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EhRabB mobilises the EhCPADH complex through the actin cytoskeleton during phagocytosis of Entamoeba histolytica

2019; Wiley; Volume: 21; Issue: 10 Linguagem: Inglês

10.1111/cmi.13071

1462-5822

Rosario Javier‐Reyna, Sarita Montaño, Guillermina Garcı́a-Rivera, Mario A. Rodríguez, Arturo González‐Robles, Esther Orozco,

Heme Oxygenase-1 and Carbon Monoxide

Cellular MicrobiologyVolume 21, Issue 10 e13071 RESEARCH ARTICLEFree Access EhRabB mobilises the EhCPADH complex through the actin cytoskeleton during phagocytosis of Entamoeba histolytica Rosario Javier-Reyna, Rosario Javier-Reyna orcid.org/0000-0003-0724-7535 Departamento de Infectómica y Patogénesis Molecular, CINVESTAV-IPN, Mexico City, MexicoSearch for more papers by this authorSarita Montaño, Sarita Montaño orcid.org/0000-0002-0420-4268 Laboratorio de Bioinformática, Facultad de Ciencias Químico-Biológicas, Universidad Autónoma de Sinaloa (FCQB-UAS), Culiacán, Sinaloa, MéxicoSearch for more papers by this authorGuillermina García-Rivera, Guillermina García-Rivera orcid.org/0000-0002-1831-1806 Departamento de Infectómica y Patogénesis Molecular, CINVESTAV-IPN, Mexico City, MexicoSearch for more papers by this authorMario Alberto Rodríguez, Mario Alberto Rodríguez orcid.org/0000-0003-3998-9779 Departamento de Infectómica y Patogénesis Molecular, CINVESTAV-IPN, Mexico City, MexicoSearch for more papers by this authorArturo González-Robles, Arturo González-Robles orcid.org/0000-0002-3749-3756 Departamento de Infectómica y Patogénesis Molecular, CINVESTAV-IPN, Mexico City, MexicoSearch for more papers by this authorEsther Orozco, Corresponding Author Esther Orozco esther@cinvestav.mx orcid.org/0000-0002-8360-2968 Departamento de Infectómica y Patogénesis Molecular, CINVESTAV-IPN, Mexico City, Mexico Correspondence Esther Orozco, Departamento de Infectómica y Patogénesis Molecular, CINVESTAV-IPN, Av. IPN 2508, San Pedro Zacatenco, Mexico City, Mexico. Email: esther@cinvestav.mxSearch for more papers by this author Rosario Javier-Reyna, Rosario Javier-Reyna orcid.org/0000-0003-0724-7535 Departamento de Infectómica y Patogénesis Molecular, CINVESTAV-IPN, Mexico City, MexicoSearch for more papers by this authorSarita Montaño, Sarita Montaño orcid.org/0000-0002-0420-4268 Laboratorio de Bioinformática, Facultad de Ciencias Químico-Biológicas, Universidad Autónoma de Sinaloa (FCQB-UAS), Culiacán, Sinaloa, MéxicoSearch for more papers by this authorGuillermina García-Rivera, Guillermina García-Rivera orcid.org/0000-0002-1831-1806 Departamento de Infectómica y Patogénesis Molecular, CINVESTAV-IPN, Mexico City, MexicoSearch for more papers by this authorMario Alberto Rodríguez, Mario Alberto Rodríguez orcid.org/0000-0003-3998-9779 Departamento de Infectómica y Patogénesis Molecular, CINVESTAV-IPN, Mexico City, MexicoSearch for more papers by this authorArturo González-Robles, Arturo González-Robles orcid.org/0000-0002-3749-3756 Departamento de Infectómica y Patogénesis Molecular, CINVESTAV-IPN, Mexico City, MexicoSearch for more papers by this authorEsther Orozco, Corresponding Author Esther Orozco esther@cinvestav.mx orcid.org/0000-0002-8360-2968 Departamento de Infectómica y Patogénesis Molecular, CINVESTAV-IPN, Mexico City, Mexico Correspondence Esther Orozco, Departamento de Infectómica y Patogénesis Molecular, CINVESTAV-IPN, Av. IPN 2508, San Pedro Zacatenco, Mexico City, Mexico. Email: esther@cinvestav.mxSearch for more papers by this author First published: 20 June 2019 https://doi.org/10.1111/cmi.13071Citations: 7AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract Movement and phagocytosis are clue events in colonisation and invasion of tissues by Entamoeba histolytica, the protozoan causative of human amoebiasis. During phagocytosis, EhRab proteins interact with other functional molecules, conducting them to the precise cellular site. The gene encoding EhrabB is located in the complementary chain of the DNA fragment containing Ehcp112 and Ehadh genes, which encode for the proteins of the EhCPADH complex, involved in phagocytosis. This particular genetic organisation suggests that the three corresponding proteins may be functionally related. Here, we studied the relationship