Data Sheet 1_Involvement of HemI, an ECF sigma factor, in hemin acquisition and antibiotic susceptibility in Stenotrophomonas maltophilia.pdf

BackgroundHemin is a major source of iron for pathogens in infectious niches. The FecIRA-like surface signaling cascade is a common regulatory system for iron acquisition by pathogens. This system consists of a FecA-like TonB-dependent transporter (TBDT), a FecR-like inner membrane anti-sigma factor, and a FecI-like extracytoplasmic function (ECF) sigma factor. Beyond iron acquisition, FecIRA-like systems have been reported to regulate additional physiological processes. The known hemin acquisition system in Stenotrophomonas maltophilia includes HemA, a TBDT; HemU, an inner membrane transporter; and the TonB1–ExbB1–ExbD1a–ExbD1b complex, a multi-subunit motor that energizes HemA. Fur and HemP are the primary regulators involved in hemin utilization. In this study, we identified a novel FecIRA-like regulatory system, i.e., HemI–HemR–HemAD.
MethodsThe regulatory role of HemI was examined using promoter–xylE transcriptional fusion constructs and real-time PCR. Mutants associated with the hemI–hemR–hemAD operon were generated and evaluated for iron utilization, swimming motility, oxidative stress tolerance, and antibiotic susceptibility.
ResultsThe hemI–hemR–hemAD operon was repressed by Fur–Fe2+ under iron-replete conditions. Its expression was partially derepressed under iron depletion and further derepressed in the presence of hemin; however, the operon showed no autoregulation. HemI was essential for hemin acquisition. The overexpression of hemI in the S. maltophilia KJ strain increased the susceptibility to levofloxacin (LVX) and trimethoprim–sulfamethoxazole (SXT). All S. maltophilia isolates examined displayed increased minimum inhibitory concentrations (MICs) for ceftazidime (CAZ) and minocycline (MIN) under the iron-depleted and hemin-available conditions; notably, the changes in the MICs of LVX and SXT were strain-dependent.
ConclusionHemI, a novel ECF sigma factor, not only regulates hemin acquisition but also contributes to antibiotic susceptibility under iron-limited and hemin-available conditions.

【背景】血红素（hemin）是病原体在感染微生境中的主要铁源。类FecIRA的表面信号级联是病原体获取铁的常见调控系统，该系统由类FecA的TonB依赖性转运蛋白（TonB-dependent transporter, TBDT）、类FecR的内膜抗σ因子，以及类FecI的胞质外功能σ因子（extracytoplasmic function, ECF）组成。除铁摄取外，已有研究报道类FecIRA系统可调控额外的生理过程。嗜麦芽窄食单胞菌（Stenotrophomonas maltophilia）中已知的血红素摄取系统包括：HemA（一种TBDT）、HemU（一种内膜转运蛋白），以及为HemA供能的多亚基动力复合物TonB1–ExbB1–ExbD1a–ExbD1b。铁摄取调节因子Fur（Ferric uptake regulator）与HemP是参与血红素利用的主要调控因子。本研究中，我们鉴定出一种新型类FecIRA调控系统，即HemI–HemR–HemAD。
【方法】本研究采用启动子-xylE转录融合构建体与实时荧光定量PCR（real-time PCR），解析了HemI的调控作用。我们构建了hemI–hemR–hemAD操纵子相关突变株，并对其铁利用能力、泳动能力、氧化应激耐受性与抗生素敏感性进行了评价。
【结果】在铁充足条件下，hemI–hemR–hemAD操纵子受Fur–Fe²⁺的抑制。铁匮乏条件下该操纵子的表达被部分去抑制，而在血红素存在时进一步去抑制；但该操纵子未表现出自我调控现象。HemI是血红素摄取所必需的。在嗜麦芽窄食单胞菌KJ菌株中过表达hemI，可提升其对左氧氟沙星（levofloxacin, LVX）与甲氧苄啶-磺胺甲恶唑（trimethoprim–sulfamethoxazole, SXT）的敏感性。所有受试嗜麦芽窄食单胞菌分离株在铁匮乏且血红素存在的条件下，对头孢他啶（ceftazidime, CAZ）与米诺环素（minocycline, MIN）的最低抑菌浓度（minimum inhibitory concentrations, MICs）均升高；值得注意的是，左氧氟沙星与甲氧苄啶-磺胺甲恶唑的MIC变化呈现菌株依赖性。
【结论】HemI作为一种新型ECF σ因子，不仅可调控血红素摄取，还可在铁受限且血红素存在的条件下影响抗生素敏感性。