Protection from hydrogen peroxide stress relies mainly on AhpCF and KatA2 in Stenotrophomonas maltophilia

Background: Aerobically-grown bacteria can be challenged by hydrogen peroxide stress from endogenous aerobic metabolism and exogenously generated reactive oxygen species. Catalase (Kat), alkyl hydroperoxidase (Ahp), and glutathione peroxidase (Gpx) systems are major adaptive responses to H₂O₂ stress in bacteria. Stenotrophomonas maltophilia is a ubiquitous Gram-negative bacterium equipped with four Kats (KatA1, KatA2, KatMn, and KatE), one Ahp (AhpCF), and three Gpxs (Gpx1, Gpx2, and Gpx3). Here, we systematically investigated how the eight H₂O₂ scavenging genes differentially contribute to the low-micromolar levels of H₂O₂ generated from aerobic metabolism and high-millimolar levels of H₂O₂ from exogenous sources. Methods: Gene expression was assessed and quantified by reverse transcription-PCR (RT-PCR) and real time quantitative PCR (qRT-PCR), respectively. The contribution of these enzymes to H₂O₂ stress was assessed using mutant construction and functional investigation. Results: Of the eight genes, katA2, ahpCF, and gpx3 were intrinsically expressed in response to low-micromolar levels of H₂O₂ from aerobic metabolism, and the expression of katA2 and ahpCF was regulated by OxyR. AhpCF and KatA2 were responsible for alleviating aerobic growth-mediated low concentration H₂O₂ stress and AhpCF played a critical role for stationary-phase cells. KatA2 was upregulated to compensate for AhpCF in the case of ahpCF inactivation. After exposure to millimolar levels of H₂O₂, katA2 and ahpCF were upregulated in an OxyR-dependent manner. KatA2 was the critical enzyme for dealing with high concentration H₂O₂. Loss-of-function of KatA2 increased bacterial susceptibility to high concentration H₂O₂. Conclusions: AhpCF and KatA2 are key enzymes protecting S. maltophilia from hydrogen peroxide stress.