of EhRabB with EhCPADH and actin during phagocytosis. First, we obtained the EhRabB 3D structure to carry out docking analysis to predict the interaction sites involved in the EhRabB protein and the EhCPADH complex contact. By confocal microscopy, transmission electron microscopy, and immunoprecipitation assays, we revealed the interaction among these proteins when they move through different vesicles formed during phagocytosis. The role of the actin cytoskeleton in this event was also confirmed using Latrunculin A to interfere with actin polymerisation. This affected the movement of EhRabB and EhCPADH, as well as the rate of phagocytosis. Mutant trophozoites, silenced in EhrabB gene, evidenced the interaction of this molecule with EhCPADH and strengthened the role of actin during erythrophagocytosis. 1 INTRODUCTION Entamoeba histolytica is the protozoan causative of intestinal amoebiasis and hepatic abscess in humans. It infects around 50 million people worldwide and provokes approximately 100,000 deaths per year (W.H.O., 1997). Amoebiasis is the third most common cause of death due to parasitic diseases, after malaria and schistosomiasis (Gunther, Shafir, Bristow, & Sorvillo, 2011; Verma, Srivastava, & Datta, 2018). This parasite has two phases in its life cycle: the cysts, the infective and resistant form, and the trophozoites, the invasive form. Trophozoites are motile and very fragile cells to the environmental conditions. Trophozoites constitutively generate pseudopodia, necessary for movement. Permanently, they form vesicles, involved in internal trafficking, secretion, and endocytosis. Three cellular processes are considered essential for the parasite virulence: adherence to target cell, target cell destruction, and phagocytosis. Phagocytosis is one of the main functions expressed during destruction of the epithelia by the trophozoites. Membrane proteins participating in these events have been already identified, among them the Gal/GalNac lectin (Petri, Haque, & Mann, 2002), the EhCPADH complex (Garcia-Rivera et al., 1999), the KERP1 protein (Perdomo et al., 2016), and others (Garcia-Rivera et al., 1999; Laughlin & Temesvari, 2005; Pillai & Kain, 2005). EhCPADH is formed by the proteins EhCP112 (446 amino acids) and EhADH (687 amino acids), which belongs to the ALIX family. EhADH binds to EhCP112 by the Bro1 domain (9–349 residues), which makes contact with the region located at 442 to 479 residues of the protease (Montano et al., 2017). Once trophozoites have adhered, the bombardment of molecules to the target cells is speeded, giving as a result the appearance of pores in target membrane (Leippe, 1997). At the same time, in the trophozoites, the fission and fusion of vesicles is accelerated to initiate the sorting of ingested molecules for recycling or digestion. The EhCPADH complex is involved in vesicle trafficking. As in other eukaryotes, in trophozoites, cytoskeleton is fundamental for vesicle transport, cell structure, and shape maintenance and motility (Franker & Hoogenraad, 2013; Kjos, Vestre, Guadagno, Borg Distefano, & Progida, 2018; Tolic-Norrelykke, 2008). In this concerted movement, EhRab-GTPase proteins transport functional molecules to different cellular compartments (Javier-Reyna et al., 2012; Mitra, Saito-Nakano, Nakada-Tsukui, Sato, & Nozaki, 2007). In eukaryotes, Rab GTPases modulate the vesicular transport through the cytoskeleton (Franker & Hoogenraad, 2013; Kjos et al., 2018; Tolic-Norrelykke, 2008). They are molecular switches that present conformational changes associated with the GTP/GDP-binding cycle. Active Rab-GTP joins to effector proteins, whereas, inactive Rab-GDP has low affinity for they (Kotyada et al., 2018; Verma et al., 2018). Saito-Nakano, Loftus, Hall, and Nozaki (2005) identified 91 putative Rab proteins in E. histolytica (EhRab). All of them conserve the GTP-binding consensus sequences (GDXXVGKT, DTAGQE, GNKXD, and SAK; Pereira-Leal & Seabra, 2000). According to in silico analyses, these authors found that 22 of 91 EhRab exhibited more than 40% sequence identity to yeast and human Rabs. The remaining 69 EhRabs display low homology with Rabs of other organisms, but 30 of them show more than 40% identity to one or more of these