Mycobacterial Esx-3 requires multiple components for iron acquisition.
Mycobacterial Esx-3 requires multiple components for iron acquisition.
复制标题
分枝杆菌 Esx-3 需要多种成分来获取铁。
作者：
Siegrist,MSloan;Steigedal,Magnus;Ahmad,Rushdy;Mehra,Alka;Dragset,MarteS;Schuster,BrianM;Philips,JenniferA;Carr,StevenA;Rubin,EricJ
VII型分泌系统在分枝杆菌和许多革兰氏阳性细菌中都是保守的。虽然在模式生物污垢分枝杆菌中，ESX-1途径是致病分枝杆菌毒力和结合所必需的，但ESX-3有助于分枝杆菌蛋白介导的铁在这些细菌中的获取。在这里，我们证明了几个ESX-3组分是在低铁条件下发挥作用所必需的，但至少有一个是污垢分枝杆菌的膜结合蛋白酶MycP3，是部分可消耗的。所有被测试的esx-3突变体，包括ΔmycP3ms突变体，都未能将天然的esx-3底物EsxHms和EsxGms输出到靶向质谱学测定的可定量水平。虽然我们能够通过基因互补恢复esx-3突变体的低铁生长，但我们发现了蛋白质输出的广泛互补水平。事实上，在我们的实验条件下，细胞外微量的EsxHms和EsxGms足以获得铁。在ΔmycP3ms突变体和一些互补的esx-3突变体中，esx-3在铁获取方面的功能明显从强健的esxGms和EsxHms分泌中分离出来，这迫使人们重新审视VII型分泌系统的结构与功能关系。虽然ESX-1是致病分支杆菌在受感染宿主内生长所必需的，但ESX-3对于体外生长是必不可少的。我们和其他人已经证明，ESX-3是铁载体介导的铁获取所必需的。在这项工作中，我们确定了对此过程做出贡献的各个ESX-3组件。就像在ESX-1系统中一样，大多数取消ESX-3蛋白输出的突变也破坏了它的功能。然而，出乎意料的是，多反应监测质谱仪(MRM-MS)对ESX-3分泌的超灵敏定量表明，在类似条件下，非常低的出口水平足以获得铁。尽管蛋白质出口显然有助于VII型功能，但这种关系并不是绝对的。
The type VII secretion systems are conserved across mycobacterial species and in many Gram-positive bacteria. While the well-characterized Esx-1 pathway is required for the virulence of pathogenic mycobacteria and conjugation in the model organism Mycobacterium smegmatis, Esx-3 contributes to mycobactin-mediated iron acquisition in these bacteria. Here we show that several Esx-3 components are individually required for function under low-iron conditions but that at least one, the membrane-bound protease MycP3of M. smegmatis, is partially expendable. All of theesx-3mutants tested, including the ΔmycP3msmutant, failed to export the native Esx-3 substrates EsxHmsand EsxGmsto quantifiable levels, as determined by targeted mass spectrometry. Although we were able to restore low-iron growth to theesx-3mutants by genetic complementation, we found a wide range of complementation levels for protein export. Indeed, minute quantities of extracellular EsxHmsand EsxGmswere sufficient for iron acquisition under our experimental conditions. The apparent separation of Esx-3 function in iron acquisition from robust EsxGmsand EsxHmssecretion in theΔmycP3msmutant and in some of the complementedesx-3mutants compels reexamination of the structure-function relationships for type VII secretion systems.IMPORTANCEMycobacteria have several paralogous type VII secretion systems, Esx-1 through Esx-5. Whereas Esx-1 is required for pathogenic mycobacteria to grow within an infected host, Esx-3 is essential for growthin vitro. We and others have shown that Esx-3 is required for siderophore-mediated iron acquisition. In this work, we identify individual Esx-3 components that contribute to this process. As in the Esx-1 system, most mutations that abolish Esx-3 protein export also disrupt its function. Unexpectedly, however, ultrasensitive quantitation of Esx-3 secretion by multiple-reaction-monitoring mass spectrometry (MRM-MS) revealed that very low levels of export were sufficient for iron acquisition under similar conditions. Although protein export clearly contributes to type VII function, the relationship is not absolute.