Mesh : Yersinia pseudotuberculosis / metabolism Iron / metabolism Type III Secretion Systems / metabolism Anaerobiosis

来  源:   DOI:10.3791/66642

Abstract:
A key virulence mechanism for many Gram-negative pathogens is the type III secretion system (T3SS), a needle-like appendage that translocates cytotoxic or immunomodulatory effector proteins into host cells. The T3SS is a target for antimicrobial discovery campaigns since it is accessible extracellularly and largely absent from non-pathogenic bacteria. Recent studies demonstrated that the T3SS of Yersinia and Salmonella are regulated by factors responsive to iron and oxygen, which are important niche-specific signals encountered during mammalian infection. Described here is a method for iron starvation of Yersinia pseudotuberculosis, with subsequent optional supplementation of inorganic iron. To assess the impact of oxygen availability, this iron starvation process is demonstrated under both aerobic and anaerobic conditions. Finally, incubating the cultures at the mammalian host temperature of 37 °C induces T3SS expression and allows quantification of Yersinia T3SS activity by visualizing effector proteins released into the supernatant. The steps detailed here offer an advantage over the use of iron chelators in the absence of iron starvation, which is insufficient for inducing robust iron starvation, presumably due to efficient Yersinia iron uptake and scavenging systems. Likewise, acid-washing laboratory glassware is detailed to ensure the removal of residual iron, which is essential for inducing robust iron starvation. Additionally, using a chelating agent is described to remove residual iron from media, and culturing the bacteria for several generations in the absence of iron to deplete bacterial iron stores. By incorporating standard protocols of trichloroacetic acid-induced protein precipitation, SDS-PAGE, and silver staining, this procedure demonstrates accessible ways to measure T3SS activity. While this procedure is optimized for Y. pseudotuberculosis, it offers a framework for studies in pathogens with similar robust iron uptake systems. In the age of antibiotic resistance, these methods can be expanded to assess the efficacy of antimicrobial compounds targeting the T3SS under host-relevant conditions.
摘要:
许多革兰氏阴性病原体的关键毒力机制是III型分泌系统(T3SS),将细胞毒性或免疫调节效应蛋白转移到宿主细胞中的针状附件。T3SS是抗微生物发现活动的靶标,因为它是细胞外可接近的,并且基本上不存在于非致病性细菌中。最近的研究表明,耶尔森氏菌和沙门氏菌的T3SS受铁和氧反应因子的调节,这是哺乳动物感染过程中遇到的重要生态位特异性信号。这里描述了一种用于假结核耶尔森氏菌的铁饥饿的方法,随后任选补充无机铁。为了评估氧气可用性的影响,这种铁饥饿过程在有氧和厌氧条件下都得到了证明。最后,在37°C的哺乳动物宿主温度下孵育培养物诱导T3SS表达并允许通过可视化释放到上清液中的效应蛋白来定量耶尔森氏菌T3SS活性。在没有铁饥饿的情况下,此处详述的步骤提供了优于使用铁螯合剂的优势,不足以诱发强烈的铁饥饿,推测是由于有效的耶尔森氏菌铁吸收和清除系统。同样,酸洗实验室玻璃器皿是详细的,以确保去除残留的铁,这对于诱导强烈的铁饥饿至关重要。此外,描述了使用螯合剂从介质中去除残余铁,并在没有铁的情况下培养细菌几代以耗尽细菌铁储备。通过纳入三氯乙酸诱导的蛋白沉淀的标准方案,SDS-PAGE,和银染色,此过程演示了测量T3SS活动的可访问方法。虽然此程序针对假结核进行了优化,它为具有类似稳健铁摄取系统的病原体研究提供了框架。在抗生素耐药的时代,这些方法可以扩展以评估在宿主相关条件下靶向T3SS的抗微生物化合物的功效。
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