New insights into host-pathogen interactions during Entamoeba histolytica liver infection

Abstract

Amoebiasis is the third worldwide disease due to a parasite. The causative agent of this disease, the unicellular eukaryote Entamoeba histolytica, causes dysentery and liver abscesses associated with inflammation and human cell death. During liver invasion, before entering the parenchyma, E. histolytica trophozoites are in contact with liver sinusoidal endothelial cells (LSEC). We present data characterizing human LSEC responses to interaction with E. histolytica and identifying amoebic factors involved in the process of cell death in this cell culture model potentially relevant for early steps of hepatic amoebiasis. E. histolytica interferes with host cell adhesion signalling and leads to diminished adhesion and target cell death. Contact with parasites induces disruption of actin stress fibers and focal adhesion complexes. We conclude that interference with LSEC signalling may result from amoeba-triggered changes in the mechanical forces in the vicinity of cells in contact with parasites, sensed and transmitted by focal adhesion complexes. The study highlights for the first time the potential role in the onset of hepatic amoebiasis of the loss of liver endothelium integrity by disturbance of focal adhesion function and adhesion signalling. Among the amoebic factors required for changed LSEC adherence properties we identified the Gal/GalNAC lectin, cysteine proteases and KERP1.

Keywords: Entamoeba; KERP1; amoebiasis; cell death; focal adhesions; integrins; liver sinusoidal endothelial cells.

Fig. 1.

Amoebic liver abscess formation in the hamster model of hepatic amoebiasis Male Syrian golden hamsters (Mesocricetus auratus) were infected intraportally with virulent parasites (8×10⁵ trophozoites per animal) according to our published protocol [7]. Livers were excised and fixed immediately after necropsy at 24 hours post-inoculation (A). Shown is a paraffin-embedded section of infected liver stained with hematoxylin to visualize the mammalian cells (in blue) and immuno-labelled with a human anti-E. histolytica antibody (in pink). Note the presence of massive infiltrates containing inflammatory cells. Over time, the inflammatory foci coalesce and form macroscopic abscesses (B). Scale bar, 100 μm

Fig. 2.

Immunolocalization of amoebic protein KERP1 during interaction of virulent trophozoites with LSEC. Confocal microscopy acquisitions showing immunolocalization of KERP1 (green) delimiting the parasite and detection of F-actin (red) the LSEC. Nuclei are stained by DAPI (blue). Scale bars (on axis labels), 5 µm. A. Virtual three-dimensional reconstruction of the stack. Blending and shadowing reveal the volumes on this two-dimensional representation obtained with Imaris software. The nuclei of two LSEC are detected. A trophozoite is seen in the upper part of the image and in the centre an amoeba is partially covered by a LSEC, which has retracted from the substrate and the neighbouring cells. A membrane protrusion emitted by the trophozoite engulfs a portion of the LSEC (arrow and intersection of the white lines in Panel B). B. The micrograph shows three orthogonal planar sections. The white lines correspond to the orthogonal projection of the planes presented aside and their intersection represents the point indicated by the arrow in Panel A. The arrows indicate the orientation of the plane and z = 0 corresponds to the focal plane acquired closest to substratum. The retracted LSEC is detected with its nucleus. The trophozoite surface in contact with the LSEC is not homogeneously enriched in KERP1, as detected at the top of the trophozoite in the xz section. The three orthogonal sections show actin from LSEC (at the intersection of the white lines) surrounded by membrane protrusions from the parasite that are strongly enriched in KERP1. Note that, in the yz section, the membrane invagination is also detected and corresponds to the area with the highest z coordinates (i.e. the most distant from the z = 0) and the greatest KERP1 density. The three-dimensional reconstruction allowed detection of this topological link

Fig. 3.

Retraction and cell death in LSEC cultures during incubation with E. histolytica. Micrographs of video-microscopy sequences show frames taken at 0, 15 and 30 min of incubation. LSEC were labelled with fluorescent CMFDA cell tracker (green). Trophozoites were then added (parasite to LSEC ratio 1:10) and observed by phase contrast microscopy, as non-fluorescent cells (white) with brightly reflecting plasma membranes. Dead cells were detected by incorporation of propidium iodide (red). Note that human cells (i.e. * and #) were in contact with an amoeba before dying. The space not occupied by cells (i.e. §) is increasing over time, indicating LSEC retraction. Scale bar, 10 µm

Fig. 4.

