Selected phage clones bind to endothelial and epithelial cells.

<p>Individual phage clones (∼10¹⁰) were added with confluent endothelial EA.hy926 or epithelial HEp-2 monolayers, or to wells containing cell medium only for 2 hr at 37°C under 5% CO₂. Phage attachment was quantified by ELISA using an anti-M13 (fd phage coat) antibody. Vector phage clone fdDOG <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003215#pntd.0003215-Coburn1" target="_blank">[37]</a> serves as a negative control. Binding is expressed as fold difference in the absorbance between monolayers vs. wells containing medium alone. Results are expressed as means ± standard errors, representative of at least two independent experiments repeated in quadruplicate. Error bars represent standard error while asterisks (*) indicate significant difference in attachment of candidate adhesins to cells compared to fdDOG (two-tailed <i>t-</i>test, *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001).</p

Localization of proteins after Triton X-114 fractionation of <i>L. interrogans</i> sv. Copenhageni.

<p><i>L. interrogans</i> was phase partitioned using Triton X-114 as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003215#s2" target="_blank">Materials and Methods</a>. Equivalents of 5×10⁸ or 2.5×10⁹ (*) spirochetes/lane of the whole cell lysate (WCL), protoplasmic cylinder (PC), aqueous (AQ) and detergent (DET) fractions were separated on 12.5 or 15% SDS-PAGE electrophoresis, transferred to PVDF membrane and probed with rabbit immune sera against periplasmic protein flagellin A1 (FlaA1), inner membrane protein LipL31, outer membrane proteins OmpL47 and OmpL1, and mouse immune sera against candidate adhesins LIC10508, LIC11574, LIC12341, and LIC13411. A duplicate gel was silver stained.</p

Oligonucleotides used in this study.

<p>Underlined are the sequences recognized by restriction enzymes <i>Xba</i>I (TCTAGA), <i>Sal</i>I (GTCGAC), <i>Bam</i>H1 (GGATCC), and <i>Pst</i>I (CTGCAG).</p><p>Oligonucleotides used in this study.</p

<i>L. interrogans</i> genes encoding known or putative surface proteins selected for binding to endothelial cells.

<p>Phage clones pre-adsorbed on ECM and subjected to three rounds of selection in EA.hy926 cells were analyzed to identify the inserted <i>L. interrogans</i> sv. Copenhageni st. Fiocruz L1–130 gene. Out of the 931 phage clones selected, 779 have <i>L. interrogans</i> DNA inserts that represent 185 unique genes. Thirty-seven of these genes contain signal sequence. Four genes shown below were prioritized for further assessment.</p><p>*During translocation, the signal sequence of lipoproteins and transmembrane OMPs are cleaved by lipoprotein signal transpeptidase (Lsp) and leader peptidase (Lep), respectively.</p><p><i>L. interrogans</i> genes encoding known or putative surface proteins selected for binding to endothelial cells.</p

Identification of Cell-Binding Adhesins of <i>Leptospira interrogans</i>

<div><p>Leptospirosis is a globally distributed bacterial infectious disease caused by pathogenic members of the genus <i>Leptospira</i>. Infection can lead to illness ranging from mild and non-specific to severe, with jaundice, kidney and liver dysfunction, and widespread endothelial damage. The adhesion of pathogenic <i>Leptospira</i> species (spp.), the causative agent of leptospirosis, to host tissue components is necessary for infection and pathogenesis. While it is well-established that extracellular matrix (ECM) components play a role in the interaction of the pathogen with host molecules, we have shown that pathogenic <i>Leptospira interrogans</i> binds to host cells more efficiently than to ECM components. Using <i>in vitro</i> phage display to select for phage clones that bind to EA.hy926 endothelial cells, we identified the putative lipoproteins LIC10508 and LIC13411, and the conserved hypothetical proteins LIC12341 and LIC11574, as candidate <i>L. interrogans</i> sv. Copenhageni st. Fiocruz L1–130 adhesins. Recombinant LIC11574, but not its <i>L. biflexa</i> homologue LBF1629, exhibited dose-dependent binding to both endothelial and epithelial cells. In addition, LIC11574 and LIC13411 bind to VE-cadherin, an endothelial cell receptor for <i>L. interrogans</i>. Extraction of bacteria with the non-ionic detergent Triton X-114 resulted in partitioning of the candidate adhesins to the detergent fraction, a likely indication that these proteins are outer membrane localized. All candidate adhesins were recognized by sera obtained from leptospirosis patients but not by sera from healthy individuals as assessed by western blot. This work has identified bacterial adhesins that are potentially involved in <i>L. interrogans</i> infection of the mammalian host, and through cadherin binding, may contribute to dissemination and vascular damage. Our findings may be of value in leptospirosis control and prevention, with the bacterial adhesins potentially serving as targets for development of diagnostics, therapeutics, and vaccines.</p></div

Recombinant <i>L. interrogans</i> candidate adhesins bind to cells.

