsfA is the first bacterial 1-Cys Prx shown to modulate host immune responses and its characterization will allow a better understanding of the role of redox signaling in host-pathogen interactions.

Author Summary

Pseudomonas aeruginosa is an important human pathogen that employs a vast arsenal of virulence factors and infects immunocompromised hosts, such as patients in intensive care units, causing pneumonia and other illnesses. Macrophages are cells in the first line of defense against pathogens in the lungs. After pathogen recognition, macrophages release pro-inflammatory cytokines to recruit other immune cells and employ a process known as oxidative burst to kill invading microbes. P. aeruginosa can counteract oxidative stress using antioxidant proteins, such as peroxiredoxins. We show here that LsfA, which belongs to the poorly characterized Prx6 subfamily of peroxiredoxins, is indeed endowed with a thiol-dependent activity that is required for full virulence. In vitro and in vivo infection models confirmed that LsfA peroxidase activity is required for the immunomodulation caused by P. aeruginosa and that its absence allows the host to overcome the infection. This study demonstrates for the first time the involvement of a bacterial Prx6 in virulence.