Table 1_Unique Leishmania mexicana clones secrete populations of extracellular vesicles with unique protein profile and variable infectious capability.xlsx

The study of extracellular vesicles has become an incredibly important field of study, but the inherent heterogeneity of these vesicles continues to make their study challenging. The genetic variability and well-documented protocols for the growth and vesicle isolation from Leishmania parasites provide a unique opportunity to compare the heterogeneity of different populations secreted by Leishmania clones. Leishmania mexicana was cultured on solid SDM agar plates and 8 clonal colonies were selected. The EVs collected from the liquid cultures of these 8 clones were assessed by NTA, TEM, and proteomic analysis. We found that all 8 clonal L. mexicana cultures were visually indistinguishable from each other and had similar growth rate, and these physical similarities extended to their EVs. However, proteomic analysis reveals that the EVs collected have unique protein profiles compared to each other and EVs isolated from a heterogeneous liquid culture of L. mexicana. We selected 3 clonal EVs for further mouse infection experiments and found that EVs from CL7 L. mexicana consistently caused reduced footpad swelling in C57BL6 mice footpads compared to EVs from CL1, CL8, and heterogenous L. mexicana. This trend was not observed when infecting Balb/C mice and C57BL6 with the parasites alone, with only CL1 L. mexicana causing significantly increased infection in Balb/c mice. Our results together show that EVs isolated from different clonal colonies of L. mexicana have distinct differences in protein cargo which can lead to varying outcomes on Leishmania infection. Further evaluation will be needed to determine the underlying mechanisms behind this and verify that differences observed in infectivity are directly caused by variations between our L. mexicana clones, especially genetic sequencing and immunoblotting to validate our results.

细胞外囊泡（extracellular vesicles, EVs）的研究已成为极具重要性的学术领域，但这类囊泡固有的异质性仍使得相关研究颇具挑战。利什曼原虫（Leishmania）的遗传变异性，以及其培养与囊泡分离的经充分验证的标准化实验方案，为比对不同利什曼原虫克隆分泌的囊泡群体异质性提供了独特契机。本研究将墨西哥利什曼原虫（Leishmania mexicana）接种于固体SDM琼脂平板进行培养，随后筛选得到8个克隆菌落。从这8个克隆的液体培养物中收集的细胞外囊泡，通过纳米颗粒跟踪分析（nanoparticle tracking analysis, NTA）、透射电子显微镜（transmission electron microscopy, TEM）以及蛋白质组学分析进行了评估。研究发现，8株墨西哥利什曼原虫克隆的培养物在外观上无法区分，且生长速率相近；这种物理形态上的相似性同样延伸至其分泌的细胞外囊泡。但蛋白质组学分析结果显示，所收集的各克隆囊泡之间，以及与从异质性墨西哥利什曼原虫液体培养物中分离的囊泡相比，均具有独特的蛋白质组图谱。我们选取3株克隆的细胞外囊泡开展后续小鼠感染实验，结果发现：与CL1、CL8株以及异质性墨西哥利什曼原虫来源的囊泡相比，CL7株墨西哥利什曼原虫分泌的囊泡始终会导致C57BL/6小鼠的足垫肿胀程度减轻。当仅用利什曼原虫感染BALB/c小鼠与C57BL/6小鼠时，并未观察到上述趋势；仅CL1株墨西哥利什曼原虫可使BALB/c小鼠的感染程度显著升高。综合来看，本研究结果表明：从不同克隆菌落分离得到的墨西哥利什曼原虫细胞外囊泡，其携带的蛋白质组分存在显著差异，这种差异可对利什曼原虫感染结局产生不同影响。后续仍需开展进一步研究，以明确该现象背后的潜在机制，并验证所观察到的感染性差异确实由本研究中的墨西哥利什曼原虫克隆之间的差异所导致——尤其需通过基因测序与免疫印迹实验对本研究结果进行验证。