Collins Cathleen A, De Mazière Ann, van Dijk Suzanne, Carlsson Fredric, Klumperman Judith, Brown Eric J
Department of Microbial Pathogenesis, Genentech Inc, South San Francisco, CA, USA.
PLoS Pathog. 2009 May;5(5):e1000430. doi: 10.1371/journal.ppat.1000430. Epub 2009 May 15.
Like several other intracellular pathogens, Mycobacterium marinum (Mm) escapes from phagosomes into the host cytosol where it can polymerize actin, leading to motility that promotes spread to neighboring cells. However, only approximately 25% of internalized Mm form actin tails, and the fate of the remaining bacteria has been unknown. Here we show that cytosolic access results in a new and intricate host pathogen interaction: host macrophages ubiquitinate Mm, while Mm shed their ubiquitinated cell walls. Phagosomal escape and ubiquitination of Mm occurred rapidly, prior to 3.5 hours post infection; at the same time, ubiquitinated Mm cell wall material mixed with host-derived dense membrane networks appeared in close proximity to cytosolic bacteria, suggesting cell wall shedding and association with remnants of the lysed phagosome. At 24 hours post-infection, Mm that polymerized actin were not ubiquitinated, whereas ubiquitinated Mm were found within LAMP-1-positive vacuoles resembling lysosomes. Though double membranes were observed which sequestered Mm away from the cytosol, targeting of Mm to the LAMP-1-positive vacuoles was independent of classical autophagy, as demonstrated by absence of LC3 association and by Atg5-independence of their formation. Further, ubiquitination and LAMP-1 association did not occur with mutant avirulent Mm lacking ESX-1 (type VII) secretion, which fail to escape the primary phagosome; apart from its function in phagosome escape, ESX-1 was not directly required for Mm ubiquitination in macrophages or in vitro. These data suggest that virulent Mm follow two distinct paths in the cytosol of infected host cells: bacterial ubiquitination is followed by sequestration into lysosome-like organelles via an autophagy-independent pathway, while cell wall shedding may allow escape from this fate to permit continued residence in the cytosol and formation of actin tails.
与其他几种细胞内病原体一样，海分枝杆菌（Mm）从吞噬体逃逸到宿主细胞质中，在那里它可以聚合肌动蛋白，产生促进向邻近细胞扩散的运动性。然而，只有约25%内化的Mm形成肌动蛋白尾，其余细菌的命运一直未知。在此我们表明，细胞质进入导致了一种新的复杂的宿主-病原体相互作用：宿主巨噬细胞使Mm泛素化，而Mm则脱落其泛素化的细胞壁。吞噬体逃逸和Mm的泛素化在感染后3.5小时之前迅速发生；与此同时，泛素化的Mm细胞壁物质与宿主来源的致密膜网络混合，出现在细胞质细菌附近，表明细胞壁脱落并与裂解的吞噬体残余物相关。在感染后24小时，聚合肌动蛋白的Mm未被泛素化，而泛素化的Mm则存在于类似于溶酶体的LAMP-1阳性液泡中。尽管观察到双膜将Mm与细胞质隔离，但Mm靶向LAMP-1阳性液泡与经典自噬无关，这通过缺乏LC3关联以及其形成对Atg5的独立性得到证明。此外，缺乏ESX-1（VII型）分泌的无毒突变型Mm不会发生泛素化和与LAMP-1关联，这些突变型Mm无法从初级吞噬体逃逸；除了其在吞噬体逃逸中的功能外，ESX-1对于巨噬细胞或体外Mm的泛素化并非直接必需。这些数据表明，有毒力的Mm在感染宿主细胞的细胞质中遵循两条不同的路径：细菌泛素化后通过自噬非依赖途径被隔离到溶酶体样细胞器中，而细胞壁脱落可能允许逃脱这种命运，从而在细胞质中持续存在并形成肌动蛋白尾。
