Background

P. aeruginosa is a ubiquitous bacterium that can be found in a free form in terrestrial and aquatics habitats or as an opportunistic human pathogen causing fatal infections in immunocompromised individuals, patients with skin damage or cystic fibrosis. To defend itself from ROS generated by its strong aerobic metabolism, host phagosomal vacuoles and environmental factors, this microorganism possesses several antioxidative strategies. Among them, two monofunctional catalases (KatA and KatB) are responsible for decomposing H₂O₂ to water and O₂. KatA is the main catalase and has unique characteristics: it is unusually stable and essential to H₂O₂ resistance, osmoprotection and virulence (Hassett et al., 2000; Lee et al., 2005). It has been suggested that the stability of KatA is one of the main factors for the high level activity under normal growth conditions, and for this reason, katA has been regarded as a constitutively expressed gene in P. aeruginosa (Heo et al., 2010). However, it has been reported that KatA activity is induced in the stationary growth phase (up to 10-fold) and by increased levels of H₂O₂ (Brown et al., 1995; Suh et al., 1999; Heo et al., 2010). Moreover, katA expression has been demonstrated to be modulated by the global regulator OxyR and Quorum Sensing, whose activation depends on increased levels of H₂O₂ and high cell density, respectively (Hassett et al., 1999; Heo et al., 2010). KatB is only detected in the presence of H₂O₂ or paraquat and is partially involved in resistance to oxidative stress (Brown et al., 1995; Lee et al., 2005).

Solar ultraviolet-A (UVA) radiation is one of the main environmental stress factors for P. aeruginosa. Given the oxidative nature of UVA damage, we studied the role of catalases in defense of this microorganism against radiation. We demonstrated that KatA is essential in the optimal response against lethal doses of UVA, both in planktonic cells and biofilms (Costa et al., 2010; Pezzoni et al., 2014). In addition, we reported that low doses of UVA increase KatA and KatB activity and that this regulation occurs at the transcriptional level (Pezzoni et al., 2016). This phenomenon is relevant since it constitutes an adaptive mechanism that prevents cell damage by subsequent exposure to lethal doses of UVA, H₂O₂, or sodium hypochlorite (Pezzoni et al., 2016).

In the course of our studies, it became necessary to do an in-depth analysis of catalase activity. The total catalase activity in cell extracts was quantified by following spectrophotometrically the decomposition of H₂O₂, according to Aebi (1984). However, this assay cannot distinguish between KatA and KatB activities. To analyze individual catalase activity, we implement the method proposed by Wayne and Díaz (1986). In brief, crude cell extracts are loaded onto non-denaturing polyacrylamide gels (PAGE), and both catalases are separated by their differential electrophoretic motility; colorless bands of catalase activity are revealed by incubation of the gel with H₂O₂ and subsequent addition of a ferric chloride-potassium ferricyanide solution. The principle of this method involves the reaction of H₂O₂ with potassium ferricyanide (III) by reducing it to ferrocyanide (II); the peroxide is oxidized to O₂. Ferric chloride reacts with ferrocyanide (II) to form an insoluble blue pigment. Because of the action of catalase on H₂O₂ decomposition, areas where this enzyme is active develop as clear bands in a blue gel (Patnaik et al., 2013). Additional papers were consulted to fine-tune this technique (Brown et al., 1995; Hassett et al., 1999; Elkins et al., 1999). The studies were performed with the prototypical P. aeruginosa strain PAO1 and isogenic derivatives PW8190 (katA::IslacZ/hah) and PW8769 (katB::IslacZ/hah) carrying mutations into katA and katB, respectively. Mutant strains devoid for KatA or KatB are useful to analyze the role of each enzyme in response to stress and as controls in PAGE catalase assays.

In this protocol, we describe how to detect individual catalase activity by PAGE using cell extracts from P. aeruginosa. Because of the particular characteristics of KatA (high abundance and stability), its detection does not present major difficulties. On the contrary, KatB detection is elusive, so that two changes were applied to the conventional technique: a protein extraction reagent was used instead of sonication to prepare the cell extracts, and the electrophoresis was performed at 4 °C. Based on these assays, it was concluded that KatB is an unstable enzyme, a fact that should be taken into account in quantitative or qualitative catalase assays under inducing (oxidative) conditions.