Dataset Information

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 ?-lactamase-negative genetic background. Collectively, upon challenge with ?-lactam, the inducibly 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.

SUBMITTER: Huang YW

PROVIDER: S-EPMC6437473 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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

Huang Yi-Wei YW Huang Hsin-Hui HH Huang Kai-Hung KH Chen Wei-Chien WC Lin Yi-Tsung YT Hsu Cheng-Chih CC Yang Tsuey-Ching TC

Antimicrobial agents and chemotherapy 20190327 4

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 β-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. malt ...[more]

PMID: 30745379

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Project description:Stenotrophomonas maltophilia, a nonfermenting Gram-negative rod, is frequently isolated from the environment and is emerging as a multidrug-resistant global opportunistic pathogen. S. maltophilia harbors eight RND-type efflux pumps that contribute to multidrug resistance and physiological functions. Among the eight efflux pumps, SmeYZ pump is constitutively highly expressed. In our previous study, we demonstrated that loss-of-function of the SmeYZ pump results in pleiotropic phenotypes, including abolished swimming motility, decreased secreted protease activity, and compromised tolerance to oxidative stress and antibiotics. In this study, we attempted to elucidate the underlying mechanisms responsible for ΔsmeYZ-mediated pleiotropic phenotypes. RNA-seq transcriptome analysis and subsequent confirmation with qRT-PCR revealed that smeYZ mutant experienced an iron starvation response because the genes involved in the synthesis and uptake of stenobactin, the sole siderophore of S. maltophilia, were significantly upregulated. We further verified that smeYZ mutant had low intracellular iron levels via inductively coupled plasma mass spectrometry (ICP-MS). Also, KJΔYZ was more sensitive to 2,2'-dipyridyl (DIP), a ferrous iron chelator, in comparison with the wild type. The contribution of SmeYZ, SmeDEF, and SbiAB pumps to stenobactin secretion was suggested by qRT-PCR and further verified by Chrome Azurol S (CAS) activity, iron source utilization, and cell viability assays. We also demonstrated that loss-of-function of SmeYZ led to the compensatory upregulation of SbiAB and SmeDEF pumps for stenobactin secretion. The overexpression of the SbiAB pump resulted in a reduction in intracellular iron levels, which may be the key factor responsible for the ΔsmeYZ-mediated pleiotropic phenotypes, except for antibiotic extrusion. IMPORTANCE Efflux pumps display high efficiency of drug extrusion, which underlies their roles in multidrug resistance. In addition, efflux pumps have physiological functions, and their expression is tightly regulated by various environmental and physiological signals. Functional redundancy of efflux pumps is commonly observed, and mutual regulation occurs among these functionally redundant pumps in a bacterium. Stenotrophomonas maltophilia is an opportunistic pathogen that shows intrinsic multi-drug resistance. In this study, we demonstrated that SmeYZ, SbiAB, and SmeDEF efflux pumps of S. maltophilia display functional redundancy in siderophore secretion. Inactivation of smeYZ led to the upregulation of smeDEF and sbiAB. Unexpectedly, sbiAB overexpression resulted in the reduction of intracellular iron levels, which led to pleiotropic defects in smeYZ mutant. This study demonstrates a previously unidentified connection between efflux pumps, siderophore secretion, and intracellular iron levels in S. maltophilia.

Dataset Information

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

Publications

AmpI Functions as an Iron Exporter To Alleviate β-Lactam-Mediated Reactive Oxygen Species Stress in <i>Stenotrophomonas maltophilia</i>.

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