Proteomic characterization and label-free comparison of outer membrane vesicles from Francisella tularensis subsp. holarctica strain FSC200 isolated from different cultivation conditions

Francisella tularensis is a Gram-negative, facultative intracellular bacterium, causing a severe disease known as tularemia. It is known to secrete unusually shaped nanotubular outer membrane vesicles (OMV) loaded with number of virulence factors and immunoreactive proteins. In this study the nanotubes were purified from a clinical isolate of subsp. holarctica strain FSC200. We here provide a comprehensive proteomic characterization of OMV isolated from bacteria grown under different cultivation conditions simulating the diverse conditions of F. tularensis life cycle. These included conditions mimicking the environment inside the mammalian host during inflammation: oxidative stress, low pH and high temperature (42 °C); and by contrast, low temperature (25 °C) that mimicked the external milieu and agar plate cultivation. We observed several-fold increase in vesiculation rate at high temperature and low pH but the differences were not only in the amount of secreted OMV but particularly in their protein cargo. Amongst the proteins preferentially selectively packed into OMV under conditions mimicking mammalian host many were previously described as virulence factors connected to the unique intracellular trafficking of Francisella. Protein changes revealed also high probability of alterations of the bacterial surface on the level of lipopolysaccharide, polysaccharide capsule and phospholipids.

土拉弗朗西斯菌（Francisella tularensis）是一类革兰氏阴性（Gram-negative）兼性胞内细菌，可引发重症土拉菌病（tularemia）。研究表明，该菌能够分泌形态特殊的纳米管状外膜囊泡（outer membrane vesicles, OMV），囊泡内装载有多种毒力因子与免疫活性蛋白。本研究从土拉弗朗西斯菌holarctica亚种（subsp. holarctica）临床分离株FSC200中纯化得到此类纳米管状结构，并对在多种模拟土拉弗朗西斯菌生命周期环境的培养条件下生长的细菌所分泌的OMV开展了全面的蛋白质组学表征。所模拟的环境分为两类：一类为模拟哺乳动物宿主炎症期胞内环境的条件，包括氧化应激、低pH值与高温（42℃）；另一类为模拟自然外界环境的低温（25℃）与琼脂平板培养条件。研究结果显示，高温与低pH条件下的囊泡分泌速率提升数倍，且两类条件下的差异不仅体现在OMV的分泌总量上，更显著反映在其蛋白质载荷的组成差异中。在模拟哺乳动物宿主的培养条件下，优先选择性富集于OMV的蛋白中，有多种此前被报道为与土拉弗朗西斯菌独特胞内转运过程相关的毒力因子。蛋白质组学分析揭示的蛋白变化还提示，细菌表面的脂多糖、多糖荚膜与磷脂成分可能发生显著改变。