Department of Biotechnology and Laboratory Science in Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan.
School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan.
Microbiol Spectr. 2022 Jun 29;10(3):e0244821. doi: 10.1128/spectrum.02448-21. Epub 2022 Jun 1.
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 -mediated pleiotropic phenotypes. RNA-seq transcriptome analysis and subsequent confirmation with qRT-PCR revealed that 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 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 -mediated pleiotropic phenotypes, except for antibiotic extrusion. 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 led to the upregulation of and . Unexpectedly, overexpression resulted in the reduction of intracellular iron levels, which led to pleiotropic defects in mutant. This study demonstrates a previously unidentified connection between efflux pumps, siderophore secretion, and intracellular iron levels in S. maltophilia.
嗜麦芽寡养单胞菌是一种不发酵革兰氏阴性杆菌，常从环境中分离出来，是一种具有多重耐药性的全球机会性病原体。嗜麦芽寡养单胞菌含有八种 RND 型外排泵，有助于产生多重耐药性和发挥生理功能。在这八种外排泵中，SmeYZ 泵组成型高度表达。在我们之前的研究中，我们证明了 SmeYZ 泵的功能丧失会导致多种表型，包括游泳运动能力丧失、分泌蛋白酶活性降低以及对氧化应激和抗生素的耐受性降低。在这项研究中，我们试图阐明导致介导的多种表型的潜在机制。RNA-seq 转录组分析和随后的 qRT-PCR 验证表明，突变体经历了铁饥饿反应，因为参与合成和摄取嗜麦芽寡养单胞菌唯一铁载体 Stenobactin 的基因显著上调。我们进一步通过电感耦合等离子体质谱法 (ICP-MS) 验证了突变体的细胞内铁含量较低。此外，与野生型相比，KJΔYZ 对 2,2'-二吡啶 (DIP)（一种亚铁螯合剂）更敏感。通过 qRT-PCR 并进一步通过 Chrome Azurol S (CAS) 活性、铁源利用和细胞活力测定验证了 SmeYZ、SmeDEF 和 SbiAB 泵对 Stenobactin 分泌的贡献。我们还证明了 SmeYZ 的功能丧失导致 SbiAB 和 SmeDEF 泵为 Stenobactin 分泌的代偿性上调。SbiAB 泵的过表达导致细胞内铁水平降低，这可能是介导的多种表型的关键因素，除了抗生素外排。外排泵对药物外排具有高效性，这是它们在多药耐药性中的作用基础。此外，外排泵具有生理功能，其表达受各种环境和生理信号的严格调节。外排泵的功能冗余是常见的，并且这些功能冗余泵在细菌中相互调节。嗜麦芽寡养单胞菌是一种机会性病原体，表现出固有多重耐药性。在这项研究中，我们证明了嗜麦芽寡养单胞菌的 SmeYZ、SbiAB 和 SmeDEF 外排泵在铁载体分泌中具有功能冗余。SmeYZ 的失活导致 和 的上调。出乎意料的是，SbiAB 的过表达导致细胞内铁水平降低，导致突变体出现多种缺陷。这项研究表明，在嗜麦芽寡养单胞菌中，外排泵、铁载体分泌和细胞内铁水平之间存在以前未识别的联系。
