Leptospira interrogans causes quantitative and morphological disturbances in adherens junctions and other biological groups of proteins in human endothelial cells

Pathogenic Leptospira transmits from animals to humans, causing the zoonotic life-threatening infection called leptospirosis. This infection is reported worldwide with higher risk in tropical regions. Symptoms of leptospirosis range from mild illness to severe illness such as liver damage, kidney failure, respiratory distress, meningitis, and fatal hemorrhagic disease. Invasive species of Leptospira rapidly disseminate to multiple tissues where this bacterium damages host endothelial cells, increasing vascular permeability. Despite the burden in humans and animals, the pathogenic mechanisms of Leptospira infection remain to be elucidated. The pathogenic leptospires adhere to endothelial cells and permeabilize endothelial barriers in vivo and in vitro. In this study, human endothelial cells were infected with the pathogenic L. interrogans serovar Copenhageni or the saprophyte L. biflexa serovar Patoc to investigate morphological changes and other distinctive phenotypes of host cell proteins by fluorescence microscopy. Among those analyzed, 17 proteins from five biological classes demonstrated distinctive phenotypes in morphology and/or signal intensity upon infection with Leptospira. The affected biological groups include: 1) extracellular matrix, 2) intercellular adhesion molecules and cell surface receptors, 3) intracellular proteins, 4) cell-cell junction proteins, and 5) a cytoskeletal protein. Infection with the pathogenic strain most profoundly disturbed the biological structures of adherens junctions (VE-cadherin and catenins) and actin filaments. Our data illuminate morphological disruptions and reduced signals of cell-cell junction proteins and filamentous actin in L. interrogans-infected endothelial cells. In addition, Leptospira infection, regardless of pathogenic status, influenced other host proteins belonging to multiple biological classes. Our data suggest that this zoonotic agent may damage endothelial cells via multiple cascades or pathways including endothelial barrier damage and inflammation, potentially leading to vascular hyperpermeability and severe illness in vivo. This work provides new insights into the pathophysiological mechanisms of Leptospira infection.

Introduction infection at the cellular and molecular levels are poorly understood. It has been demonstrated that the pathogenic L. interrogans or its proteins adhere to endothelial cells and permeabilize endothelial cell monolayers in vitro [23,24].
At the molecular level, genetic tools for Leptospira work less efficiently than for many other bacteria [25], making studies of Leptospira biology challenging. Even when Leptospira mutants are constructed, there are few available efficient methods to elucidate the pathogenic mechanisms [26]. Most often, the mutants have been examined for attenuated phenotypes in the mortality of animals or altered histological perturbations of organs from infected animals, such as hamsters [26][27][28][29][30], guinea pigs [30,31], transgenic mice [32,33], and zebrafish [34,35]. Because wild-type mice and rats are carrier animals for leptospires, these animals are used as negative controls to study pathogenicity, e.g. to examine leptospire colonization without disease [26,36].
As in vitro assays to screen Leptospira strains, cell attachment and transmigration through polarized epithelial cells have been used [16,37]. The epithelial translocation of leptospires does not alter the transepithelial electrical resistance [37], so the resistance measurement itself is not informative. Human umbilical vein endothelial cells (HUVEC) are also used to test recombinant leptospiral proteins for changes in host protein expression or cell-junction permeability [24, 38,39]. To study Leptospira pathogenicity more intensely, other in vitro screening systems remain to be explored.
In this study, morphological changes and other distinctive phenotypes in Leptospirainfected human endothelial cells were investigated. Antibodies and reagents recognizing human proteins were screened by fluorescence microscopy. Most proteins analyzed demonstrated little change in Leptospira-infected endothelial cells. Yet, 17 host proteins from five biological classes demonstrated distinctive phenotypes in the morphology and/or signal intensity upon infection: 1) extracellular matrix, 2) intercellular adhesion molecules and cell surface receptors, 3) intracellular proteins, 4) cell-cell junction proteins, and 5) a cytoskeletal protein.
The most prominent phenotype of pathogenic L. interrogans sv. Copenhageni-infected cells was the loss of the adherens junction proteins VE-cadherin and p120-, alpha-, and beta-catenins from the original site at intercellular junctions. Copenhageni infection also influenced the actin cytoskeleton as well as a tight junction protein, ZO-1. Infection with both pathogenic and non-pathogenic Leptospira strains altered other host proteins belong to multiple biological classes, although the pathogenic strain caused more intense changes. This work provides the insights in biological and pathological effects of Leptospira infection.

Methods
Leptospira strains and growth conditions L. interrogans serovar Copenhageni strain Fiocruz L1-130 (pathogen), L. interrogans sv. Canicola strain Moulton (pathogen), and L. biflexa sv. Patoc strain Patoc 1 (non-pathogen) were purchased from ATCC. Bacteria were grown at 30˚C in Ellinghausen-McCullough-Johnson-Harris (EMJH) medium supplemented with 1% rabbit serum [40,41]. The viability, motility, and general morphology of strains were periodically checked using darkfield microscopy. When bacterial cultures reached 1 to 2 × 10 8 cells/ml, bacteria were used for infection or subcultured in fresh medium. The bacterial cell number was determined using a Petroff-Houser chamber under darkfield microscopy prior to infection. Bacterial cultures of 8 passages or less were used for all experiments. All procedures involving Leptospira were performed in a biosafety cabinet.

