Table 1.
Reagents for immunofluorescence microscopy.
Table 2.
Overall effect of Leptospira infection on host proteins.
Fig 1.
Effect of Leptospira infection on extracellular matrix proteins in endothelial cells detected by immunofluorescence microscopy.
(A) collagen type IV, (B) decorin, and (C) laminin in HMEC-1 and HDLEC are shown in green. The nuclei are stained in blue for all panels. Scale bars represent 50 μm. Quantified signal intensity of the host protein is indicated in the right-hand graphs (mean +/- SD, p-value is indicated below each graph, the independent p-values shown as an asterisk are compared to uninfected cells).
Fig 2.
Effect of Leptospira infection on ICAMs and cell surface receptors in endothelial cells.
(A) ICAM-1 (4-fold longer exposure time was used for uninfected HDLEC), (B) ICAM-2, (C) CD-36, and (D) vascular endothelial growth factor-receptor 2 (VEGF-R2) in HMEC-1 and HDLEC are shown in green. The nuclei are stained in blue for all panels. Scale bars represent 50 μm. Quantified signal intensity of the host protein is indicated in the right-hand graphs (mean +/- SD, p-value is indicated below each graph, the independent p-values shown as an asterisk or a double-dagger are compared to uninfected cells).
Fig 3.
Effect of Leptospira infection on intracellular proteins in endothelial cells detected by immunofluorescence microscopy.
(A) vascular endothelial growth factor (VEGF), (B) small GTPase RhoA, and (C) integrin-linked kinase (ILK) in HMEC-1 and HDLEC are shown in green. The nuclei are stained in blue for all panels. Scale bars represent 50 μm. Quantified signal intensity of the host protein is indicated in the right-hand graphs (mean +/- SD, p-value is indicated below each graph, the independent p-values shown as an asterisk, a dagger, or a double-dagger are compared to uninfected cells).
Fig 4.
Effect of Leptospira infection on adherens junction proteins in endothelial cells detected by immunofluorescence microscopy.
(A) VE-cadherin, (B) p120 catenin, (C) alpha-catenin, and (D) beta-catenin in HMEC-1 and HDLEC are shown in green. The nuclei are stained in blue for all panels. Scale bars represent 50 μm. Quantified signal intensity of the host protein is indicated in the right-hand graphs (mean +/- SD, p-value is indicated below or inside the graph, the independent p-values shown as an asterisk or a double-dagger for Copenhageni are compared to uninfected and Patoc-infected cells).
Fig 5.
Effect of Leptospira infection on tight junction and gap junction proteins in endothelial cells.
(A) tight junction protein, zonula occludens-1 (ZO-1) and (B) gap junction protein, connexin 43 (connexin) in HDLEC are shown in green. The nuclei are stained in blue for all panels. Scale bars represent 50 μm. Quantified signal intensity of the host protein is indicated in a right-hand graph (mean +/- SD, p-value is indicated below each graph, the independent p-value shown as an asterisk for Copenhageni is compared to uninfected and Patoc-infected cells).
Fig 6.
Effect of Leptospira infection on actin filaments in endothelial cells detected by immunofluorescence microscopy.
Actin filaments in HMEC-1 and HDLEC are shown in green. The nuclei are stained in blue for all panels. Scale bars represent 50 μm. Quantified signal intensity of the host protein is indicated in the right-hand graph (mean +/- SD, p-value is indicated below the graph, the independent p-value shown as an asterisk for Copenhageni is compared to uninfected and Patoc-infected cells).
Fig 7.
Features of intercellular junctions in endothelial cells.
The tight junction consists of claudin, occludin, and intracellular zonula occludens (ZO) proteins. ZO-1, ZO-2, and ZO-3 directly associate with actin filaments. Pathogenic Leptospira infection disrupted only the ZO-1 structure in tight junctions. The adherens junction is comprised of VE-cadherin, p120 catenin (p120), alpha-catenin (α-cat), beta-catenin (β-cat), and nectin. Alpha-catenin is a molecular switch that interacts with either the cadherin/beta-catenin complex or actin filaments. Our data indicate that the primary target of pathogenic Leptospira is the VE-cadherin/catenins complex in adherens junctions.