Fuentes Ignacio, Parra Francisco, Rojas Diego, Silva Andrés, Nevermann Jan, Otero María Carolina, Gil Fernando, Calderón Iván L, Fuentes Juan A
Laboratorio de Genética y Patogénesis Bacteriana, Centro de Investigación de Resiliencia a Pandemias, Facultad de Ciencias de la Vida, Universidad Andres Bello, Santiago 8370186, Chile.
Doctorado en Biotecnología, Facultad de Ciencias de la Vida, Universidad Andres Bello, Santiago 8370186, Chile.
Int J Mol Sci. 2025 Apr 29;26(9):4223. doi: 10.3390/ijms26094223.
serovar Typhi ( Typhi) produces outer membrane vesicles (OMVs) that remain comparatively underexplored as potential biotechnological tools. Here, we investigated how hypervesiculating Typhi mutants (Δ and Δ) can be engineered to load and deliver the fluorescent reporter protein mCherry, targeting human epithelial cells and the murine immune system. Deletions in and led to distinct OMV phenotypes characterized by higher vesicle production and altered cargo composition, underscoring the impact of disrupted membrane integrity and envelope stress on OMV biogenesis. By fusing mCherry with the Typhi OmpA signal peptide (SP), we achieved robust and functionally intact intravesicular packaging in all strains. Flow cytometry and confocal microscopy revealed that the Δ mutant exhibited particularly high cargo loading in the OMV fraction and pronounced mCherry delivery to epithelial cells, highlighting the potential of hypervesiculation to enhance OMV-based protein transport. However, immunization studies in mice showed that wild-type OMVs, despite carrying less mCherry than their hypervesiculating counterparts, induced the strongest anti-mCherry IgG responses. These findings indicate that, at least under these conditions, antigen loading alone is not sufficient to fully determine immunogenicity. Instead, the intrinsic composition or adjuvant-like properties of OMVs play a pivotal role in driving robust immune activation. Our results establish Typhi OMVs, especially when genetically modified with a Sec-dependent targeting signal (SP), as versatile platforms for heterologous protein delivery. Although hypervesiculation facilitates increased protein encapsulation and delivery to epithelial cells, native OMVs appear to better preserve and/or present antigens for effective immunogenic responses in vivo. These insights set the stage for further optimization of Typhi OMVs in vaccine development and protein therapeutics, where balancing cargo loading with immunostimulatory features may be key to achieving maximal efficacy.
伤寒杆菌（Typhi）产生的外膜囊泡（OMVs）作为潜在的生物技术工具，其研究相对较少。在此，我们研究了如何构建高囊泡化的伤寒杆菌突变体（Δ和Δ），以装载并递送荧光报告蛋白mCherry，靶向人类上皮细胞和小鼠免疫系统。和基因的缺失导致了不同的OMV表型，其特征是囊泡产量更高且货物组成改变，这突出了膜完整性破坏和包膜应激对OMV生物发生的影响。通过将mCherry与伤寒杆菌OmpA信号肽（SP）融合，我们在所有菌株中实现了强大且功能完整的囊泡内包装。流式细胞术和共聚焦显微镜显示，Δ突变体在OMV组分中表现出特别高的货物装载量，并将mCherry显著递送至上皮细胞，凸显了高囊泡化增强基于OMV的蛋白质运输的潜力。然而，对小鼠的免疫研究表明，野生型OMV尽管携带的mCherry比其高囊泡化对应物少，但诱导了最强的抗mCherry IgG反应。这些发现表明，至少在这些条件下，仅抗原装载不足以完全决定免疫原性。相反，OMV的内在组成或佐剂样特性在驱动强大的免疫激活中起关键作用。我们的结果确立了伤寒杆菌OMV，特别是当用依赖Sec的靶向信号（SP）进行基因改造时，作为异源蛋白质递送的通用平台。尽管高囊泡化有助于增加蛋白质包封并递送至上皮细胞，但天然OMV似乎能更好地保留和/或呈递抗原，以在体内引发有效的免疫反应。这些见解为伤寒杆菌OMV在疫苗开发和蛋白质治疗中的进一步优化奠定了基础，在这些领域中，平衡货物装载与免疫刺激特性可能是实现最大功效的关键。
