EhNPC1 and EhNPC2 Proteins Participate in Trafficking of Exogenous Cholesterol in <i>Entamoeba histolytica</i> Trophozoites: Relevance for Phagocytosis

<div><p><i>Entamoeba histolytica</i>, the highly phagocytic protozoan causative of human amoebiasis lacks the machinery to synthesize cholesterol. Here, we investigated the presence of NPC1 and NPC2 proteins in this parasite, which are involved in cholesterol trafficking in mammals. Bioinformatics analysis revealed one <i>Ehnpc1</i> and two <i>Ehnpc2</i> genes. EhNPC1 appeared as a transmembrane protein and both EhNPC2 as peripheral membrane proteins. Molecular docking predicted that EhNPC1 and EhNPC2 bind cholesterol and interact with each other. Genes and proteins were identified in trophozoites. Serum pulse-chase and confocal microscopy assays unveiled that after trophozoites sensed the cholesterol source, EhNPC1 and EhNPC2 were organized around the plasma membrane in a punctuated pattern. Vesicles emerged and increased in number and size and some appeared full of cholesterol with EhNPC1 or EhNPC2 facing the extracellular space. Both proteins, but mostly EhNPC2, were found out of the cell associated with cholesterol. EhNPC1 and cholesterol formed networks from the plasma membrane to the nucleus. EhNPC2 appeared in erythrocytes that were being ingested by trophozoites, co-localizing with cholesterol of erythrocytes, whereas EhNPC1 surrounded the phagocytic cup. EhNPC1 and EhNPC2 co-localized with EhSERCA in the endoplasmic reticulum and with lysobisphosphatidic acid and EhADH (an Alix protein) in phagolysosomes. Immunoprecipitation assays confirmed the EhNPC1 and EhNPC2 association with cholesterol, EhRab7A and EhADH. Serum starved and blockage of cholesterol trafficking caused a low rate of phagocytosis and incapability of trophozoites to produce damage in the mouse colon. <i>Ehnpc1</i> and <i>Ehnpc2</i> knockdown provoked in trophozoites a lower intracellular cholesterol concentration and a diminished rate of phagocytosis; and <i>Ehnpc1</i> silencing also produced a decrease of trophozoites movement. Trafficking of EhNPC1 and EhNPC2 during cholesterol uptake and phagocytosis as well as their association with molecules involved in endocytosis strongly suggest that these proteins play a key role in cholesterol uptake.</p></div

3D structures of NTD-EhNPC1, EhNPC2a and EhNPC2b and molecular dockings between them and with cholesterol.

<p>(A) NTD-EhNPC1 model (1 to 250 amino acids) predicted by RaptorX server was compared with the crystal of human NTD-NPC1 protein (HsNPC1) (23 to 254 amino acids). (B) Docking simulation of EhNPC1 with cholesterol performed using the AutoDock Tools V1.5.6 program. SSD: sterol sensing domain. NTD: amino terminal domain. (C, D) The EhNPC2a and EhNPC2b 3D structures (full-length amino acid sequences) were compared between them and with the NPC2 <i>B</i>. <i>taurus</i> crystal (BtNPC2). (E) Docking simulation of EhNPC2a and EhNPC2b with cholesterol. Arrows: amino acids tail at the amino terminus that it is not present in the crystal of <i>Btaurus</i> NPC2. Squares at the right in B and E show the amino acids involved in the protein-cholesterol interaction. (F) Docking simulation of EhNPC1 and EhNPC2a binding. ΔG: binding energy. Blue lines in dockings: plasmatic membrane. NT: amino terminus. CT: carboxy terminus.</p

Erythrophagocytosis of trophozoites cultured in TYI-S, TYI and TYI plus U18.

<p>Trophozoites were incubated ON at 37°C in TYI-S, TYI and TYI plus U18. (A) Cholesterol concentration in the respective trophozoites was measured as described in material and methods. (B) Laser confocal microscopy showing the morphology and the cholesterol localization by filipin staining. Nuclei were counterstained with propidium iodide (pi). Images were false colored to obtain a better contrast. Ph c: phase contrast images, arrows: cytoplasmic dots. (C) Rate of erythrophagocytosis spectrophotometrically measured by hemoglobin (Hb) concentration inside trophozoites.</p

Expression and localization of EhNPC1 and EhNPC2 in trophozoites.

<p>(A) <i>Ehnpc1</i>, <i>Ehnpc2a</i> and <i>Ehnpc2b</i> full-length genes were PCR amplified using specific primers and genomic DNA. (B) RT-PCR amplification of transcript fragments using specific primers and cDNA. c: Controls without gDNA or with mRNA as template. (C) The relative expression of the three genes was measured by RT-qPCR in trophozoites, using as a control the <i>40s ribosomal</i> S2 protein gene. ** p<0.01. (D, E) Total extracts of <i>E</i>. <i>histolytica</i> were separated by 10% SDS-PAGE and analyzed by western blot assays using pre-immune serum (PS) or rabbit α-EhNPC1 (D) or rat α-EhNPC2 (E) antibodies. (F) Representative images of laser confocal microscopy of PFA-fixed trophozoites using rabbit α-EhNPC1 or rat α-EhNPC2 or mouse α-Gal/GalNAc lectin antibodies. (G) Controls using only secondary antibodies. Ph c: phase contrast images. (H) Protein localization of EhNPC1 and EhNPC2 in plasma membrane (pm), cytoplasm (c) or nucleus (n). Counts were performed in 50 cells. (I) Pearson coefficient (PC) correlation measured in at least 15 confocal images, indicating co-localization of EhNPC1 or EhNPC2 with Gal/GalNAc lectin in the entire cell and in the plasma membrane. Laser sections = 0.5 μm.</p