proteins. The other 39 (EhRabA, EhRabB, EhRabH, and EhRabX1-36) displayed low homology to Rab from other organisms or to any other member of EhRab family (Saito-Nakano et al., 2005). EhRabB (192 amino acids) participates in phagocytosis and may be involved in the regulation of vesicle docking and fusion (Juarez-Hernandez et al., 2013; Rodriguez et al., 2000). The EhrabB gene is located in the complementary chain of the DNA fragment containing the Ehcp112 and Ehadh genes (Juarez-Hernandez et al., 2013; Rodriguez et al., 2000). Furthermore, their regulatory sequences overlap (Flores-Soto, Azuara-Liceaga, Lopez-Camarillo, & Orozco, 2005; Romero-Diaz et al., 2007). Organisation of EhrabB, Ehcp112, and Ehadh genes in the genome could suggest that the roles of the encoding proteins may be somehow linked; however, the functional relationship of the three proteins is not well known. Here, we investigated the participation of EhRabB and actin cytoskeleton in the mobilisation of the EhCPADH complex during phagocytosis. Our results evidenced the participation of actin in this event. Disruption of cytoskeleton by Latruculin A (LAT A) interfered with the mobilisation of EhRabB and EhCPADH to the cell membranes. In addition, we also revealed that silencing of the EhrabB gene decreases the ability of trophozoites to the phagocytos red blood cells (RBCs). Elucidation of molecular interactions during the trophozoites attack will give clues to better understand the invasion mechanism of the parasite and find pathways to block it and defeat amoebiasis. 2 RESULTS 2.1 Three-dimensional structure of EhRabB protein As the EhrabB (AF127375), Ehcp112 (AF172320), and Ehadh (AF127375.2) genes are forming a cluster in the E. histolytica genome (Figure 1a), we investigated the relationship of EhRabB and the EhCPADH complex, during phagocytosis by in silico and experimental analysis. Table 1 (Figure 1b) shows the promoter sequences of Ehadh Ehcp112 and EhRabB sequences, as well as the position of the main promoter motives to evidence their overlapping with the sequences belonging to the open reading frame of the three genes. Figure 1Open in figure viewerPowerPoint Tertiary structure of EhRabB protein and its relationship with the EhCPADH complex. (a) Schematic representation of the Ehcp112, Ehadh, and EhrabB genes containing locus. Numbers indicate relative positions to their respective ATG start codons. (b) Table showing the main motives in the promoter region, their sequence and position. (c) Schematic representation (upper panel) and tertiary structures (lower panel) of EhRabB, EhCP112, and EhADH proteins. (d) Zoom of the peptide selected to generate anti-EhRabB antibodies (left). Tertiary structure of the EhCPADH complex (middle). Zoom of the EhCPADH complex region recognised by the monoclonal antibody Mab5 (right) (e) Western blot on total Entamoeba histolytica proteins probed with anti-EhCPADH, anti-EhADH, anti-EhCP112, and anti-EhRabB. PS: pre-immune sera, corresponding to each one of the antibodies To initiate this analysis, we first predicted the in silico 3D model of EhRabB in its active form (GTP-bound), using the I-TASSER server and performing a 100-ns simulation. The 3D model was validated by Ramachandran plot, which indicated the refinement of the system after MD simulation (Figure S1a,b). After validation, the EhRabB 3D structure showed a typical GTPase fold. EhRabB is a globular protein with four α-helices (instead of five that have the majority of Rab proteins) and six β-strands (Lee, Mishra, & Lambright, 2009; Figure 1c, Table S1). Molecular dynamics (MD) simulation of the 100-ns trajectory through RMSD of EhRabB showed that the protein reached the equilibrium after 60 ns (Figure S1C). The Rg values exhibited two principal expansion regions after 24 and 44 ns that remained after 60 ns (Figure S1D), coinciding with the equilibration after 60 ns showed in RMSD values. The RMFS values presented two main peaks with high fluctuation zones, one of them, located at the residues Q29 to D42, with the highest value in V35 with 6.86 Ǻ. The other peak, at T62 to Q80, exhibited the maxim value at I71 with 4.89 Ǻ. The other fluctuation zone was located from G167 to C192 amino acids (Figure S1E). Part of this region, from E178 to C192, was used to synthesie a peptide and produce anti-EhRabB antibodies (Figure 1a,c; Rodriguez et al., 2000). 