Internalized <i>E</i>. <i>granulosus</i> AgB colocalizes with protein endocytic markers in RH cells.

<p>Alexa Fluor 633-conjugated Tfn was used as a marker of clathrin-mediated endocytosis and Alexa Fluor 555-conjugated Ctx-B was used as a marker of caveolae/raft-mediated endocytosis. Confocal microscopy images of RH cells incubated with 40 μg/ml AgB and 1 μg/ml Ctx-B (A) or 50 μg/ml Tfn (B) are presented. AgB was detected using polyclonal antibodies against AgB8/1, 2 and 4 subunits and a secondary anti-rabbit IgG Alexa Fluor 488 conjugated antibody (green). Arrows indicate colocalization points. Cell nuclei were labeled with DAPI (blue). The endocytic markers are shown in red.</p

Antigen B from <i>Echinococcus granulosus</i> enters mammalian cells by endocytic pathways

<div><p>Background</p><p>Cystic hydatid disease is a zoonosis caused by the larval stage (hydatid) of <i>Echinococcus granulosus</i> (Cestoda, Taeniidae). The hydatid develops in the viscera of intermediate host as a unilocular structure filled by the hydatid fluid, which contains parasitic excretory/secretory products. The lipoprotein Antigen B (AgB) is the major component of <i>E</i>. <i>granulosus</i> metacestode hydatid fluid. Functionally, AgB has been implicated in immunomodulation and lipid transport. However, the mechanisms underlying AgB functions are not completely known.</p><p>Methodology/Principal findings</p><p>In this study, we investigated AgB interactions with different mammalian cell types and the pathways involved in its internalization. AgB uptake was observed in four different cell lines, NIH-3T3, A549, J774 and RH. Inhibition of caveolae/raft-mediated endocytosis causes about 50 and 69% decrease in AgB internalization by RH and A549 cells, respectively. Interestingly, AgB colocalized with the raft endocytic marker, but also showed a partial colocalization with the clathrin endocytic marker. Finally, AgB colocalized with an endolysosomal tracker, providing evidence for a possible AgB destination after endocytosis.</p><p>Conclusions/Significance</p><p>The results indicate that caveolae/raft-mediated endocytosis is the main route to AgB internalization, and that a clathrin-mediated entry may also occur at a lower frequency. A possible fate for AgB after endocytosis seems to be the endolysosomal system. Cellular internalization and further access to subcellular compartments could be a requirement for AgB functions as a lipid carrier and/or immunomodulatory molecule, contributing to create a more permissive microenvironment to metacestode development and survival.</p></div

<i>E</i>. <i>granulosus</i> AgB uptake by mammalian cells in culture.

<p>Immunofluorescence assay was performed on NIH-3T3, A549, J774 and RH cells exposed to 40 μg/ml AgB for 4 h, and mock treated cells (Control). AgB was labeled with polyclonal antibodies against AgB8/1, 2 and 4 subunits and an Alexa Fluor 488-conjugated secondary antibody (green). Nuclei and cytoskeleton were stained with DAPI (blue) and Alexa Fluor 594-conjugated phalloidin (red), respectively. Images are median optical sections from z-stacks obtained by confocal microscopy. Inserts correspond to two-fold digital magnification of boxed areas. Arrows indicate vesicular-like distribution of internalized AgB. Scale bar, 10 μm.</p

Caveolae/raft-mediated endocytosis is the main route involved in <i>E</i>. <i>granulosus</i> AgB internalization by A549 and RH cells.

<p>Indicated cells were incubated in medium alone (Ctrl), with 100 μg/ml genistein (GNT), or with 5 μg/ml chlorpromazine (CPZ) for 30 min and then exposed to 40 μg/ml AgB for 1.5 h. AgB was detected using anti-AgB polyclonal antibodies followed by anti-rabbit IgG Alexa Fluor 488 conjugated antibody (green). Cell nuclei were labelled with DAPI (blue). <i>A</i> and <i>B</i>, Inhibition of caveolae/raft-mediated endocytosis by genistein reduces AgB internalization. <i>C</i> and <i>D</i>, inhibition of clathrin-mediated endocytosis pathway by chlorpromazine does not cause a significant decrease in uptake of AgB. The quantitative data presented in B and D are measurements from three experiments with RH cells and two with A549 cells. Arbitrary units correspond to immunofluorescence intensity calculated as total immunofluorescence in the cell divided by the area of the cell. Error bars indicate SEM. *p = 0.037, ***p = 0.0004 according to Student’s t-test.</p

MTT reduction assay.

