Phenotypes based on subclade classification.

Leptospirosis is a global zoonotic disease caused by pathogenic species of the genus Leptospira. Leptospira species are classified into two major clades (pathogenic, P, and saprophytic, S), and four subclades (P1, P2, S1, and S2), with the P1 subclade further divided into high virulence (P1+) and low virulence (P1-) groups. While previous studies have associated P1 + species to greater virulence in the host, phenotypic characterization across clades, particularly regarding dissemination and cell barrier disruption, remains limited. In this study, sixteen strains of pathogenic and saprophytic Leptospira representing subclades P1 + , P1-, P2, and S1 were evaluated in vitro to assess association with human endothelial cells, disruption of host VE-cadherin localization in adherens junctions, and immune response as measured by cytokine and chemokine release. Our findings indicate that VE-cadherin disruption correlates with P1 + species and the presence of virulence-associated genes. Additionally, bacterial association with host cells correlates with the loss of VE-cadherin localization in adherens junctions. In vitro Leptospira interaction with endothelial cells induced production of chemokine and cytokines, most prominent in the P1 + clade and correlating with the presence of virulence-associated genes. Using an in vivo murine model of hematogenous dissemination to assess tissue tropism, live Leptospira were cultured from relevant tissues of animals inoculated with most of the strains tested and bacterial burdens were quantified to measure adhesion to tissues. Four of the six P1 + strains exhibited significantly higher tissue burdens in kidney, liver, and bladder at one hour post-inoculation compared to other Leptospira species. Together, these results suggest that endothelial cell interactions may be a key phenotypic marker for virulence classification in Leptospira. Further defining these interactions may therefore provide insights into interventions to combat this potentially fatal disease.钩端螺旋体病（Leptospirosis）是一种由钩端螺旋体属（Leptospira）致病菌种引发的全球性人畜共患病。钩端螺旋体可分为两大进化枝（clade）：致病型（P）与腐生型（S），并进一步划分为四个亚进化枝：P1、P2、S1与S2；其中P1亚进化枝还可细分为高毒力（P1+）与低毒力（P1-）组别。既往研究已将P1+菌种与宿主更高毒力表现相关联，但跨进化枝的表型特征分析——尤其是在播散能力与细胞屏障破坏方面——仍较为有限。本研究选取覆盖P1+、P1-、P2与S1亚进化枝的16株致病型与腐生型钩端螺旋体菌株，开展体外实验以评估其与人血管内皮细胞的结合情况、宿主黏着连接中血管内皮钙粘蛋白（VE-cadherin）定位的破坏情况，以及通过细胞因子与趋化因子释放量衡量的免疫应答水平。研究结果显示，血管内皮钙粘蛋白的破坏程度与P1+菌种及毒力相关基因的存在呈正相关。此外，病原菌与宿主细胞的结合能力与黏着连接中血管内皮钙粘蛋白定位的丢失存在关联。体外实验中，钩端螺旋体与内皮细胞的相互作用可诱导趋化因子与细胞因子的产生，该效应在P1+进化枝中最为显著，且与毒力相关基因的存在密切相关。本研究采用血源性播散小鼠模型（murine model）评估组织嗜性，对多数受试菌株接种后的动物相关组织进行活钩端螺旋体培养，并通过定量细菌载量以衡量其对组织的黏附能力。结果显示，6株P1+菌株中有4株在接种1小时后的肾脏、肝脏与膀胱组织中，细菌载量显著高于其他钩端螺旋体菌种。综合以上结果，本研究提示内皮细胞相互作用或可作为钩端螺旋体毒力分类的关键表型标记物。进一步明确此类相互作用机制，或可为对抗这一潜在致死性疾病的干预手段提供新思路。