2.2 EhRabB protein co-localises with the EhCPADH complex, and both are translocated during phagocytosis The EhCPADH complex was identified by the Mab5 monoclonal antibody (Arroyo & Orozco, 1987) that react poorly with EhCP112 (id: AAF04255.1) and EhADH (id: AAF04256.1) separated proteins (Figure 1e). In addition, we also used specific antibodies against each one of these proteins (Banuelos, Garcia-Rivera, Lopez-Reyes, & Orozco, 2005; Garcia-Rivera et al., 1999). Western blot experiments using total extracts of trophozoites confirmed that each antibody recognises a specific band of the predicted molecular weight (Figure 1e). It has been previously reported that independently, both, the EhCPADH complex and EhRabB (id: AAF37308.1) protein re-localise to different cellular compartments after interaction of trophozoites with RBCs (Garcia-Rivera et al., 1999; Rodriguez et al., 2000). To investigate whether EhRabB and EhCPADH move together, we performed confocal microscopy assays of trophozoites during erythrophagocytosis (Figure 2a,b), using the specific antibodies presented in Figure 1e. In basal conditions (absence of RBCs), EhCPADH was mainly detected in the plasma membrane, whereas EhRabB was observed in spots (vesicles) in the cytoplasm. At this time, we identified few sites of co-localisation, which were evident on the base of pseudopodia-like structures (Figure 2a, 0 min, 2bA). After 2 min of interaction with RBCs, EhCPADH was still observed in the plasma membrane. Interestingly, at this time, EhRabB suffered an evident mobilisation from the cytoplasm to the plasma membrane, co-localising with the EhCPADH complex in few points (Figure 2a, 2 min). At 15 min of interaction, EhRabB appears in vesicles, and fluorescent signal for EhCPADH was more intense, principally in a cytoplasmic ring close to the plasma membrane (Figure 2a, 15 min). Here, protein co-localisation was detected in a major extension in the cytoplasm near to the internal plasma membrane (Figure 2a, 15 min). At this time, RBCs adhered to the trophozoite membrane, and those in the process of ingestion were observed surrounded by EhRabB (Figure 2a, 15 min, red fluorescence). In the particular image of Figure 2a, RBCs also appeared slightly illuminated by the anti-EhCPADH antibody (Figure 2a, 15 min; green fluorescence), due to the laser section selected, but in others, EhCPADH also surrounded the trophozoites. Ingested RBCs were decorated by both antibodies (Figure 2a, 15 min). After 30 min of interaction, anti-EhCPADH and anti-EhRabB antibodies clearly illuminated the ingested RBCs (Figure 2a, 30 min; 2bC). In the phagocytic cups, co-localisation of both proteins was stronger (Figure 2a, 30 min, 2bD). At longer times (60 min), the EhCPADH complex returned to the plasma membrane, whereas EhRabB was observed mainly in the cytoplasm, around the ingested RBCs and weakly co-localising with EhCPADH near to the plasma membrane (Figure 2a, 60 min). Signal produced by anti-EhCPADH antibody was also found in the remaining phagocytic cups (Figure 2a, 60 min). Magnified images in Figure 2b show some of the main events described in these experiments. Pearson's coefficient (obtained from 20 laser section of 20 trophozoites) evidenced that co-localisation of EhCPADH and EhRabB was higher in plasma membrane than in total cells, at all times quantified. However, co-localisation was clearer at 15 min (Figure 2c), suggesting that at certain times but not always, these proteins might be in contact, mainly in the internal plasma membrane to be mobilised during phagocytosis. Figure 2Open in figure viewerPowerPoint Confocal microscopy of trophozoites after erythrophagocytosis using anti-EhCPADH complex and anti-EhRabB antibodies. (a) Representative images of EhCPADH complex (green) and EhRabB protein (red) at different times of phagocytosis. Solid arrows: EhCPADH and EhRabB at the plasma membrane. Arrowheads: EhCPADH and EhRabB co-localisation in the phagocytic cups. Segmented arrows: EhCPADH and EhRabB co-localisation in ingested RBCs. (b): Magnification of regions marked by squares in merge + phase contrast