Model for the passage of E. histolytica through the liver sinusoidal barrier. A. Schematic representation of a liver sinusoid. Red blood cells circulate in the sinusoidal lumen lined by fenestrated LSEC (L; blue). LSEC adhere at FA plates (orange) to the ECM (gray lines) present in the Disse’s space (DS) and in contact which the hepatocytes (H). Stellate and Kupffer cells are not represented. The arrow indicates the direction of the blood flow. Note that the drawing is not in scale. B. During early stages of hepatic amoebiasis, E. histolytica trophozoites (yellow) obstruct hepatic sinusoid capillaries and induce LSEC retraction (indicated by arrowheads). The amoeba (10–50 µm) being bigger than the sinusoid diameter (5–7 µm), trophozoites exert mechanical forces on the endothelium. In addition, several virulence factors and adhesion molecules (green) facilitate the loss of FA complexes accelerating LSEC retraction. C. Obstruction caused by amoebae reduces the blood flow, locally creating ischemia and decreasing concentrations of oxygen and nutrients. As a consequence, the oxidative stress for the amoebae is diminished, LSEC death by apoptosis and necrosis (purple nucleus, grey cytoplasm) is induced and the inflammatory response initiated by immune cells (light blue). D. Retraction and cell death allow the amoeba to penetrate into the liver parenchyma in which it induces hepatocyte death. Phagocytosis of red blood cells, apoptotic bodies and necrotic debris provides nutrients and energy to the trophozoites. The immune response is developing

Fig. 5.

E. histolytica interference with cell adhesion signalling may induce cell death. The scheme summarizes the levels at which E. histolytica may interfere with target cell integrin-mediated cell adhesion signalling, here LSEC. Several relevant virulence factors are presented: Gal/GalNAc lectin and KERP-1 [44]. Host cell receptors for both amoebic molecules remain to be identified. Amoebic β2-integrin like molecule (β2-like EhR) shown to bind to intercellular adhesion molecules ICAM-1 and -2 [45] whose expression is induced in activated endothelial cells. RGD motif containing proteins, like the ECM component fibronectin expressed by LSEC, may serve as binding sites for β1 integrin-like fibronectin receptor β1EhFNR which shares a high degree of homology with the intermediate Gal/GalNAc lectin subunits Igl1 and Igl2 ([35] and references therein). Interaction may reinforce trophozoite adherence and eventually compete with integrins for RGD ligands, thus interfering with LSEC adhesion signalling. The immuno-dominant amoeba protein M17 [46] may be involved in recognition of phosphatidyl-serine (PS) exposed at the surface of apoptotic cells, which are preferentially phagocytosed by amoebae. Pore-forming amoebapore proteins (AP) though involved in lysis of other host cells and ALA, are not essential for LSEC apoptosis and death. The calcium-binding proteins Grainin-1 and -2 (Gr) are found in secreted granules, but their potential role in LSEC killing has not been elucidated so far. Cysteine proteases, among which CP-A5, present at the trophozoite surface or released into the extracellular medium, are implicated in ECM degradation. The pro-form of CP-A5, localized in secreted and surface-bound fractions, contains a RGD sequence that confers to this protein a protease activity-independent additional function in adherence and host cell inflammatory response induction; binding to integrin αvβ3 from colonic enterocyte cells has been found [34], potential receptors on LSEC are not characterized. Activation of vascular endothelial cell G-protein coupled receptors, like the protease-activated receptors (PAR), by pathogen-encoded CPs have been described for several microorganisms, including African trypanosome parasites. Anchorage-dependent survival of adherent cells relies on the activity of the integrin-mediated signal transduction pathway, regulating cell survival/death signalling activities and cytoskeleton organization. LSEC death results from abrogation of adhesion signalling leading to diminished adhesion, to inactivation of survival and to activity of cell death pathways. Loss of adhesion may in turn trigger apoptosis (anoikis). Elements intervening in E. histolytica contact-dependent apoptosis of LSEC were inferred from data obtained with the human lymphoma line Jurkat [–49]. Upon contact with trophozoites, the protein tyrosine phosphorylation level rapidly decreases and the concentration of free intracellular calcium drastically rises. The initial trigger for both events remain yet unknown. Calcium-dependent activation of calpain in turn activates caspase-3 responsible for the execution of the apoptotic program. Calpain activity may also account for necrotic cell death and diminished protein phosphorylation levels, by cleavage-dependent activation of protein tyrosine phosphatase PTP1B. Amoeba-induced activity of calpain, caspase-3 and PTP1B and several other protein tyrosine phosphatases as well as increased calcium levels may negatively modulate integrin/FA pathway activity