Borrelia burgdorferi growth conditions
In this study, the wild-type B. burgdorferi B31-A3 strain was used as a control bacterium. B. burgdorferi was grown in Barbour-Stoenner-Kelly (BSKII) medium [42] at 33˚C to a density of 1 × 10 8 cells/ml. Prior to each infection, the presence of genomic plasmids was confirmed in each culture by PCR [43,44].

Human endothelial cells and growth conditions
The dermal endothelial cell line human microvascular endothelial cells (HMEC-1), was originally a gift from Dr. E. Ades and Dr. T. J. Lawley (Emory School of Medicine and the Centers for Disease Control and Prevention). This cell line is currently available from ATCC. HMEC-1 were cultured in MCDB 131 (Gibco) supplemented with 10 mM L-glutamine, 10 ng/ml epidermal growth factor (Corning), 1 μg/ml hydrocortisone (Sigma), 15% Hyclone Fetal bovine serum (FBS, Thermo), and 25 mM HEPES (Gibco). Two types of primary cells, human dermal lymphatic endothelial cells (HDLEC) and human dermal microvascular endothelial cells (HDMEC), were purchased from ScienCell. These primary cells were cultured in endothelial cell medium (ECM) with the endothelial cell growth supplement (ECGS) and 5% FBS (all from ScienCell), according to the vendor's protocol. All human cells were grown at 36.5˚C under 5% CO 2 . For infection, passage of endothelial cells was limited to 20 or less for HMEC-1 and 13 or less for HDLEC and HDMEC. In preliminary experiments, we confirmed the morphological similarity of HDMEC to the cell line HMEC-1. For screening, HMEC-1 was selected for use as a stable microvascular endothelial cell type, and for some experiments was compared to HDLEC, which possesses morphologically well-organized intercellular junctions.

Infection of endothelial cells with Leptospira strains
Human endothelial cells were grown on sterile coverslips placed in wells of 12-well plates. Seeding numbers used are 4.2 x 10 5 /well for HMEC-1 and 2.2 x 10 5 /well for HDLEC for 2-day growth. Cells were checked under a brightfield microscope for confluence and maturation of intercellular junctions prior to infection. Cells were washed with phosphate-buffered saline (PBS) once, placed in cell culture medium (supplemented MCDB 131 or ECM), and infected with L. interrogans sv. Copenhageni or L. biflexa sv. Patoc in EMJH (similar volumes of EMJH were added to the uninfected control cells) at a multiplicity of infection (MOI) of 20 for 24 h at 36.5˚C in 5% CO 2 . Infection with B. burgdorferi B31-A3 was performed under the same conditions as for Leptospira. Infected endothelial cells were fixed with 2% para-formaldehyde for 15 min and then rinsed with PBS three times prior to immunofluorescence procedures. All procedures were performed in a biosafety cabinet.

Immunofluorescence microscopy
The infected and then fixed cells were rinsed with PBS and then either directly used for immunofluorescence procedures without permeabilization or treated with 0.1% Triton X-100 in PBS for 15 min for permeabilization, depending on the cell localization of a host protein or the specificity of an antibody (shown in Table 1). The samples were blocked with 3% bovine serum albumin (BSA) in PBS for 1 h and then incubated in a primary antibody diluted in 3% BSA/PBS for 1 h. For this study, antibodies and reagents were titrated and optimized for immunofluorescence microscopy analyses. The primary antibodies for which data are shown and the dilution factor used are listed in Table 1.
Unbound primary antibody was washed away with 3% BSA/PBS three times prior to incubation with either an anti-mouse-IgG or anti-rabbit-IgG antibody conjugated with Alexa Fluor 488 (Molecular Probes) for detection. After 1 h incubation, the unbound secondary antibody was rinsed away with 3% BSA/PBS twice and then with PBS twice. Filamentous actin was labeled with Alexa Fluor 488-conjugated phalloidin (Table 1) for 20 min and washed with 3% BSA/PBS twice and then with PBS twice. Coverslips were mounted on glass slides using ProLong Diamond containing DAPI (Molecular Probes). The mountant was cured in the dark for 12 h or longer before sealing of the coverslips with nail polish.
Fluorescence microscopy images were acquired by a Nikon Eclipse Ti-U inverted microscope equipped with a CoolSNAP ES2 CCD camera (Photometrics) and a multifluorescent Sedat Quad ET filter set (multichroic splitter, Chroma) using the 20× Plan Apo objective lens (N.A. 0.75, Nikon). NIS-Elements software (Nikon) was used for image acquisition, processing, and analysis. Scale bars represent 50 μm.

Quantification of fluorescence signal intensity and statistical analysis
In each microscopy experiment, at least 3 to 5 images were acquired, and the experiment was independently repeated at least 3 times for each host protein tested. For the quantification of signal intensity, raw images of each host protein in endothelial cells were processed in NIS-Elements software (Nikon) as follows: adjusting the color values of indexed-color pixels, converting to RGB format, selecting the whole field of an image as a region of interest (ROI), and obtaining the number of the mean intensity indicated in the ROI statistics in the software. Graphs of the quantified signal intensity were created in Microsoft Excel 2016. Error bars indicate standard deviations (SD) from the means. Statistical analysis was performed using two-tailed unpaired t-test in GraphPad Prism version 7.00. The p-values are indicated inside or below the graph.

