Recombinant antibody against Trypanosoma cruzi from patients with chronic Chagas heart disease recognizes mammalian nervous system

Abstract

Background: To deeply understand the role of antibodies in the context of Trypanosoma cruzi infection, we decided to characterize A2R1, a parasite antibody selected from single-chain variable fragment (scFv) phage display libraries constructed from B cells of chronic Chagas heart disease patients.

Methods: Immunoblot, ELISA, cytometry, immunofluorescence and immunohistochemical assays were used to characterize A2R1 reactivity. To identify the antibody target, we performed an immunoprecipitation and two-dimensional electrophoresis coupled to mass spectrometry and confirmed A2R1 specific interaction by producing the antigen in different expression systems. Based on these data, we carried out a comparative in silico analysis of the protein target´s orthologues, focusing mainly on post-translational modifications.

Findings: A2R1 recognizes a parasite protein of ~50 kDa present in all life cycle stages of T. cruzi, as well as in other members of the kinetoplastid family, showing a defined immunofluorescence labeling pattern consistent with the cytoskeleton. A2R1 binds to tubulin, but this interaction relies on its post-translational modifications. Interestingly, this antibody also targets mammalian tubulin only present in brain, staining in and around cell bodies of the human peripheral and central nervous system.

Interpretation: Our findings demonstrate for the first time the existence of a human antibody against T. cruzi tubulin capable of cross-reacting with a human neural protein. This work re-emphasizes the role of molecular mimicry between host and parasitic antigens in the development of pathological manifestations of T. cruzi infection.

Keywords: Chagas disease; Digestive system; Molecular mimicry; Phage-display; Post-translational modification; Tubulin.

Fig. 1

Features of anti-T. cruzi A2R1 antibody isolated from scFv immune phage display libraries constructed from B cells of patients with cChHD. (a) Agarose gel electrophoresis (2%) of PCR amplified products of heavy chain (VH) and light chain (VL) genes and A2R1 gene. Fragment sizes are indicated on the left. (b) Western-blot analysis of periplasmic expression of scFv A2R1. Molecular weight markers (in kDa) are shown on the left. (c) Comparison of VH and VL sequences of scFv A2R1 with their respective human germline sequences according IMGT. FWR: framework; CDR: complementarity determining region. (d) Western-blot of total lysates from T. cruzi and its different stages with scFv A2R1. Total cell lysates (30 µg) of amastigote (AM), trypomastigote (TR) and epimastigote (EP) were loaded in each lane and processed by immunoblotting. Molecular weight markers (in kDa) are shown on the left. (e) Immunofluorescence of different stages of T. cruzi incubated with scFv A2R1 (green) and counterstained with DAPI (nucleic acid stain, blue). The third and fourth columns show the merged and bright field images of the parasites, respectively. Scale bar: 0.5 μm.

Fig. 2

Identification of A2R1´s target antigen. (a) Parasite lysate was immunoprecipitated with chim mA2R1 covalently coupled to NHS-Activated magnetic beads or beads only (negative control). Bound proteins were eluted and subjected to SDS-PAGE (12%), followed by protein staining using colloidal Coomassie Blue G-250. The arrow indicates the band excised and subjected to LC–MS/MS analysis. (b) The 2-D gel of T. cruzi flagellar proteins was stained with colloidal Coomassie Blue G-250 (left panel) or transferred to nitrocellulose and revealed with scFv A2R1 (right panel). The arrows denote the spots analysed by MALDI-TOF. Molecular weight markers (in kDa) are indicated on the left.

Fig. 3

Reactivity of scFv A2R1 against tubulin expressed in L. tarentolae. Western-blot of samples corresponding to the supernatant of L. tarentolae expressing TcATUB (LtA; 20 µl), supernatant of wild type L. tarentolae (LtW; 20 µl) and total extracts from T. cruzi (Tc; 3.5 µg) or E. coli expressing TcATUB-GST (EcA; 2 µg). The nitrocellulose membranes were incubated with scFv A2R1 (a), anti-α tubulin (α-Tub) (b), anti-acetylated α-tubulin (Acetyl-α-Tub) antibodies (c) or sera from donor #1 and #2 (d). Molecular weight markers (in kDa) are indicated on the left.

