Identification of cell-binding adhesins of Leptospira interrogans

Abstract

Leptospirosis is a globally distributed bacterial infectious disease caused by pathogenic members of the genus Leptospira. 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 Leptospira 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 Leptospira interrogans binds to host cells more efficiently than to ECM components. Using in vitro 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 L. interrogans sv. Copenhageni st. Fiocruz L1-130 adhesins. Recombinant LIC11574, but not its L. biflexa 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 L. interrogans. 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 L. interrogans 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.

Figure 1. Schematic of analysis of phage clones that bind to endothelial EA.hy926 monolayers.

Following three rounds of selection for phage clones that bind endothelial cells, 931 clones were sequenced and analyzed by BLAST. Seven hundred seventy-nine phage clones have DNA inserts of L. interrogans open reading frames, corresponding to 185 unique genes. Using prediction programs SignalP 3.0 and LipoP 1.0, 37 of the 185 identified genes contain signal peptide at the N-terminus of the predicted protein product.

Figure 2. Selected phage clones bind to endothelial and epithelial cells.

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 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 t-test, *P<0.05, **P<0.01, ***P<0.001).

Figure 3. Recombinant L. interrogans candidate adhesins bind to cells.

Recombinant forms of L. interrogans 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 t-test, *P<0.05, ***P<0.001). Panel C shows concentration-dependent attachment of LIC11574 to endothelial and epithelial cells.

Figure 4. L. interrogans candidate adhesin LIC11574, but not its L. biflexa ortholog, LBF1629, binds to cells.

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 t-test, *P<0.05, ***P<0.001). Results show that LBF1629 (the L. biflexa ortholog of LIC11574) does not have cell-binding activity.

Figure 5. L. interrogans candidate adhesins LIC11574 and LIC13411 bind to the host protein VE-cadherin.

L. interrogans proteins, LIC11574 and LIC13411 were expressed as fusions to MBP (diluted 1×10⁻⁵–0.1 µM) and incubated for 1 hr with 0.01 µM VE-cadherin or control BSA immobilized on a Linbro plate. The unbound recombinant proteins were washed off and bound proteins were quantified by ELISA using an anti-MBP antibody. Attachment to VE-cadherin is expressed as the OD reading at 655 nm minus BSA and no receptor (or “plastic”) binding. Results indicate that attachment of LIC11574 (K _D = 7.6±2.5 nM) and LIC13411 (K _D = 0.4±0.2 nM) to VE-cadherin, but not to BSA, is dose-dependent and saturable.

Figure 6. Localization of proteins after Triton X-114 fractionation of L. interrogans sv. Copenhageni.

L. interrogans was phase partitioned using Triton X-114 as described in Materials and Methods. 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.