Leptospira-mediated changes in endothelial cells
Infection with pathogenic Leptospira strains and serovars causes vascular leakage in the tissues and organs of the host organism by increasing the permeability of endothelial layers [8][9][10][11][12]. The mechanisms of this disruption of the endothelial barrier at pathological and molecular levels are unclear. In this study, we used two types of cultured human endothelial cells to investigate which host proteins are affected during infection with pathogenic L. interrogans strains, compared to the non-pathogenic, saprophytic strain, L. biflexa sv. Patoc.
For primary screening, the endothelial line HMEC-1 was used due to the faster growth rate and stability of this cell line. The phenotypes of interest detected in HMEC-1 were confirmed and further characterized by infection of primary endothelial cells, human dermal lymphatic endothelial cells (HDLEC). Advantages to the use of HDLEC are: 1) cell size is large, 2) cell structure is generally flat without overlapped cell edges, and 3) the well-defined structure of cell-cell junctions. Endothelial cells were infected with leptospires at a multiplicity of infection (MOI) of 20 for 24 h throughout the screening process of candidate proteins (Methods). Leptospires remained motile throughout the 24 h co-incubation. During incubation, we did not observe a hallmark of apoptosis, nuclear condensation or fragmentation in infected endothelial cells (S5 Fig, DAPI). To investigate the effect of Leptospira infection on endothelial cells, we screened antibodies and reagents to detect any changes in biological structures of human proteins by immunofluorescence microscopy.
After repeated screening, we found that the signal intensity or overall morphology of most of the host proteins tested were not significantly influenced by infection with either pathogenic or non-pathogenic leptospires. The lack of change could be a result of the irrelevance of the host protein to Leptospira infection or could be due to technical issues, such as the epitope position(s) in the tested protein or the antibody specificity for immunofluorescence microscopy. Among the analyzed reagents, we identified that the signal intensity and/or cellular morphology of 17 host proteins were affected by Leptospira infection (Table 2). These 17 human proteins, belonging to 5 biological groups, are the focus of this study.

Extracellular matrix (ECM) proteins-Collagen type IV, decorin, and laminin
We first analyzed several extracellular matrix (ECM) proteins that are known to be the targets of many of the Leptospira adhesins [18][19][20][21]. In this study, L. interrogans sv. Copenhageni infection was found to influence three ECM proteins. One is collagen type IV, which is one of the most abundant ECM proteins and is located exclusively in the basement membrane [45]. Collagen type IV provides a scaffold for cell structural stability and also plays a role in interaction of cells with underlying basement membranes, critical for cell adhesion [45]. Compared to uninfected endothelial cells, Copenhageni infection increased the signal intensity by 1.5-to 2-fold with concomitant morphological changes leading to formation of puncta ( Fig 1A,  Table 2). The signal increase was minor in L. biflexa sv. Patoc-infected cells,~1.3-fold ( Fig 1A). The signal increase and punctate morphology were also observed with another type of collagen, type VI (S1A Fig). Decorin is another ECM protein affected by infection. Decorin is a small leucine-rich proteoglycan that associates with fibrillar collagen type I [46,47]. After Leptospira infection, the signal intensity of decorin slightly increased by 1.2-to 1.4-fold, in combination with increases in puncta in both Patoc-and Copenhageni-infected endothelial cells ( Fig 1B, Table 2). These data indicate that the changes of decorin signal and morphology are caused by Leptospira infection in general, and are not specific to the pathogenic strain.
Laminin is a glycoprotein, a major component of basal lamina located in the basement membrane [48]. In micrographs of uninfected endothelial cells, laminin displayed an intricate net-shaped structure (Fig 1C, uninfected). When cells were infected with Copenhageni, the laminin network appeared to form bundles of small, rolled up, rope-like nets ( Fig 1C). There was a minor reduction of the signal intensity in Copenhageni-infected HDLEC but not in HMEC-1 ( Fig 1C). In Patoc-infected cells, there were only subtle structural rearrangements or changes in the signal intensity of laminin ( Fig 1C). These data suggest that the rearrangement of the laminin structure is a pathogenic Copenhageni-specific phenotype.
Fibronectin is one of most abundant ECM proteins in tissues, along with collagen and laminin. There was no detectable change in fibronectin morphology upon infection with either Copenhageni or Patoc, although the signal intensity of fibronectin was slightly increased (S1B Fig).  Intercellular adhesion molecules and cell surface receptors-ICAM-1, ICAM-2, CD-36, and VEGF-receptor 2 In addition to ECM proteins, other host cell surface proteins may be involved in Leptospira infection or pathogenicity. To test this hypothesis, we examined intercellular adhesion molecules (ICAMs) and other cell surface receptors. ICAMs belong to the immunoglobulin superfamily, and participate in inflammatory responses [49]. Compared to uninfected endothelial cells, the signal intensities of ICAM-1 and ICAM-2 were elevated by infection with either L. interrogans sv. Copenhageni or L. biflexa sv. Patoc (Fig 2A and 2B CD36 is a fatty acid/scavenger receptor, and is involved in microvascular endothelial cell migration and metastasis [50,51]. Leptospira infection increased the signal intensity of CD36 by 1.3-to 1.8-fold, and was slightly higher in Copenhageni-infected than Patoc-infected HMEC-1 ( Fig 2C). Infection also elevated the CD36 signal in HDLEC, although the difference between Copenhageni-and Patoc was miniscule, and mainly caused by the increase in punctate morphology ( Fig 2C, S2B Fig). These data suggest that this CD36 phenotype is induced by Leptospira infection with both pathogenic and non-pathogenic strains.
