Identification of a Neospora caninum microneme protein (NcMIC1) which interacts with sulfated host cell surface glycosaminoglycans

Abstract

The invasive stages of apicomplexan parasites enter their host cells through mechanisms which are largely conserved throughout the phylum. Host cell invasion is divided into two distinct events, namely, adhesion onto the host cell surface and the actual host cell entry process. The former is mediated largely through microneme proteins which are secreted at the onset of establishing contact with the host cell surface. Many of the microneme proteins identified so far contain adhesive domains. We here present the genomic and corresponding cDNA sequences coding for a 460-amino-acid (aa) microneme protein in Neospora caninum tachyzoites which, due to its homology to MIC1 in Toxoplasma gondii (TgMIC1), was named NcMIC1. The deduced NcMIC1 polypeptide sequence contains an N-terminal signal peptide of 20 aa followed by two tandemly internal repeats of 48 and 44 aa, respectively. Integrated into each repeat is a CXXXCG sequence motif reminiscent of the thrombospondin-related family of adhesive proteins. The positioning of this motif is strictly conserved in TgMIC1 and NcMIC1. The C-terminal part, comprised of 278 aa, was expressed in Escherichia coli, and antibodies affinity purified on recombinant NcMIC1 were used to confirm the localization within the micronemes by immunofluorescence and immunogold transmission electron microscopy of tachyzoites. Immunohistochemistry of mouse brains infected with tissue cysts showed that expression of this protein is reduced in the bradyzoite stage. Upon initiation of secretion by elevating the temperature to 37 degrees C, NcMIC1 is released into the medium supernatant. NcMIC1 binds to trypsinized, rounded Vero cells, as well as to Vero cell monolayers. Removal of glycosaminoglycans from the host cell surface and modulation of host cell surface glycosaminoglycan sulfation significantly reduces the binding of NcMIC1 to the host cell surface. Solid-phase binding assays employing defined glycosaminoglycans confirmed that NcMIC1 binds to sulfated glycosaminoglycans.

FIG. 1.

Schematic presentation of NcMIC1 and TgMIC1 genomic sequences. The numbers refer to the overall lengths and positions of the intron-exon splice sites. The positioning of the domains of high similarity (>70%) with respect to the nucleotide sequence is indicated with black bars.

FIG. 2.

Deduced polypeptide sequence of NcMIC1 and alignment with the homologous protein in T. gondii (TgMIC1). Hydrophobic signal peptide sequences are marked in italics. Internal repeats (TgRPT1/2 and NcRPT1/2) are underlined. Conserved cysteine residues are indicated with asterisks, potential N-glycosylation sites are marked with gggg, and CXXXCG motifs are boxed. The TSP-like domain (14) is underlined with a solid bar.

FIG. 3.

Immunoblots of N. caninum tachyzoite fractions obtained by extraction of parasites with the nonionic detergent Triton X-114. SDS-PAGE was carried out under reducing conditions with whole tachyzoite extract (lanes 1), Triton X-114-insoluble proteins (lanes 2), Triton X-114-soluble hydrophilic proteins (lanes 3), and hydrophobic detergent-soluble proteins (lanes 4). Lane 5, soluble hydrophilic proteins separated under nonreducing conditions; lane 6, E. coli extract expressing recNcMIC1 separated under reducing conditions. Immunoblots were stained with either anti-N. caninum antiserum or antibodies affinity purified on recNcMIC1. Numbers on the left indicate molecular masses in kilodaltons.

FIG. 4.

Immunofluorescent staining of N. caninum tachyzoites obtained from in vitro cultures (infected Vero cells [a] and isolated tachyzoites [b] and of an N. caninum tissue cyst in a paraffin-embedded brain tissue section obtained from an infected mouse (c to f). Panels a and b were labeled with anti-recNcMIC1 antibodies and fluorescein isothiocyanate-conjugated anti-rabbit immunoglobulin G, followed by staining with anti-N. caninum antiserum and tetramethyl rhodamine isocyanate-conjugated anti-rabbit immunoglobulin G. In panel a, nuclei are stained with Hoechst 23558. Panel c shows DNA staining of a tissue cyst using Hoechst 23558, panel d shows the absence of staining with anti-recNcMIC1 antibodies, and panel e shows that labeling with MAb CC2 is confined to the cyst wall. In panel f, the overlay is presented.

FIG. 5.

Immunogold TEM of LR-White-embedded N. caninum tachyzoites, either located extracellularly (a) or residing within the parasitophorous vacuole (b). Sections were stained with anti-recNcMIC1 antibodies and secondary goat anti-rabbit antibodies conjugated to 10-nm-diameter gold particles. Note preferential staining of the micronemes at the apical portion of the cells.

FIG. 6.

Immunoblots of proteins which are secreted from N. caninum tachyzoites. Tachyzoites were maintained at either 4 or 37°C for 10 min in EBSS. Western blots of secreted fractions were stained with anti-recNcMIC1 antibodies.

FIG. 7.

Immunoblots of coprecipitation assays using prefixed Vero cells incubated in the presence of Triton X-100-soluble extracts of N. caninum tachyzoites. Immunoblots were stained with anti-N. caninum antiserum, anti-recNcMIC1 antibodies, and anti-NcGRA7 antibodies. Lanes P (pellet), fraction cosedimenting with Vero cells; lanes SN (supernatant), nonbound proteins.

FIG. 8.

(a) Binding of live N. caninum tachyzoites and NcMIC1 to Vero cell monolayers. Untreated, NaClO₃-treated, and β-xyloside-treated Vero cell monolayers were incubated with freshly purified N. caninum tachyzoites or with the secreted fraction of N. caninum tachyzoites. Bound tachyzoites were detected using anti -N. caninum antiserum, and bound NcMIC1 was detected using anti-recNcMIC1 antibodies. Note the reduction in parasite adherence as well as NcMIC1 binding to Vero cells following treatments which affect the host cell surface glycosaminoglycan composition. OD, optical density; Trt, treated; Con, control. Error bars indicate standard deviations. (b) Binding of NcMIC1 to defined, solid-phase-bound glycosaminoglycans. Secreted fractions were incubated with heparin (Hep), dextran (Dex), dextran sulfate (Dexsul), and chondroitin sulfates A, B, and C (CSA, CSB, and CSC, respectively). Bound NcMIC1 was detected using anti-recNcMIC1 antibodies. Note the preferential binding of NcMIC to the highly sulfated glycosaminoglycan dextran sulfate, but only low binding is detected with dextran and chondroitin sulfate A.