AmpI Functions as an Iron Exporter To Alleviate -Lactam-Mediated Reactive Oxygen Species Stress in Stenotrophomonas maltophilia

Abstract

Stenotrophomonas maltophilia is an organism with a remarkable capacity for drug resistance with several antibiotic resistance determinants in its genome. S. maltophilia genome codes for L1 and L2, responsible for intrinsic -lactam resistance. The Smlt3721 gene (denoted ampI), located downstream of the L2 gene, encodes an inner membrane protein. The existence of an L2 gene-ampI operon was verified by reverse transcription-PCR (RT-PCR). For aerobically grown S. maltophilia KJ, inactivation of ampI downregulated siderophore synthesis and iron acquisition systems and upregulated the iron storage system, as demonstrated by a transcriptome assay, suggesting that AmpI is involved in iron homeostasis. Compared with the wild-type KJ, an ampI mutant had an elevated intracellular iron level, as revealed by inductively coupled plasma mass spectrometry (ICP-MS) analysis, and increased sensitivity to H2O2, verifying the role of AmpI as an iron exporter. The -lactam stress increased the intracellular reactive oxygen species (ROS) level and induced the expression of the L1 gene and L2 gene-ampI operon. Compared to its own parental strain, the ampI mutant had reduced growth in -lactam-containing medium, and the ampI mutant viability was improved after complementation with plasmid pAmpI in either a -lactamase-positive or -lacta-mase-negative genetic background. Collectively, upon challenge with -lactam, the in-ducibly expressed L1 and L2 -lactamases contribute to -lactam resistance by hydrolyzing -lactam. AmpI functions as an iron exporter participating in rapidly weakening -lactam-mediated ROS toxicity. The L1 gene and L2 gene-ampI operon enable S. maltophilia to effectively cope with -lactam-induced stress.