Vascular endothelial growth factor-receptor 2 (VEGF-R2) is another Leptospira-influenced cell surface protein. This VEGF-specific receptor is involved in the proliferation of vascular endothelial cells and the regulation of the endothelial barrier function [52]. Leptospira infection with both Copenhageni and Patoc elevated the VEGF-R2 signal, more so in HMEC-1 (1.5-to 2-fold) than the slight increase (1.1-to 1.4-fold) in HDLEC ( Fig 2D, S2C Fig). Again, these signal elevations in VEGF-R2 were induced by both pathogenic and non-pathogenic Leptospira species.

Intracellular proteins-VEGF, RhoA, and ILK (integrin-linked kinase)
Although the screening of cell surface proteins was originally our focus, we also examined several intracellular proteins. We found three intracellular host proteins that were affected by Leptospira infection ( Table 2). One protein was vascular endothelial growth factor (VEGF), which plays roles in the control of vascular endothelial cell proliferation and vascular permeability [53]. Infection with either L. interrogans sv. Copenhageni or L. biflexa sv. Patoc slightly elevated the fluorescence signal of VEGF by 1.1-to 1.9-fold with puncta formation (Fig 3A). The signal increase caused by the pathogenic Copenhageni was higher than by the nonpathogenic Patoc in HMEC-1 but there was no difference between the changes caused by the two Leptospira strains in HDLEC ( Fig 3A).
A second Leptospira-affected protein, the small GTPase RhoA, is an important molecule that regulates the assembly of the actin cytoskeleton and the remodeling of cell junction proteins. RhoA activity accompanied by actin remodeling can lead to a loss of endothelial barrier integrity [52,[54][55][56]. In both endothelial cell types we tested, the fluorescence intensity of small GTPase RhoA was slightly elevated by Copenhageni (1.3-to 1.4-fold) and Patoc (1.1-to 1.2-fold) infection ( Fig 3B). The signal increase was slightly higher when cells were infected with Copenhageni than with Patoc ( Fig 3B). An increase in punctate morphology of the RhoA signal was also observed in infected cells, but not specific to the Leptospira species (Fig 3B).
Another intracellular protein affected by Leptospira infection is integrin-linked kinase (ILK). ILK associates with integrins as a regulator of integrin-mediated signaling, correlating with multiple cellular functions such as cell proliferation, migration, adhesion, and vascular integrity [57][58][59]. The intensity of ILK signal was slightly higher (1.1-to 1.5-fold) with puncta formation when HMEC-1 were infected with leptospires regardless of pathogenic status ( Fig  3C).
Overall, Leptospira-mediated changes were found in intracellular host proteins that have roles in the regulation of cell proliferation, endothelial barrier integrity, and actin remodeling, but differences between the pathogenic and non-pathogenic strains were not always apparent.

Cell-cell junction proteins
In endothelial cells, intercellular connections are formed through multiple adhesive structures, regulating the passage of blood constituents and circulating cells to the underlying tissues [60,61]. Pathological conditions of endothelial paracellular permeability lead to severe or fatal organ dysfunction [60,61], similar to the symptoms in severe leptospirosis patients. To determine the effect of Leptospira infection on cell-cell junctions, we examined the transmembrane proteins and cytosolic adaptor proteins of three major intercellular junction types: 1) adherens junction, 2) tight junction, and 3) gap junction.
Adherens junction-VE-cadherin, p120 catenin, alpha-catenin, and beta-catenin. Adherens junctions (or zonula adherens, intermediate junctions) are composed of adhesion protein complexes located at the basal side of cell-cell junctions, playing a significant role in endothelial barrier function [60][61][62][63]. VE-cadherin (vascular endothelial cadherin, also known as cadherin 5 or CD144) is exclusively expressed in endothelial cells, and is essential for the formation of adherens junctions and the endothelial barrier [60,63]. This transmembrane protein forms homodimers on the cell surface, interconnecting neighboring endothelial cells [62,64,65]. The cytoplasmic face of VE-cadherin associates directly with p120 catenin and betacatenin, and indirectly with alpha-catenin and the actin cytoskeleton, forming the adherens junction as a stable intercellular-and intracellular-structure [62,66].
In our experiments, VE-cadherin was clearly localized at intercellular junctions of uninfected cells, especially in HDLEC (Fig 4A, uninfected). When endothelial cells were infected with L. interrogans sv. Copenhageni, the fluorescence intensity of VE-cadherin was dramatically reduced by 40 to 60% as compared to that of uninfected cells, and was largely lost at the original localization at cell-cell junctions ( Fig 4A, Table 2). In comparison, infection with L. biflexa sv. Patoc or pathogenic B. burgdorferi B31-A3 did not demonstrate either prominent signal reduction or mislocalization of VE-cadherin ( Fig 4A, S9 Fig). 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). https://doi.org/10.1371/journal.pntd.0005830.g002 Intracellular adapter proteins that associate with VE-cadherin at the adherens junction include p120 catenin, alpha-catenin, and beta-catenin [62,67]. p120 catenin and beta-catenin  directly interact with the cytoplasmic face of VE-cadherin while alpha-catenin indirectly associates as a component of the adherens junction complex [62,66]. p120 catenin possesses a key role in VE-cadherin expression, internalization, and membrane localization as well as the regulation of endothelial permeability [63,68]. Infection with the pathogenic Copenhageni reduced the fluorescence intensity of p120 catenin by 30 to 40% of the signal detected in uninfected endothelial cells (Fig 4B, S4A Fig, Table 2). As observed with VE-cadherin, Copenhageni infection disrupted the localization of p120 catenin at intercellular junctions (Fig 4B, S4A  Fig). Infection with the non-pathogenic Patoc did not alter the junctional localization p120 catenin (Fig 4B, S4A Fig).