<p>Recombinant forms of <i>L. interrogans</i> candidate adhesins expressed as fusions to MBP, along with β-galactosidase (β-gal) were diluted to 0.3 µM and incubated with confluent monolayers of endothelial HMEC-1 (Panel A) or epithelial HEp-2 (Panel B) cells for 1 hr at 37°C, 5% CO₂. The unbound proteins were removed by washing and the bound adhesins were detected by ELISA using anti-MBP antibody. Results were expressed as the difference in the absorbance reading between wells with or without cells. The data shown are means ± standard errors from 8 independent assays with n = 8–28. Asterisks (*) indicate significant difference in binding when compared to the MBP fusion to β-gal, the negative control (two-tailed <i>t-</i>test, *<i>P</i><0.05, ***<i>P</i><0.001). Panel C shows concentration-dependent attachment of LIC11574 to endothelial and epithelial cells.</p

<i>L. interrogans</i> candidate adhesin LIC11574, but not its <i>L. biflexa</i> ortholog, LBF1629, binds to cells.

<p>MBP fusions of LIC11574 and LBF1629, along with control β-galactosidase (β-gal) were diluted to 0.3 µM and incubated with confluent monolayers of HMEC-1 endothelial (Panel A) or HEp-2 epithelial (Panel B) cells for 1 hr at 37°C, 5% CO₂. The unbound proteins were removed by washing and the bound adhesins were detected by ELISA using anti-MBP antibody. Results are expressed as the difference in the absorbance reading between wells with or without cells. The data shown are means ± standard errors from 8 independent assays with n = 16–28. Asterisks (*) indicate significant difference in binding when compared to the MBP fusion to β-gal, the negative control (two-tailed <i>t-</i>test, *<i>P</i><0.05, ***<i>P</i><0.001). Results show that LBF1629 (the <i>L. biflexa</i> ortholog of LIC11574) does not have cell-binding activity.</p

Cellular localization of <i>L. interrogans</i> enolase.

<p>(A) Subcellular localization of enolase. <i>L. interrogans</i> whole cells (WC) were separated into outer membrane (OM) and protoplasmic cylinder (PC) fractions, resolved by SDS-PAGE, and immunoblotted with antibodies specific for enolase or proteins known to localize in the OM (LipL32) or in the inner membrane (LipL31). (B) Enolase is secreted extracellularly. Viability of the <i>L. interrogans</i> culture was determined by microscopy, and the supernatant was collected from a culture of intact viable cells. The samples were filtered, concentrated, and analyzed by 2D gel electrophoresis followed by immunoblotting assays using antibodies against enolase (upper panel) or a subcellular protein LipL31 (lower panel). </p

Plasminogen (Pg) binds <i>L. interrogans</i> enolase and activates plasmin.

<p><u>The error bars indicate the standard deviations from three independent experiments performed in triplicate</u>. * <i><u>P</u></i><u> < 0.05</u>. (A) <u>Pg binds to <i>L. interrogans</i> in a concentration-dependent manner. Microtiter plates were coated with fixed cells and incubated with various concentrations of human plasminogen (hPg)</u>. (B) <u><i>L. interrogans</i> converts Pg into plasmin in the presence of tissue plasminogen (tPA). Well-bound <i>L. interrogans</i> cells were incubated with hPg (1µg) and a chromogenic substrate (D-valyl-L-lysyl-<i>p</i>-nitroaniline hydrochloride) in the presence or absence of tPA and a known Pg inhibitor, a lysine analogue, ε-ACA</u>. (C) <u>A Pg inhibitor</u> blocks <i>L. interrogans</i>-hPg interaction. Microtiter plates were coated with fixed <i>L. interrogans</i> and increasing concentrations (0 to 100 mM) of ε-ACA were incubated with a fixed amount (1µg) of hPg. (D) Fibrinolytic activity of Pg-bound <i>L. interrogans</i>. Panels represent <i>L. interrogans</i> cells in the presence or absence of hPg and tPA. Arrow denotes fibrinolytic activity of spirochetes only in the presence of both hPg and tPA.</p

Enolase can be detected on <i>L. interrogans</i> surfaceand specifically interacts with outer membrane proteins.

<p>(<b>A</b>) Detection of enolase on the surface of intact <i>L. interrogans</i>. Microtiter plates were coated in the absence or presence of intact or lysed <i>L. interrogans</i> (10⁹/ml) and incubated with enolase antibody. Bound antibody was detected using HRP-labeled secondary antibodies and TMB substrate for color development, which was recorded at <i>A</i>₄₅₀. A known surface-exposed and outer membrane protein, LipL32, and a subsurface inner membrane protein, LipL31, were used as controls. (<b>B</b>) Interaction of enolase with OM proteins. A fixed amount (1µg) of solubilized proteins from isolated OM vesicles were coated on microtiter plates and assessed for binding with increasing amount of recombinant enolase, as described in panel B. The binding of enolase to immobilized OM proteins reached saturation at 5 µg, as there is a significant increase (P < 0.001) in individual OD values between 0-5 µg, while the difference between 5 and 10 µg values is nonsignificant (P > 0.05). (<b>C</b>) Recognition of recombinant enolase by infected hamster serum as assessed by immunoblotting. Recombinant enolase was probed with antibodies produced in immunized mice or antiserum collected from hamster infected with <i>L. interrogans</i>. The arrow indicates the position of enolase. Migration of protein standards is shown to the left.</p