images. e: erythrocytes. Bar = 10 μm. (c) Pearson's coefficient of EhCPADH and EhRabB co-localisation in total cells and in plasma membrane. (**) P < .01 (***) P < .001 To further investigate the EhRabB and EhCPADH complex relationship during phagocytosis, we performed immunoelectron microscopy assays using secondary antibodies labelled with gold particles of different size (anti-EhRabB, 30 nm and anti-EhCPADH, 10 nm). In basal conditions, TEM images evidenced the presence of EhRabB and EhCPADH near to the plasma membrane (Figure 3aA). At this time, the majority of the proteins were not close to each other. Number of associated proteins increased through phagocytosis (Figure 3aB–F). At early times, we could distinguish gold-labelled antibodies of the two different sizes near each other (Figure 3aB,C). In some cases, they were also observed together in the plasma membrane (Figure 3aB), in membranes of vacuoles (Figure 3aC,D), and inside structures that could correspond to multivesicular bodies (MVBs; Figure 3aE). Intriguingly, after 15 and 30 min of phagocytosis, EhCPADH and EhRabB association was also detected in the phagocytosed RBCs (Figure 3aF). Finally, at longer times (60 min), EhCPADH and EhRabB returned to the plasma membrane, where in some cases, antibodies showed them associated (Figure 3aG). Controls using only the secondary antibodies gave no signal (Figure 3a, Control). Figure 3Open in figure viewerPowerPoint Localisation of EhCPADH and EhRabB of trophozoites at different erythrophagocytosis times by transmission electron microscopy. (a) Thin sections (0.5 μm) of trophozoites were incubated with mouse anti-EhCPADH and rabbit anti-EhRabB antibodies, and then, with gold-labelled anti-mouse and anti-rabbit secondary antibodies (10 and 30-nm gold particles, respectively). Segmented squares indicate the magnified areas at the bottom of each image. PM: plasma membrane, V: vacuoles/vesicles, MVB: multivesicular bodies like structures, e: erythrocytes. Control trophozoites using only secondary antibodies. Bar = 0.5 μm. (b) Number of gold-labelled EhCPADH and EhRabB proteins by μm2. (c) Association of EhCPADH and EhRabB in different cellular compartments To better determine the dynamic of the proteins movement, gold-labeled anti-EhCPADH or anti-EhRabB antibodies were quantified in TEM images of trophozoites (Figure 3b; 30 thin sections). Number of particles corresponding to EhCPADH and EhRabB varied through the phagocytosis kinetics. The highest number of both proteins was observed after 10 min of phagocytosis. At this time, the labelled EhCPADH complex increased from 15 to 140 gold-labeled particles/μm2; whereas gold-labelled EhRabB protein augmented from 2 to 80 particles/μm2. At 20 min, the number of both proteins decreased to 65 and 3 particles/μm2 for EhCPADH and EhRabB, respectively (Figure 3b). Then, we analysed the proximity of both molecules. Association was taken as positive when they appeared at 5 nm or less distance. In these experiments, the highest percentage of EhRabB and EhCPADH association was observed after 15 to 60 min of phagocytosis. At 15 and 20 min, 11% of the total labelled proteins appeared in contact. From this percentage, 9% was found in vesicles. These results are in concordance with the confocal microscopy images in which we observed the highest co-localisation of both proteins after 15 min of phagocytosis (Figure 2a–c). In these experiments, the number of associated particles was low, in comparison with the increase of free proteins after 10 min of phagocytosis. This finding could be due to the fact that this type of association is a highly dynamic event, making the capture difficult in a given image of the associated proteins, as reported (Masi, Cicchi, Carloni, Pavone, & Arcangeli, 2010; Nooren & Thornton, 2003) for several proteins involved in cellular trafficking. Confocal microscopy and TEM assays strongly suggested that EhCPADH and EhRabB amount varies through phagocytosis. To corroborate this, we performed western blot assays using trophozoite lysates obtained after different times of erythrophagocytosis. EhRabB protein increased at 5 and 10 min, and then after 15 min, the amount of protein remained without significant alteration, whereas EhCPADH