<p>A549 (black bars) and RH (gray bars) cells were treated for 24 h with the indicated concentrations of <i>E</i>. <i>granulosus</i> AgB. Cell viability is expressed as percentage of MTT reduction measured for untreated cells and assumed as 100% (horizontal dashed line). Error bars correspond to the SEM values of five independent experiments.</p

<i>E</i>. <i>granulsous</i> AgB reaches the endolysosomal system after endocytosis by RH cells.

<p>DiI-labelled AgB (red) at 40 μg/ml and Lysosensor DND-189 (green) at 1.5 μM were added to the cell culture media and left incubate for 1.5 h. Median sections from z-stacks of confocal images are shown. Arrows indicate colocalization points.</p

Differentially expressed proteins.

<p>The proteins that were identified as exclusive in at least two replicates or the proteins that were differentially expressed with significant T-Test values (<i>p</i>≤0.05) are presented.</p><p>^aFunctional classification determined by eggNOG.</p><p>^<i>b</i>Protein accession numbers according to GeneDB (<a href="http://www.genedb.org/" target="_blank">www.genedb.org/</a>).</p><p>^<i>c</i>Protein accession numbers according to NCBI (<a href="http://www.ncbi.nlm.nih.gov/" target="_blank">www.ncbi.nlm.nih.gov/</a>).</p><p>^dAverage NSAF for SSD replicates.</p><p>^eAverage NSAF for NSD replicates.</p><p>Differentially expressed proteins.</p

Comparative analysis of NSPs from <i>E</i>. <i>granulosus</i> PSCs after the induction of strobilar development.

<p>(A) Functional categories of total identified NSPs. Percentages of identified proteins in each functional category are indicated. (O) Post-translational modification, protein turnover, and chaperones; (C) Energy production and conversion; (Z) Cytoskeleton; (U) Intracellular trafficking, secretion, and vesicular transport; (S) Function unknown; (K) Transcription; (J) Translation, ribosomal structure and biogenesis; (A) RNA processing and modification; (F) Nucleotide transport and metabolism; (E) Amino acid transport and metabolism; (T) Signal transduction mechanisms; (D) Cell cycle control, cell division, chromosome partitioning; (G) Carbohydrate transport and metabolism; (Q) Secondary metabolites biosynthesis, transport, and catabolism; (L) Replication, recombination and repair; (P) Inorganic ion transport and metabolism; (M) Cell wall/membrane/envelope biogenesis. The distribution of level 3 biological processes for SSD and NSD-exclusive and up-regulated proteins. (B) A heat map from NSPs with high (red) or low (green) expression levels between the SSD and NSD groups.</p

Detection of NSPs from <i>in vitro</i> cultured <i>E</i>. <i>granulosus</i> PSCs.

<p>The coomassie-stained proteins and UV detected TAMRA-labeled NSPs from the PSCs incubated for 72 h in the presence (A and B) or absence (C and D) of AHA, respectively.</p

<i>In toto</i> visualization of proteins synthesized by <i>E</i>. <i>granulosus</i> PSCs induced to strobilar development.

<p>(A) NSPs were specifically visualized in the presence of AHA and Alexa Fluor 488 Alkyne. The other images represent the DAPI nuclei staining, the bright field merges of DAPI and Alexa Fluor 488 antibody staining and the bright field images. (B) AHA+/Alexa-, (C) AHA-/Alexa+ and (D) AHA-/Alexa- did not show significant fluorescence or autofluorescence (400x). (E) The quantification of NSP (Alexa Fluor 488) and nucleic acid regions (DAPI) fluorescence. a.u., arbitrary units.</p