Alpha-catenin functions as a molecular switch through exclusively associating with either an cadherin/beta-catenin complex or actin filaments, coordinating actin organization and remodeling [66,69]. The signal intensity of alpha-catenin was significantly decreased by Copenhageni infection even though alpha-catenin associates with VE-cadherin indirectly ( Fig  4C, S4B Fig). The fluorescence signal was reduced to approximately 50% of the signal detected in uninfected endothelial cells, and alpha-catenin was almost invisible at the cell periphery ( Fig  4C, S4B Fig). As observed in VE-cadherin and p120 catenin, there was little influence by infection with the saprophyte Patoc strain (Fig 4C, S4B Fig).
Beta-catenin is a multifunctional protein that localizes at either the cadherin adhesive complex in cell-cell junctions or at the T-cell factor (TCF)-transcriptional complex in the nucleus of multiple types of cells, including endothelial cells [65,70]. VE-cadherin/beta-catenin complexes are involved in the regulation of endothelial cell survival as well as vascular patterning and permeability [71,72]. In uninfected cells, beta-catenin was localized at the cell-cell junction and in the nucleus as expected (Fig 4D, see single-color format in S5 Fig). In cells infected with Copenhageni, beta-catenin was reduced at the cell-cell junction, although the localization in the nucleus was unchanged and remained at the levels in uninfected or Patoc-infected cells (Fig 4D, S4C Fig and S5 Fig). Overall, the fluorescence signal of beta-catenin decreased by 20-30% as compared to uninfected cells (Fig 4D, S4C Fig). The signal reduction was not as intense as VE-cadherin, p120 catenin, or alpha-catenin, which is likely the result of the unchanged fluorescence signal in the nucleus regardless of Leptospira infection.
Nectin is another type of an adherens junction protein that functions independently from VE-cadherin complexes, and can be located near tight junctions during junctional development [64,65]. While VE-cadherin and its adaptor protein catenins at the adherens junction were largely decreased by Copenhageni infection (Fig 4, S4 Fig) These data suggest that pathogenic Leptospira specifically targets the adherens junctions containing the VE-cadherin/catenins complex.
Tight junction-zonula occludens-1 (ZO-1). Another group of adhesive junctional structures is tight junctions (occluding junctions or zonula occludens), which are generally located at the apical side of cell-cell junctions as compared to adherens junctions [62,64]. Major types of tight junction proteins include claudin, occludin, and zonula occludens-1 (ZO-1) [64,73]. Claudin and occludin are transmembrane proteins, directly involved in junctional adhesion [62,64]. We examined the effect of Leptospira infection on claudin 5 and occludin. Claudin 5 is specifically produced in endothelial cells and the expression is partially controlled by VEcadherin complexes [65]. During infection with either L. interrogans sv. Copenhageni and L. biflexa sv. Patoc, there was little or no change in morphology or the signal intensity of claudin 5 or occludin (S7 Fig). ZO-1 is a peripheral membrane protein located at the cytoplasmic side of the plasma membrane [64,73]. In uninfected HDLEC, ZO-1 was apparently localized at cell-cell junctions ( Fig  5A, uninfected). In contrast to the results with claudin 5 and occludin (S7 Fig), Copenhageni infection clearly disrupted the localization of ZO-1 from the cell periphery ( Fig 5A, Table 2). The fluorescence signal of ZO-1 indicated relocalization of this protein from cell-cell junctions to intracellular locations, as the total signal intensity decreased only slightly (Fig 5A). Patoc infection did not influence the intercellular junction localization or signal intensity of ZO-1 ( Fig 5A). In summary, the intracellular tight junction protein ZO-1 was mislocalized by the pathogenic Leptospira species but the transmembrane proteins claudin 5 and occludin, which play an essential role in junctional adhesion, were not affected.
Gap junction-Connexin. A gap junction is formed as junctional channels between neighboring cells by oligomers of specific integral membrane proteins, such as connexins [74,75]. The gap junctional proteins play roles in subcellular microdomain signaling as well as the regulation of intercellular communication through passage of ions and small molecules [74,75]. The gap junction protein we examined is connexin 43, which is the most ubiquitously distributed of this class of proteins in mammalian cells [74].