increased since 2-min contact with RBCs (Figure 4a). Actin protein presented no changes through the phagocytosis, and it was used as a control to normalise the data for quantification. Densitometry analysis confirmed that EhRabB increases twofold after 5 min, whereas EhCPADH more than twofold after 2 min, threefold after 5 min, and fourfold at 10 and 15 min. After this time, the amount of the protein decreased but remained higher than at basal conditions (Figure 4b). Figure 4Open in figure viewerPowerPoint EhCPADH and EhRabB proteins during phagocytosis. (a) Western blot of EhCPADH, EhRabB, and EhActin of total proteins from trophozoites of 0 to 60 min of phagocytosis. (b) Densitometry analysis of bands in a, normalised against EhActin, using Image J software. Data are the mean ± standard error obtained from assays in triplicate. (*) P < .05, (**) P < .01 (***) P < .001 2.3 EhRabB and EhCPADH proteins localise in acidic vesicles after phagocytosis Electroimmuno-localisation assays suggested that the EhRabB and EhCPADH complex were located in MVB-like structures after phagocytosis (Figure 3). To get further evidence of this, we used Lysotracker as a marker to identify the acidic vesicles and antibodies to localise the proteins (Figure 5). The results showed that at 0 time, Lysotracker appeared in small points in the cytoplasm, as reported (Castellanos-Castro et al., 2016). However, at 30 min, Lysotracker was located in MVB-like vesicles and in phagosomes containing RBCs, together with EhRabB and the EhCPADH complex, co-localising in many vesicles (Figure 5). MVB-like structures were also evident at 60 min of phagocytosis (Figure 5). EhCPADH was more abundant than EhRabB in these compartments. By this fact, we hypothesised that at least part of the complex is driven for degradation. In contrast, a part of EhRabB could be degraded by other pathways, as it was suggested by Hernandes-Alejandro et al. (2013). Interestingly, MVB-like structures presented internal vesicles that could correspond to the intraluminal vesicles. Figure 5Open in figure viewerPowerPoint Localisation of EhCPADH and EhRAbB in acidic vesicles. Trophozoites were incubated with RBCs as in Figure 2 and processed for confocal microscopy. After incubation with anti-EhCPADH and anti-EhRabB antibodies, followed by secondary antibodies, cells were incubated with Lysotracker. EhCPADH complex (green), EhRabB (blue), and Lysotracker (red). Arrowheads: MVB-like structures, e: erythrocyte. Bar = 10 μm. Lower panels: zoom of the MVB-like structures marked with squares in 60-min images 2.4 EhCPADH and EhRabB are mobilised to distinct cellular compartments during phagocytosis Altogether, the results exposed above suggested that EhRabB and EhCPADH could be associated at plasma membranes (PM) or other internal membranes (IM). To investigate this, we obtained membrane fractions from trophozoites incubated with RBCs for different times, and samples were examined for the presence of these proteins. TEM images showed that fractions were indeed enriched in PM or IM, respectively (Figure 6a). Western blot assays evidenced that in basal conditions, the EhCPADH complex was similarly distributed in PM and IM; at 15 min, the complex appeared enriched in PM in comparison with IM; at 30 min, it appeared diminished in PM but enriched in IM. Whereas, EhRabB protein was more abundant in IM at 0 and 15 min, but no significant differences were detected at 30 min (Figure 6b,c). EhActin was present in all samples, and it was used as internal loading control (Figure 6b,c). These results together suggest that the presence of the RBCs stimulates the protein mobilisation in cellular membranes. Additionally, these results gave further evidence that the EhCPADH complex and EhRabB protein are membrane associated proteins. Figure 6Open in figure viewerPowerPoint Subcellular distribution of EhCPADH, EhRabB, and actin in trophozoites during erythrophagocytosis. (a): Electron microscopy images of plasma membrane and internal membrane fractions, prepared by the procedure described in materials and methods. Bar = 0.5 μm. (b) Western blot of plasma membrane and internal membrane at different times of phagocytosis. (c) Densitometry analysis of band in B. (*) P < .05, (**) P < .01, (***) P < .001 2.5 Actin participates in the mobilisation of EhRabB and EhCPADH Actin, as a part of the cytoskeleton, might be associated to EhRabB and EhCPADH, probably, during the mobilisation of these proteins through cell membranes. To investigate this, we performed erythrophagocytosis experiments, and the proteins were localised by confocal microscopy using the specific antibodies and rhodamine-phalloidin for polymerised actin. EhRabB and EhCPADH appeared distributed as described in Figure 2; whereas EhActin exhibited a cortical dissemination and also appeared in small dots in the cytoplasm, as described (Hernandez-Cuevas, Jhingan, Petropolis, Vargas, & Guillen, 2018; Lopez-Contreras et al., 2013; Marion, Laurent, & Guillen, 2005). However, once phagocytosis started (5 min), the EhCPADH complex and the EhRabB protein mobilised towards the plasma membrane, and EhActin was re-localised to the sites of contact with the erythrocytes (Figure 7a). In some sites, small co-localisation points between actin and the other proteins were distinguished. The complex delimitated the phagocytic channel (Figure 7a, 5 min, green fluorescence), actin appeared surrounding the adhered and partially ingested RBCs, and EhRabB, around some of the ingested RBCs co-localising with EhActin and EhCPADH (Figure 7a 5 min, blue fluorescence). Later, (10 min), a higher co-localisation of the three proteins was clear in RBCs that were in the process of being phagocytosed and in some that were already inside the trophozoites. At longer times (20 min), EhCPADH appeared close to the plasma membrane, around the ingested RBCs and in the phagocytic channel, mainly in the cup, whereas EhActin illuminated the internal channel through which the RBCs were internalised. In these images, EhRabB decorated the internal part of the channel, co-localising with EhActin and EhCPADH in different areas (Figure 7a 20 min, blue fluorescence). These results suggest that the EhCPADH, EhRabB, and EhActin move in concert, actively participating from the phagocytic cup formation up to the end of the digestive process of the ingested RBCs. A cartoon in Figure 7b simulates the steps that were observed for the three proteins in these events. Figure 7Open in figure viewerPowerPoint Localisation of EhActin, EhRabB, and EhCPADH during the phagocytosis of Entamoeba histolytica. (a) Representative images of laser confocal assays showing EhCPADH complex (green), EhRabB protein (blue), and EhActin (red). Arrowheads: EhCPADH, EhRabB, and EhActin co-localisation. Bar = 10 μm. phc: phagocytic cups. e: erythrocytes. Lower panels: zoom of the squared regions in a. (b) Cartoon of the main steps of phagocytosis The assumption of the cytoskeleton participation in the EhRabB and EhCPADH complex mobilisation might be strengthened, assuming that, if the actin cytoskeleton becomes disorganised, EhCPADH and EhRabB would not be adequately mobilised during phagocytosis. To support this hypothesis, we used LAT A to destabilise the actin filaments (Castillo-Romero et al., 2010). Viability of trophozoites after LAT A treatment was 95%, analysed by trypan blue exclusion. Culturing of the treated trophozoites in fresh medium after washing out the LAT A evidenced that they presented a similar rate of growth than untreated cells, corroborating their viability. Then, LAT A-treated trophozoites were incubated with RBCs and processed for confocal microscopy. After 5 min of phagocytosis, LAT A-treated trophozoites looked rounded and without RBCs ingested, although few erythrocytes were adhered to trophozoites (Figure 8a). In contrast, the nontreated trophozoites looked pleomorphic and with different number of ingested and adhered RBCs, as shown in Figure 7a. EhActin lost its typical cortical location, and it was trapped in the cytoplasm without reaching the plasma membrane (Figure 8a). Interestingly, at all times studied, EhRabB and EhCPADH also lost their cellular distribution, in comparison with nontreated trophozoites. EhActin, EhRabB, and EhCPADH were unable to reach the plasma membrane, and they accumulated inside the cell (Figure 8a). Experiments in parallel were carried out to quantify the adherence efficiency and rate of erythrophagocytosis of LAT