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Abstract：
Outer membrane vesicles (OMVs) are nano-sized proteoliposomes discharged from the cell envelope of Gram-negative bacteria. OMVs normally contain toxins, enzymes and other factors, and are used as vehicles in a process that has been considered a generalized, evolutionarily conserved delivery system among bacteria. Furthermore, OMVs can be used in biotechnological applications that require delivery of biomolecules, such as vaccines, remarking the importance of their study. Although it is known that Salmonella enterica serovar Typhi (S. Typhi), the etiological agent of typhoid fever in humans, delivers toxins (e.g., HlyE) via OMVs, there are no reports identifying genetic determinants of the OMV biogenesis in this serovar. In the present work, and with the aim to identify genes participating in OMV biogenesis in S. Typhi, we screened 15,000 random insertion mutants for increased HlyE secretion. We found 9 S. Typhi genes (generically called zzz genes) determining an increased HlyE secretion that were also involved in OMV biogenesis. The genes corresponded to ompA, nlpI, and tolR (envelope stability), rfaE and waaC (LPS synthesis), yipP (envC), mrcB (synthesis and remodeling of peptidoglycan), degS (stress sensor serine endopeptidase) and hns (global transcriptional regulator). We found that S. Typhi Δzzz mutants were prone to secrete periplasmic, functional proteins with a relatively good envelope integrity. In addition, we showed that zzz genes participate in OMV biogenesis, modulating different properties such as OMV size distribution, OMV yield and OMV protein cargo.
摘要：
外膜囊泡(OMV)是从革兰氏阴性细菌的细胞包膜排出的纳米大小的蛋白脂质体。OMV通常含有毒素，酶和其他因素，并在一个被认为是广义的过程中用作车辆，细菌之间进化保守的递送系统。此外，OMV可用于需要输送生物分子的生物技术应用，比如疫苗，强调他们学习的重要性。尽管众所周知，伤寒沙门氏菌（S。Typhi),人类伤寒的病原体，递送毒素(例如，HlyE)通过OMV，没有报道确定该血清型中OMV生物发生的遗传决定因素。在目前的工作中,为了鉴定参与伤寒沙门氏菌OMV生物发生的基因，我们筛选了15,000个随机插入突变体增加的HlyE分泌。我们发现了9个伤寒沙门氏菌基因（通常称为zzz基因），它们决定了HlyE分泌的增加，这些基因也参与了OMV的生物发生。这些基因对应于ompA，nlpI,和tolR(信封稳定性)，rfaE和waaC（LPS合成），yipP(envC)，mrcB（肽聚糖的合成和重塑），degS（应激传感器丝氨酸内肽酶）和hns（全局转录调节因子）。我们发现伤寒沙门氏菌Δzzz突变体容易分泌周质，具有相对良好的包膜完整性的功能蛋白。此外,我们发现ZZZ基因参与OMV的生物发生,调节不同的属性，如OMV大小分布，OMV产量和OMV蛋白货物。
