# Distinct strategies of epithelial cell barrier disruption by Leptospira interrogans isolated from human patients in Okinawa, Japan

## TL;DR

This study shows how two Leptospira strains from Okinawa disrupt kidney cell barriers using different strategies, which could help in understanding and treating leptospirosis.

## Contribution

The study reveals distinct infection mechanisms of two Leptospira strains in disrupting epithelial barriers through different effects on junction proteins and cytoskeleton.

## Key findings

- Oki53 and Oki65 strains both reduce TEER in infected cells, indicating epithelial barrier disruption.
- Oki53-induced effects are fully prevented by proteasomal and caspase inhibitors, unlike Oki65.
- Epithelial barrier integrity depends on both afadin-F-actin and α-catenin-F-actin links.

## Abstract

Leptospirosis is a bacterial infection common in tropical and subtropical regions, which causes feverish conditions. Although approximately half of human leptospirosis cases in Japan are reported in Okinawa, a subtropical area, the pathogenic mechanisms of clinical isolates from this region remain unknown. This study aimed to identify the infection mechanisms of L. interrogans isolates from Okinawa (Oki-strains) in human renal proximal tubule epithelial cells (RPTECs).

The transepithelial electrical resistance (TEER) measurements of 11 Oki-strains in infected renal proximal tubule epithelial cells (RPTECs) showed that all strains caused a decrease in TEER by 48 hours post-infection. Imaging analysis of RPTECs infected with two selected strains (Oki53 and Oki65) revealed that both strains induced the displacement of adherens junction (AJ) proteins E-cadherin, α-catenin, afadin, and nectin-2 and cytoskeletal F-actin disorganization. However, western blotting analysis revealed that AJ protein levels were not reduced, except for afadin—an important protein for linking F-actin to AJs. Chemical inhibition revealed that the proteosome inhibitors MG132 and bortezomib and pan-caspase inhibitor Z-VAD-FMK prevented the Oki53-induced TEER decrease, AJ protein mislocalization, afadin degradation, and F-actin disorganization. However, in Oki65-infected RPTECs, the inhibitors partially prevented these effects. Thus, the AJ-F-actin link and epithelial barrier were fully preserved in Oki53-infected RPTECs pretreated with these inhibitors at 24 h post-infection (~TEER 130% of initial TEER), whereas in Oki65-infected cells, the AJ-F-actin link was maintained only partially (~TEER 70%).

Our findings suggest that the maintenance of epithelial barrier integrity requires both the afadin-F-actin and α-catenin-F-actin links, and that Oki53 and Oki65 employ distinct strategies to disrupt the AJ-F-actin link and, thus, the epithelial barrier.

Leptospirosis is a bacterial infection common in tropical and subtropical areas, including Okinawa, Japan. Pathogenic Leptospira colonize animal kidneys and are released in urine. In humans, colonization sometimes causes kidney failure. This study aimed to identify the infection mechanisms in L. interrogans isolates from Okinawa on human renal proximal tubule epithelial cells (RPTECs). All 11 clinical isolates induced a reduction in transepithelial electrical resistance (TEER), an indicator of cell-cell adhesion strength, in infected RPTECs by 48 h post-infection. Moreover, two selected strains, Oki53 and Oki65, induced adherens-junction protein displacement (afadin, α-catenin, E-cadherin, and nectin-2), afadin degradation, and cytoskeleton F-actin disorganization. In addition, proteasomal and pan-caspase inhibitors completely prevented these effects in Oki53-infected RPTECs, but only partially prevented the TEER decrease and afadin degradation in Oki65-infected cells. Consequently, the connection between junction proteins and the cytoskeleton (the AJ–F-actin link) and the overall epithelial barrier were fully preserved in Oki53-infected cells when pretreated with these inhibitors, but only partially preserved in Oki65-infected cells. These findings suggest that maintaining the epithelial barrier integrity depends on both the afadin–F-actin and α-catenin–F-actin links, and that different strains of Leptospira disrupt this barrier via different strategies.