Fig. 4

Co-localization of scFv A2R1 with anti-α/β tubulin antibodies in T. cruzi. (a) Confocal microscopy of T. cruzi epimastigotes parasites incubated with scFv A2R1 (green, first column) and rat anti-α tyrosinated (α-Tub) or mouse anti-β tubulin (β-Tub) antibodies (red, second column). The third column shows the overlay image. Right panel: Western-blot showing that both commercial antibodies recognize a protein of ~50 kDa presented in parasite lysate (30 µg/lane). (b) Detergent-extracted flagella from T. cruzi epimastigotes were co-stained with scFv A2R1 (green, first column), α- or β-tubulin antibodies (red, second column) together with DAPI (blue, third column). The fourth column shows the overlay image. Scale bar: 0.5 μm.

Fig. 5

Reactivity of scFv A2R1 with T. cruzi in infected cells. (a) Representative immunofluorescence of HeLa cells infected with T. cruzi and incubated with scFv A2R1 (green). DNA and actin filaments staining were performed with DAPI (blue) and phalloidin-rhodamine (red), respectively. Scale bar: 0.5 μm. (b) Immunochemistry assay of mouse skin tissue infected with T. cruzi amastigotes (Tulahuen strain). The arrow shows the scFv A2R1 signal. Upon DAB staining, sections were counterstained with haematoxylin. Scale bar: 50 μm.

Fig. 6

Reactivity of scFv A2R1 against mammalian tissues. (a) Binding of scFv A2R1 against rat (r) and mouse (m) organ tissue extracts or T. cruzi lysate (Tc) evaluated by ELISA (upper panel) and Western-blot (lower panel). The data are expressed as the OD_{450 nm} (means ± SD; n = 3). Differences between means of binding to different lysates vs to BSA were evaluated by one-way ANOVA followed by Tukey's multiple comparison tests. **** P < 0.0001; *** P < 0.001. For Immunoblotting, 30 µg of protein corresponding to different rat or mouse organ tissue lysate was loaded per lane. The expression of actin was used as a loading control. Molecular weight markers (in kDa) are indicated on the right. Wb: whole brain; Ce: cerebellum; Sc: spinal cord; Bm: bone-marrow; Ki: kidney; Sp: spleen; Lv: liver; He: heart; Lg: lung; Li: large intestine; Si: small intestine; Sm: skeletal muscle; Ln; lymph node; Ov: ovary; Ts: testis; H/C: hippocampus/cortex; Cx: cortex; Ob: olfactory bulb; Md: midbrain; Hs: hypothalamus; Sb: striated body; Pg: pituitary gland. (b) Competition ELISA performed with T. cruzi proteins coated-plates in the presence of increasing concentrations of mouse (m) and rat (r) organ tissue extracts. Results are expressed as mean percent inhibition values ± SD (n = 3). (c) Upper panel: Immunofluorescence of embryonic hypothalamus murine cells (cell line N43/5) using scFv A2R1 (green) and anti-β tubulin antibody (red). Scale bar: 0.5 μm. Lower panel: Representative histograms showing fixed-cells incubated with scFv A2R1 and mouse anti-VSV antibodies (gray line), with anti-VSV antibody only (solid gray) or unstained as negative control (solid blue).

Fig. 7

Immunohistochemical staining of human brain and peripheral nervous system. Representative images of paraffin-embedded sections of tissues from refractory epilepsy patients or healthy individuals challenged with scFv A2R1 and sera from donor patients. After deparaffinization, tissue sections were incubated with scFv A2R1 (a-h; j), serum from donor #2 (i) or from a non-infected donor (k). Tissue coverslips were developed with DAB as a substrate and counterstained with haematoxylin. Scale bars: 100 µm (left column); 200 µm (inset in right column). A: temporal lobe; B: frontal lobe; C: hippocampus; D: diencephalon; E: left colon; F: right colon; G: esophagus; H: cecal appendix; I: olfactory bulb; J: skin of murine chagoma provoked by T. cruzi Tulahuen strain (positive control); K: left colon (negative control). No staining pattern was observed with sera from other donors that were included to prepare the scFv libraries.

Fig. 8

Multiple sequence analysis of post-translational modification sites and C-terminal regions of tubulin from mammals and trypanosomatids. Sequence alignment corresponding to the PTM sites (+/- 2 amino acids) and C-terminal regions of α- and β-tubulins from mammalian isotype and trypanosomatid chains. Multiple sequences were aligned for the trypanosomatid α- (N = 100) and β- (N = 83) tubulins, but as the featured regions show 100% conservation, only a single sequence is being displayed. The amino acid positions are displayed below the aligned sequences.