To investigate the gap-junction structure in endothelial cells, HDLEC cells, which possess well-organized cell junctions, were used. In uninfected cells, connexin 43 localized at intercellular junctions in a punctate signal pattern, suggesting a less well-organized cell junction morphology (Fig 5B, uninfected), as compared to adherens junctions (Fig 4) and tight junctions (ZO-1, Fig  5A). L. interrogans sv. Copenhageni infection decreased the signal intensity of connexin 43 more than 20% as compared to uninfected cells, especially at the cell periphery ( Fig 5B, Table 2). L. biflexa sv. Patoc infection demonstrated translocation of connexin 43 from the cell-cell junctions to intracellular locations, but the overall signal intensity was not significantly influenced ( Fig 5B). Cytoskeletal protein-Filamentous actin L. interrogans sv. Copenhageni infection apparently disrupted multiple proteins of the adherens junction, the tight junction protein ZO-1, and the gap junction protein connexin (Figs 4 and 5). The adherens junction and tight junction proteins directly or indirectly interact with actin filaments to stabilize the cellular structure and cell junctions [72,73]. To examine if Leptospira infection influences actin filaments (microfilaments) in endothelial cells, filamentous actin was labeled with Alexa Fluor-conjugated phalloidin. In permeabilized, uninfected HMEC-1, actin filaments were visualized as net-shaped structures, spreading ubiquitously in the cell (Fig 6, uninfected). When this cell type was infected with Copenhageni, the signal intensity of actin filaments decreased to~50% of uninfected cells (Fig 6, Table 2). L. biflexa sv. Patoc infection demonstrated no reduction in signal intensity (Fig 6).
In another type of endothelial cells, HDLEC, the morphology of actin filaments appeared well-organized, with long straight filaments rather than the net-shaped morphology (Fig 6A,  uninfected). In contrast to the reduction of the actin signal in Copenhageni-infected HMEC-1, the signal decrease in HDLEC was minor (Fig 6). Instead, Copenhageni infection induced a morphological rearrangement of actin filaments: intense localization of filamentous actin at the cell periphery and reduction of stress fibers inside the cell (Fig 6, Table 2). Patoc infection induced slightly more stress fibers but did not affect the overall morphology and signal intensity of filamentous actin in HDLEC (Fig 6). Throughout our screening of host proteins, only this protein demonstrated host cell-type specific changes in morphology and phenotype.
We also examined another cytoskeletal structure, the microtubule, by immune-labeling the most critical protein, alpha-tubulin (Table 1). There was no detectable change in morphology or signal-intensity in HMEC-1 or HDLEC infected with either Copenhageni or Patoc (S8 Fig). These data suggest that the pathogenic Leptospira specifically modifies the actin filaments of the cytoskeleton.

Discussion
To investigate the pathogenic effects of Leptospira infection of human endothelial cells, we utilized immunofluorescence microscopy to screen for changes in host protein abundance and distribution. Seventeen proteins indicated minor to major changes in Leptospira-infected endothelial cells. These 17 proteins are classified into five biological groups: 1) extracellular matrix, 2) intercellular adhesion molecules and cell surface receptors, 3) intracellular proteins, 4) cell-cell junction proteins, and 5) a cytoskeletal protein.
The most prominent phenotype of infection with pathogenic L. interrogans sv. Copenhageni was the dramatically reduced multiple adherens junction proteins and one of the tight junction proteins (ZO-1), a gap junction protein (connexin) as well as filamentous actin (summarized in Table 2). Infection with Leptospira, regardless of the strain's pathogenicity or ability to harm host cells, increased the signal intensity of some of the ECM proteins, ICAMs, cell surface receptors, and intercellular proteins ( Table 2). In general, the signal increase was more intense when endothelial cells were infected with pathogenic L. interrogans sv. Copenhageni than the saprophyte strain, L. biflexa sv. Patoc (Table 2).
Among ECM proteins, collagen type IV, decorin, and laminin were influenced by Leptospira infection (Fig 1). It has been reported that outer membrane protein(s) of pathogenic Leptospira can increase the production of an ECM protein, collagen type IV [76]. In this study, the signal elevations of collagen and decorin were caused by both pathogenic and non-pathogenic leptospires (Fig 1A and 1B). Kassegne et al. reported that L. interrogans possesses a collagenase, which is involved in the invasion and transmission of the pathogenic species [77]. Morphological changes we observed, formation of puncta as a result of infection (Fig 1A), may be a result of protein degradation by collagenase activity, which could expose epitopes for antibody binding, increasing the fluorescence signal. For laminin, the structural rearrangement was specifically caused by the pathogenic Copenhageni (Fig 1C). The net-shape structure of laminin appeared rolled up, forming thick bundles with little change in total signal intensity, suggesting that this phenotype could be a secondary effect of the Copenhageni-mediated disassembly of adherens junctions.
Leptospira infection influenced several cell-surface proteins/receptors, ICAM-1, ICAM-2, CD36, and VEGF-receptor 2 (Fig 2, S2 Fig, Table 2). We observed a significant signal increase of ICAMs in both HMEC-1 and HDLEC when infected with leptospires and the increase was more prominent with Copenhageni in HMEC-1 (Fig 2). Because ICAMs cluster at intercellular junctions distinct from the adherens junction and tight junction [61], the elevation of ICAMs is likely an independent phenomenon from the disruption of adherens junctions by pathogenic Leptospira. In pulmonary leptospirosis patients, an increase in ICAM-1 expression was detected in the alveolar septa and pulmonary vessels [7]. However, it was also reported that there was no significant change in ICAM-1 cell surface expression in HUVEC after 24 h and 48 h infection as detected by horseradish peroxidase reaction [78]. This difference may be due to the sources of the cells or specific experimental conditions, as other work has shown that leptospiral lipopolysaccharide (LPS) and outer membrane proteins increase ICAM-1 expression in HUVECs [38,39]. During early stages of leptospirosis, leptospiral LPS and outer membrane lipoproteins induce inflammation primarily via Toll-like receptor 2 (TLR2) and in some experiments, via TLR4 activation [33,34,[79][80][81]. Interestingly, pathogenic Leptospira infection induces pro-inflammatory reactions in human (susceptible to leptospirosis) but activates antiinflammatory pathways in mice (not susceptible to clinical leptospirosis) [18,36]. Thus, the phenotype of ICAM increase in human endothelial cells may be a result of inflammatory reaction induced by TLR-mediated signaling pathways and other pro-inflammatory responses.
