Figure 1.
Mapping of SAP genes in the chromosomal bands of T. cruzi.
Chromosomal bands of clone CL Brener and the CL strain were separated by PFGE, stained with ethidium bromide, transferred to nylon membranes and hybridized with the SAP-CD 32P-labeled 513 bp fragment. The sizes of the chromosomal bands are shown in Mb on the right, and the chromosomal-band nomenclature described by Cano et al. [21] is shown on the left in Roman numerals.
Table 1.
SAP transcripts isolated from epimastigotes, metacyclic trypomastigotes and extracellular amastigotes of the T. cruzi CL strain by RT-PCR amplification.
Figure 2.
Expression of SAP proteins in the developmental forms of the T. cruzi CL strain.
(A) The levels of SAP transcripts in epimastigotes (Epi) and metacyclic trypomastigotes (Meta) were estimated by qRT-PCR using primers that amplified a conserved 135 bp fragment shared by all SAP genes. The values, which were calculated after normalization with GAPDH transcripts and using epimastigotes as the reference sample (SAP/GAPDH ratio = 1), are the means ± standard deviations of four independent experiments performed in triplicate. The difference between epimastigotes and metacyclic trypomastigotes was significant (*p<0.0001). (B) SAP expression was determined by quantitative western blot using total protein extracts from epimastigotes and metacyclic trypomastigotes (15 µg protein/lane) reacted with MAb-SAP (diluted 1∶100). As loading control, α-tubulin was used. (C) Difference in size of SAP variants expressed in the different T. cruzi developmental forms. Total protein extracts from epimastigotes (3.0××107 cells), metacyclic trypomastigotes (1.0×108 cells), extracellular amastigotes (3.0×107 cells) and tissue culture-derived trypomastigotes (1.0×108 cells) were separated by SDS-PAGE, transferred to nitrocellulose membranes and incubated with MAb-SAP (diluted 1∶100). The relative molecular masses (kDa) of the immunoreactive proteins are shown on the right.
Figure 3.
Cellular distribution of SAP proteins in the developmental forms of the T. cruzi CL strain.
Epimastigotes, metacyclic trypomastigotes, extracellular amastigotes and tissue culture-derived trypomastigotes were fixed with 4% paraformaldehyde, permeabilized with saponin and incubated with anti-SAP polyclonal antibodies diluted 1∶50, followed by incubation with Alexa Fluor 488-conjugated anti-rabbit IgG (green). The figure also shows the colocalization of anti-SAP with concanavalin_A in epimastigotes (red) and with MAb-2C2 in extracellular amastigotes (red). Parasite DNA was stained with DAPI (blue). Scale bar, 5 µm.
Figure 4.
Release of SAP proteins into the extracellular medium.
(A) Metacyclic trypomastigotes (CL strain) were incubated overnight in PBS at 28°C (1.0×108 parasites/mL). After centrifugation, the conditioned medium (CM) was filtered and analyzed by western blot using MAb-SAP (diluted 1∶100). (B) Epimastigotes and metacyclic trypomastigotes (Dm28c) were incubated for 6 h at 28°C in DMEM or TAU3AAG (1.0×108 parasites/mL), respectively. After centrifugation the conditioned medium was filtered and submitted to ultracentrifugation according to the protocol described by Bayer-Santos et al. [14]. Vesicles and soluble-protein fractions (2 μg of protein from each fraction) were analyzed by western blot using MAb-SAP (diluted 1∶100) or a monoclonal antibody against the flagellar calcium-binding protein (FCaBP). The relative molecular masses (kDa) of the immunoreactive proteins are shown on the right. V2, fraction enriched in plasma membrane-derived vesicles/ectosomes; V16, fraction enriched in exosomes; and VF, vesicle-free fraction enriched in soluble proteins.
Table 2.
SAP proteins secreted into the extracellular medium by epimastigotes and metacyclic trypomastigotes (clone Dm28c) identified by mass spectrometry.
Figure 5.
Cell adhesion and lysosome exocytosis-inducing properties of SAP-CE associated with T. cruzi metacyclic trypomastigote internalization.
(A) Increasing amounts of the purified recombinant protein SAP-CE or GST were added to 96-well plates covered with HeLa cells. After fixation and washes in PBS, the cells were incubated with MAb-SAP (diluted 1∶100) and with anti-mouse IgG peroxidase conjugate. The bound protein was revealed by o-phenylenediamine. Values are the means ± standard deviations of triplicates. (B) HeLa cells were incubated for 30 min with or without the recombinant protein SAP-CE or GST (40 μg/mL) and then incubated with metacyclic forms. After incubation for 1 h, cells were washed in PBS, fixed, and stained with Giemsa. The number of internalized parasites was counted in 500 cells. The values represent the means ± standard deviations of three independent experiments performed in duplicate. SAP-CE significantly inhibited parasite invasion (*p<0.05). (C) Semi-confluent HeLa cell monolayers were incubated in absence or in the presence of GST or the purified recombinant protein SAP-CE (20 μg/mL) for 60 min. The supernatant was collected and the release of β-hexosaminidase measured. Exocytosis was expressed as a percentage of the total β-hexosaminidase activity (supernatant + cell extract). Values are the means ± standard deviations of four independent experiments performed in duplicate. β-hexosaminidase activity was significantly higher in the presence of SAP-CE (*p<0.05). (D) HeLa cells were incubated with or without the purified recombinant protein SAP-CE (20 μg/mL) and processed for indirect immunofluorescence using anti-Lamp-2 antibody and Alexa Fluor 488-conjugated anti-mouse IgG (green), phalloidin-TRITC (red) for actin visualization and DAPI (blue) for DNA. Scale bar, 10 µm.