Protein Phosphorylation in Serine Residues Correlates with Progression from Precancerous Lesions to Cervical Cancer in Mexican Patients

Protein phosphorylation is a posttranslational modification that is essential for normal cellular processes; however, abnormal phosphorylation is one of the prime causes for alteration of many structural, functional, and regulatory proteins in disease conditions. In cancer, changes in the states of protein phosphorylation in tyrosine residues have been more studied than phosphorylation in threonine or serine residues, which also undergo alterations with greater predominance. In general, serine phosphorylation leads to the formation of multimolecular signaling complexes that regulate diverse biological processes, but in pathological conditions such as tumorigenesis, anomalous phosphorylation may result in the deregulation of some signaling pathways. Cervical cancer (CC), the main neoplasm associated with human papillomavirus (HPV) infection, is the fourth most frequent cancer worldwide. Persistent infection of the cervix with high-risk human papillomaviruses produces precancerous lesions starting with low-grade squamous intraepithelial lesions (LSIL), progressing to high-grade squamous intraepithelial lesions (HSIL) until CC is generated. Here, we compared the proteomic profile of phosphorylated proteins in serine residues from healthy, LSIL, HSIL, and CC samples. Our data show an increase in the number of phosphorylated proteins in serine residues as the grade of injury rises. These results provide a support for future studies focused on phosphorylated proteins and their possible correlation with the progression of cervical lesions

Localization of EhNPC1 and EhNPC2 in trophozoites analyzed by TEM.

<p>(A) Thin sections of trophozoites were incubated with rabbit α-EhNPC1 and rat α-EhNPC2 antibodies, followed by incubation with gold labeled α-rabbit and α-rat secondary antibodies (20 and 10 nm gold particles, respectively). Squares indicate the magnified areas marked with the corresponding lower case letters. pm: plasma membrane, vl: vesicle lumen, vm: vesicle membrane, es: extracellular space, n: nucleus. (B, C) Controls using only secondary antibodies. (D) Graph showing number of EhNPC1 and EhNPC2 molecules recognized by the respective gold-labeled antibodies and their co-localization.</p

Working model of the EhNPC1 and EhNPC2 participation in cholesterol trafficking in <i>E</i>. <i>histolytica</i>.

<p>(A) Cholesterol uptake: EhNPC1 and EhNPC2 capture the cholesterol in the extracellular space and in the plasma membrane probably following the “hand-off” model (EhNPC1-cholesterol-EhNPC2-cholesterol-EhNPC1) and with the TMK39 participation. (B) Network-like structures formation: EhNPC1 and cholesterol form networks from cholesterol-containing membrane protrusions to ER, endosomes and nucleus that possibly facilitate cholesterol trafficking through EhNPC2 in a “hand-off” model. (C) Cholesterol influx: EhNPC1 and EhNPC2 associate with cholesterol and are internalized in EhRab-7A containing endosomes under basal conditions and during erythrophagocytosis. (D) EhNPC1 and EhNPC2 associate with phagolysosomal and MBVs molecules, particularly with LBPA and EhADH, which probably regulate cholesterol trafficking. (E) EhNPC1 and EhNPC2 accumulate in nucleus (n) and ER and may be distributed from there to other organelles.</p

Localization of EhNPC1 and EhNPC2 in trophozoites after an ABS pulse.

<p>Trophozoites were serum starved for 12 h by culturing in TYI medium on coverslips. Then, ABS was added as a cholesterol source at 37°C for 0.5 to 7 min. Cells were washed, fixed and incubated with filipin, rabbit α-EhNPC1 and rat α-EhNPC2 antibodies, secondary antibodies, and examined by laser confocal microscopy. Arrows: cytoplasmic dots. Squares marked with lower case letters are magnified at the right. (a-d) Membrane vacuoles containing EhNPC1 and EhNPC2. Arrows: show the plasma membrane with EhNPC2 or EhNPC1 facing the extracellular space. (e) Networks stained by α-EhNPC1 antibody. (e-j) rod and spherical structures (arrowheads) facing the extracellular space, stained mainly by α-EhNPC2 antibody. (k-m) Spherical structures of 1 to 2 μm (empty arrowheads) inside the cell. n: nucleus, Ph c: phase contrast images, nw: networks like structures.</p

Co-localization of EhNPC1 and EhNPC2 with EhSERCA and EhRab7A.

<p>Trophozoites were incubated with erythrocytes at 37°C and treated as <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006089#ppat.1006089.g007" target="_blank">Fig 7</a>. Samples were processed for confocal microscopy using α-EhNPC1, α-EhNPC2 and (A) α-EhSERCA or (C) α-EhRab7A antibodies. (a-e) Magnification of the white squares. (B, D, E) PC of the co-localization between EhNPC1 or EhNPC2 with EhSERCA (B), or with EhRab7A in the whole cell (D) or in cellular structures stained by the three antibodies (E).</p