The fatty acid/scavenger receptor CD36 is involved in the regulation of microvascular endothelial cell migration and is implicated as having a role in inflammation [50,51,82]. CD36 is also known to interact with collagens [83]. Since we observed Leptospira-mediated changes in collagen type IV and type VI (Fig 1A and S1A Fig), the signal increase phenotype of CD36 that we observed could be induced by multiple factors, such as changes in collagen or inflammatory signaling. Another receptor, VEGF-R2, specifically reacts to VEGF in controlling the growth of vascular endothelial cells. Another function of VEGF-R2 is that this receptor interacts with VE-cadherin, which physically limits the internalization of VE-cadherin from adherens junctions [52]. Overproduction of VEGF-R2 might inhibit the internalization of VEcadherin, but we observed a reduction in VE-cadherin signal without apparent internalization (Fig 4A and S3C Fig). The elevation of the VEGF-R2 signal may be a response to leptospiremediated VEGF increase rather than a direct response to Leptospira contact with endothelial cells.
Leptospira infection increased the fluorescence signal and puncta formation of three intracellular proteins, VEGF, RhoA, and ILK (Fig 3, Table 2). In addition to its function in vascular endothelial growth, VEGF promotes vascular permeability via the phosphorylation and endocytosis of VE-cadherin; this modulation is reversible [61]. There was no detectable internalization of VE-cadherin (S3C Fig) and the signal increase of VEGF was not specific to the pathogenic strain (Fig 3A), implying that any involvement of VEGF in pathogenicity is less likely. ILK is a regulator of integrin-mediated signaling to regulate cell migration, adhesions, and vascular integrity [57][58][59]. The Leptospira-mediated changes were statistically significant but modest and also detected in cells infected both Copenhageni and Patoc strains (Fig 3C).
Endothelial permeability is controlled by the opening and closing of cell-cell junctions via the rearrangement of junction proteins and cytoskeletal proteins [61]. To regulate the adherens junction organization and endothelial permeability, some small GTPases are involved [53,84]. For example, the small GTPase RhoA controls the endothelial barrier integrity via remodeling of the actin cytoskeleton and of junction proteins [52,[54][55][56]. In this study, Leptospira infection altered the actin cytoskeleton along with a moderate elevation of the RhoA signal (Figs 6 and 3B). The increase of RhoA signal was not specific to Copenhageni infection, though the signal intensity was higher than with Patoc inoculation (Fig 3B). These data suggest that the involvement of these intracellular proteins in the Leptospira pathogenicity, especially in disrupting the endothelial integrity, is relatively minor.
In this study, the most prominent L. interrogans pathogenic phenotype was the disruption of adherens junctions (Fig 4, S4 Fig). The adherens junction proteins, VE-cadherin, p120 catenin, alpha-catenin, and beta-catenin, showed drastically reduced immunofluorescence signals specifically at the cell-cell junctions (Fig 4, S4 Fig; also see Fig 7 for structural features). Because Copenhageni-infection did not disturb occludin and claudin (tight junction markers, S7 Fig), the VE-cadherin/catenin-complex at the adherens junction is likely to be the main target of pathogenic Leptospira species in endothelial cells (Fig 7). VE-cadherin is essential for adherens junction formation and barrier maintenance in endothelial cells, playing a critical role in vascular morphogenesis, especially remodeling and maturation [71,85]. The administration of the anti-VE-cadherin antibody BV13 redistributes VEcadherin molecules at adherens junctions in cultured endothelial cells and increases vascular permeability in heart and lungs of mice [60]. VE-cadherin-associated catenins are also important for the formation of the dynamic endothelial barriers. For example, VE-cadherin/betacatenin complexes are involved in the regulation of endothelial cell survival, and in VE-cadherin mutant cells, beta-catenin was not localized at intercellular junctions [71]. Inactivation of the beta-catenin gene disrupted the cell junctional organization by reduction in alpha-catenin expression and cell-adhesion strength, leading to hemorrhagic vessels [72]. Also, p120 catenin is involved in maintaining VE-cadherin expression [68]. Thus, VE-cadherin and catenins are interconnected physically and functionally to regulate the expression and interaction of these proteins, maintaining the stability and flexibility of endothelial junctional barriers required for normal biological function. Our data indicate that pathogenic Leptospira strains target these adherens junction proteins important for the endothelial barrier integrity and disassemble the junction-protein complex (Fig 7).
