Mapping of SAP genes in the chromosomal bands of <i>T. cruzi</i>.

<p>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. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083864#pone.0083864-Cano1" target="_blank">[21]</a> is shown on the left in Roman numerals.</p

Expression of SAP proteins in the developmental forms of the <i>T. cruzi</i> CL strain.

<p>(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 <i>T. cruzi</i> 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.</p

Cell adhesion and lysosome exocytosis-inducing properties of SAP-CE associated with <i>T. cruzi</i> metacyclic trypomastigote internalization.

<p>(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 <i>o</i>-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.</p

Release of SAP proteins into the extracellular medium.

<p>(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. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083864#pone.0083864-BayerSantos1" target="_blank">[14]</a>. 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.</p

SAP transcripts isolated from epimastigotes, metacyclic trypomastigotes and extracellular amastigotes of the <i>T. cruzi</i> CL strain by RT-PCR amplification.

<p>1) Accession number according to the TriTrypDB database.</p><p>(2) Localization of SAP genes based on the 41 chromosome-sized scaffolds <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083864#pone.0083864-Weatherly1" target="_blank">[25]</a>.</p><p>(3) SAP transcripts shared by epimastigotes, metacyclic trypomastigotes and extracellular amastigotes.</p><p>(4) The accession numbers represent the three copies of the same gene.</p

SAP proteins secreted into the extracellular medium by epimastigotes and metacyclic trypomastigotes (clone Dm28c) identified by mass spectrometry.

<p>(1) Accession number according to the TriTrypDB database.</p><p>(2 and 3) To validate the quality of protein identification, the following parameters were used: (2) Xcorr (CrossCorr/avg [AutoCorr offset = -75 to 75]) ≥1.5, 2.0, and 2.5, for singly, doubly and triply charged peptides, respectively. (3) DC<i>n</i> (Xcorr₁ – Xcorr₂/Xcorr₁) ≥0.1.</p><p>(4) Sample enriched in plasma membrane-derived vesicles/ectosomes.</p><p>(5) Sample enriched in exosomes.</p><p>(6) Sample enriched in soluble proteins (vesicle-free, VF).</p

Cellular distribution of SAP proteins in the developmental forms of the <i>T. cruzi</i> CL strain.

<p>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.</p

ESI-MS analysis of cell wall neutral glycolipids from Pb3 and Pb18 cultivated in the presence (pl) or absence of plasma.

<p>A, Full-scan spectra in the positive-ion mode show the identified glycolipids Hex-C16∶0-OH/d19∶2-Cer (<i>m/z</i> 848.7), Hex-C18∶0-OH/d18∶2-Cer (<i>m/z</i> 862.8), Hex-C18∶0-OH/d18∶1-Cer (<i>m/z</i> 864.9), Hex-C18∶1-OH/d19∶2-Cer (<i>m/z</i> 874.9), and Hex-C18∶0-OH/d19∶2-Cer (<i>m/z</i> 876.9). B, Tandem-MS spectrum of the major glycolipid species identified (<i>m/z</i> 876.9). <i>m/z</i>, mass to charge ratio.</p

Composition and relative abundance of major phospholipids from Pb3 and Pb18 cell wall identified by ESI-MS total-ion mapping.

<p>Only species with normalized intensity higher than 0.2 in at least one isolate is shown. ^a Normalized to the intensity of the internal standards C12∶0/C12∶0-PG (at <i>m/z</i> 609.5) and C11∶0/C11∶0-PC (at <i>m/z</i> 616.5) in the negative and positive–ion modes, respectively. ^b ND, not detected. ^c TR, trace amounts.</p

GC-MS analysis of cell wall phospholipid fatty acids from Pb3 and Pb18 cultivated in the presence (pl) or absence of plasma.

<p>Fatty acid abundance was normalized to the C16∶0 peak area.</p