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.