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. 2010 Aug 3;4(8):e778.
doi: 10.1371/journal.pntd.0000778.

Cross-reactivity of antibodies against leptospiral recurrent uveitis-associated proteins A and B (LruA and LruB) with eye proteins

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Cross-reactivity of antibodies against leptospiral recurrent uveitis-associated proteins A and B (LruA and LruB) with eye proteins

Ashutosh Verma et al. PLoS Negl Trop Dis. .

Abstract

Infection by Leptospira interrogans has been causally associated with human and equine uveitis. Studies in our laboratories have demonstrated that leptospiral lipoprotein LruA and LruB are expressed in the eyes of uveitic horses, and that antibodies directed against LruA and LruB react with equine lenticular and retinal extracts, respectively. These reactivities were investigated further by performing immunofluorescent assays on lenticular and retinal tissue sections. Incubation of lens tissue sections with LruA-antiserum and retinal sections with LruB-antiserum resulted in positive fluorescence. By employing two-dimensional gel analyses followed by immunoblotting and mass spectrometry, lens proteins cross-reacting with LruA antiserum were identified to be alpha-crystallin B and vimentin. Similarly, mass spectrometric analyses identified beta-crystallin B2 as the retinal protein cross-reacting with LruB-antiserum. Purified recombinant human alpha-crystallin B and vimentin were recognized by LruA-directed antiserum, but not by control pre-immune serum. Recombinant beta-crystallin B2 was likewise recognized by LruB-directed antiserum, but not by pre-immune serum. Moreover, uveitic eye fluids contained significantly higher levels of antiibodies that recognized alpha-crystallin B, beta-crystallin B2 and vimentin than did normal eye fluids. Our results indicate that LruA and LruB share immuno-relevant epitopes with eye proteins, suggesting that cross-reactive antibody interactions with eye antigens may contribute to immunopathogenesis of Leptospira-associated recurrent uveitis.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. LruA and LruB-antiserum reacts with lenticular and retinal tissues.
(A) Photomicrographs showing uniform homogeneous fluorescence in a section of equine lens incubated with LruA-antiserum (1∶100) but not with preserum (B) (×100). (C) Photomicrographs showing a positive fluorescence in a frozen section of equine retina incubated with LruB-antiserum (1∶100) but not with pre-immunization serum (D) (×100). Inset (×400).
Figure 2
Figure 2. Two-dimensional electrophoretic analysis of proteins in equine lenticular and retinal tissue extracts.
(A) Lens extract separated on a polyacrylamide gel stained with the fluorescent dye SYPRO-Ruby. (B) Lens proteins transferred from a second gel to nitrocellulose membrane and blotted with LruA-antiserum. The arrowheads indicate the protein spots excised from the stained gel for analysis by mass spectrometry. (C) Retinal extract separated on a polyacrylamide gel stained with SYPRO-Ruby. (D) Retinal proteins transferred from a second gel to nitrocellulose membrane and blotted with LruB-antiserum. Three protein spots (numbered 3, 4 and 5) were excised from the stained gel for analysis by mass spectrometry. Results of mass spectrometric analyses are tabulated in Table 1.
Figure 3
Figure 3. Multisequence alignment of alpha-crystallin B of horse (Equus caballus), man (Homo sapiens), cow (Bos taurus), sheep (Ovis aries) and mouse (Mus musculus) using T-COFFEE Version 5.05 [http://www.tcoffee.org].
Figure 4
Figure 4. LruA antiserum reacts with recombinant human alpha-crystallin B and purified vimentin.
(A) Immunoblot showing reactivity of LruA-specific antiserum (1∶400) with recombinant human alpha-crystallin B (1µg). (B) Pre-immune serum (1∶400) did not react with this protein. (C) Immunoblots showing reactivity of LruA-antiserum (1∶400), but not the pre-immunization serum (D), with purified vimentin (1µg). Molecular mass markers are indicated in kilodaltons.
Figure 5
Figure 5. LruB antiserum reacts with human β-crystallin B2.
(A) Immunoblots showing reactivity of LruB-antiserum (1∶400) with recombinant human β-crystallin B2(1µg). (B) Pre-immune serum (1∶400) did not react with β-crystallin B2. Molecular mass of recombinant β-crystallin B2 is 50.4 kDa. Molecular mass markers are indicated in kilodaltons.
Figure 6
Figure 6. Uveitic eye fluids contain antibody to alpha-crystallin B, vimentin and β-crystallin B2.
ELISA results showing significantly higher antibody levels of alpha-crystallin B (A), vimentin (B) and β-crystallin B2-antibodies (C) in uveitic eye fluids. ELISA plate wells were coated with 200 ng of each eye protein. After blocking, wells were sequentially incubated with uveitic or normal eye fluids diluted 1∶100 and HRP-conjugated protein G (1∶5000). Plates were developed using 3,3′,5,5′-tetramethyl benzidine substrate solution and absorbance measured at 450 nm. Presented data is a representative of at least two independent experiments with 3 or more repeats. Error bars indicate standard deviation.

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