Peroxiredoxin AhpC1 protects Pseudomonas aeruginosa against the inflammatory oxidative burst and confers virulence

Abstract

Pseudomonas aeruginosa is an opportunistic bacterium in patients with cystic fibrosis and hospital acquired infections. It presents a plethora of virulence factors and antioxidant enzymes that help to subvert the immune system. In this study, we identified the 2-Cys peroxiredoxin, alkyl-hydroperoxide reductase C1 (AhpC1), as a relevant scavenger of oxidants generated during inflammatory oxidative burst and a mechanism of P. aeruginosa (PA14) escaping from killing. Deletion of AhpC1 led to a higher sensitivity to hypochlorous acid (HOCl, IC 50 3.2 ± 0.3 versus 19.1 ± 0.2 μM), hydrogen peroxide (IC 50 91.2 ± 0.3 versus 496.5 ± 6.4 μM) and the organic peroxide urate hydroperoxide. ΔahpC1 strain was more sensitive to the killing by isolated neutrophils and less virulent in a mice model of infection. All mice intranasally instilled with ΔahpC1 survived as long as they were monitored (15 days), whereas 100% wild-type and ΔahpC1 complemented with ahpC1 gene (Δ ahpC 1 attB : ahpC1 ) died within 3 days. A significantly lower number of colonies was detected in the lung and spleen of ΔahpC1 -infected mice. Total leucocytes, neutrophils, myeloperoxidase activity, pro-inflammatory cytokines, nitrite production and lipid peroxidation were much lower in lungs or bronchoalveolar liquid of mice infected with ΔahpC1 . Purified AhpC neutralized the inflammatory organic peroxide, urate hydroperoxide, at a rate constant of 2.3 ± 0.1 × 10 6 M −1 s −1 , and only the ΔahpC1 strain was sensitive to this oxidant. Incubation of neutrophils with uric acid, the urate hydroperoxide precursor, impaired neutrophil killing of wild-type but improved the killing of ΔahpC1 . Hyperuricemic mice presented higher levels of serum cytokines and succumbed much faster to PA14 infection when compared to normouricemic mice. In summary, ΔahpC1 PA14 presented a lower virulence, which was attributed to a poorer ability to neutralize the oxidants generated by inflammatory oxidative burst, leading to a more efficient killing by the host. The enzyme is particularly relevant in detoxifying the newly reported inflammatory organic peroxide, urate hydroperoxide.

Adenanthin Is an Efficient Inhibitor of Peroxiredoxins from Pathogens, Inhibits Bacterial Growth, and Potentiates Antibiotic Activities

“…Only two polar interactions were detected in the simulations (with Gln13* and His75* residues), suggesting that the Adn hydrophobic characteristic is more important for its binding in the active site. This is relevant since several alkyl hydroperoxides that are reduced by AhpC are hydrophobic and/or bulky, − ,, and these residues may also be involved in the stabilization of biological oxidizing substrates. Additionally, we found variations between bacterial and human Prx2 in the residues involved in the Adn binding (SI Figure S3), which may explain the differences of the IC 50 values (Figure ).…”

“…While AhpC-Prx1 containing Thr (Thr-AhpC) may dissociate in dimers in a redox-dependent way, the enzymes containing Ser (Ser-AhpC) are decamers independently of the redox state. ,, Additionally, Ser-AhpC possess higher thermostability and chaperone activity when compared to Thr-AhpC . It is worth mentioning that Prx containing Ser are considerably more frequent in bacteria when compared to multicellular organisms, and some species possess both Thr-AhpC and Ser-AhpC isoforms, suggesting some functional diversification. − …”

“…Several works indicated the importance of AhpC to pathogenic bacteria and its involvement in virulence. ,,− In this context, the AhpC may represent a promising target to microbicidal drugs, but no study has systematically explored inhibitors for these bacterial enzymes. Conversely, some studies involving mammalian AhpC-Prx1 have identified inhibitor molecules, including natural compounds. − Indeed, an inhibitor named Adenanthin (Adn), a natural diterpenoid from Isodon adenantha , was described as an effective inhibitor of two cytosolic human isoforms of AhpC-Prx1 (Prx1 and Prx2).…”

Exploring functional and structural features of chemically related natural prenylated hydroquinone and benzoic acid from Piper crassinervium (Piperaceae) on bacterial peroxiredoxin inhibition

“…Bacteria resistance to antibacterial drugs may be directly related to oxidative stress since antibiotics from different classes have the common ability to generate reactive oxygen species (ROS), damaging macromolecules, especially DNA [1,2]. ROS are also produced by host immune cells to fight pathogen infections, and studies suggest that inhibition of antioxidant enzymes is deleterious to pathogens infection and establishment [3]. Bacteria cells possess several antioxidant enzymes able to decompose ROS, including the typical 2-Cys peroxiredoxins, so called AhpCs [4].…”

“…Bacteria cells possess several antioxidant enzymes able to decompose ROS, including the typical 2-Cys peroxiredoxins, so called AhpCs [4]. These enzymes are very abundant in bacteria and are considered virulence factors to some species, indicating they play an important role in pathogenesis [3,[5][6][7][8][9][10]. AhpC is the abbreviation used for alkyl hydroperoxide reductase subunit C, referring to its discovery, since the bacterial strains carrying deletions of ahpC gene were very sensitive to organic hydroperoxides [11].…”