In contrast to the drastic disturbance of the adherens junction (Fig 4, S4 Fig), there were no detectable changes in the tight junctional transmembrane proteins claudin 5 and occludin in endothelial cells infected with L. interrogans sv. Copenhageni (S7 Fig). These tight junction markers are less critical to the vascular barrier integrity since the deletion of the genes encoding claudin 5 or occludin does not influence on the vascular morphology and barrier function in mice [86,87]. In our experiments, only ZO-1, a cytosolic peripheral protein, was apparently mislocalized from tight junctions (Fig 5A). Reduction of ZO-1 was previously observed in Leptospira-infected HUVEC [24]. ZO-1 is a tight junction protein when intercellular junctions are mature but localizes at adherens junctions at an early stage of cell-cell contact [62,73,88]. In the immature cell junctions, ZO-1 can directly associate with alpha-catenin and the actin cytoskeleton [67,89] and indirectly associates with beta-catenin [90]. In the mature tight junction, ZO-1 indirectly influences the endothelial integrity via association with claudin, occuldin, and actin filaments [60,75]. The published information and our data imply that Leptospira 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.
A gap junction protein, connexin 43, was also mislocalized and showed reduced signal at cell junctions in endothelial cells when infected with pathogenic Copenhageni (Fig 5B). The non-pathogenic Patoc strain caused translocation of connexin 43 from the cell periphery to intracellular locations without losing signal intensity (Fig 5B). Connexin 43 associates with a variety of proteins located at adherens junctions and tight junctions, the cytoskeleton, and actin-binding proteins, including p120 catenin, beta-catenin, and ZO-1 [74,75]. Moreover, the gap junction is not directly involved in endothelial permeability [62]. These data imply that the gap junction is not a primary target of pathogenic Leptospira and that the phenotype we observed is likely induced as a secondary effect following the disruption of the adherens junction.
Infection with the pathogenic Copenhageni demonstrated cell-type-specific phenotypes in the actin cytoskeleton: 1) reduction of the actin signal in HMEC-1 and 2) bundling and rearrangement of filamentous actin structure in HDLEC (Fig 6). Actin filaments physically interact with multiple cell-junction proteins, which regulates the dynamic rearrangement of the actinfilament structure [62]. For instance, alpha-catenin interacts with either cadherin/beta-catenin complex or actin filaments, regulating actin assembly and organization [67,69]. The inactivation of the beta-catenin gene influences the morphology of actin filaments in endothelial cells [72], and ZO-1 regulates the cortical cytoskeleton at cell junctions [73]. The phenotypes of Copenhageni infection, a decrease in filamentous actin in HMEC-1 and translocation of the bundled-actin filaments to the cell periphery of HDLEC (Fig 6), may be induced by Copenhageni-mediated disruption of the VE-cadherin-catenin complex.
One of the functions of filamentous actin is stabilizing or reorganizing the intercellular junctions through interacting with cell-junctional proteins [62]. The cadherin-catenin complex is known to dynamically influence the actin cytoskeleton and vice versa: filamentous actin is necessary for the regulation of endothelial opening/closing in addition to the stabilization of cell-junctions [61,62]. We considered the possibility that filamentous actin is the primary target of pathogenic Leptospira infection, but the inhibition of typical actin distribution or mobilization at cell-cell junctions by cytochalasin D or jasplakinolide do not influence the dynamics of cadherin and alpha-catenin [67], suggesting that filamentous actin is unlikely to be the primary target of pathogenic Leptospira.
In physiologic conditions in vivo, dynamic and transient remodeling of intercellular junctions is well-controlled and critical to cellular maintenance, especially in endothelial cells [62,91]. However, drastic and irreversible changes in endothelial junctions contribute to pathological endothelial permeability and leakage as well as vascular network disruption [62]. Miyahara et al. identified intact cell attachment with some disturbance of intercellular junctions in hepatocytes of pre-icteric hamsters, with cell detachment plus disrupted junctional association in icteric hamsters [23]. In leptospirosis patients, proinflammatory response and vascular damage are pathologic features of leptospirosis-associated pulmonary hemorrhage syndrome or acute lung injury [11,12,25]. Thus, in the later stage of severe leptospirosis, Leptospira infection and detrimental inflammatory responses, independent of TLR activation [33], overwhelm the cellular maintenance system, leading to devastating damage to cell-cell junctions and vascular systems of the host.
We used cultured human endothelial cells to investigate how Leptospira may lead to endothelial permeability and, by inference, vascular damage seen in human patients and susceptible animals. Our study demonstrated that the primary targets of L. interrogans are intercellular junctions, primarily adherens junctions. Other host proteins affected by L. interrogans infection may be indirectly impacted by the damage to a modification of the primary targets. The changes in host proteins that were impacted by non-pathogenic Patoc, though not as robust as those impacted by Copenhageni, may be a consequence of pro-inflammatory responses induced by Leptospira LPS, cell-surface proteins, or secreted proteins.
Our systematic analyses of host proteins in Leptospira infected-human endothelial cells demonstrated pathogen-specific phenotypes in the adherens junction, filamentous actin and actin-associated proteins. Several phenotypes were observed with infection with either the pathogen or the non-pathogen in multiple biological groups. These data suggest that this zoonotic agent may damage endothelial cells via multiple cascades or pathways, potentially leading to the increased vascular permeability followed by severe illness in vivo. In addition, morphological and quantitative analyses of infected human cells by immunofluorescence microscopy constitute a reliable method to investigate the pathogenicity and biological functions of Leptospira strains and specific proteins in vitro. Further work based on these results will contribute to our understanding of pathophysiological mechanisms of Leptospira infection.