Virulence and functions of myosin II are inhibited by overexpression of light meromyosin in Entamoeba histolytica

Abstract

Several changes in cell morphology take place during the capping of surface receptors in Entamoeba histolytica. The amoebae develop the uroid, an appendage formed by membrane invaginations, which accumulates ligand-receptor complexes resulting from the capping process. Membrane shedding is particularly active in the uroid region and leads to the elimination of accumulated ligands. This appendage has been postulated to participate in parasitic defense mechanisms against the host immune response, because it eliminates complement and specific antibodies bound to the amoeba surface. The involvement of myosin II in the capping process of surface receptors has been suggested by experiments showing that drugs that affect myosin II heavy-chain phosphorylation prevent this activity. To understand the role of this mechanoenzyme in surface receptor capping, a myosin II dominant negative strain was constructed. This mutant is the first genetically engineered cytoskeleton-deficient strain of E. histolytica. It was obtained by overexpressing the light meromyosin domain, which is essential for myosin II filament formation. E. histolytica overexpressing light meromyosin domain displayed a myosin II null phenotype characterized by abnormal movement, failure to form the uroid, and failure to undergo the capping process after treatment with concanavalin A. In addition, the amoebic cytotoxic capacities of the transfectants on human colon cells was dramatically reduced, indicating a role for cytoskeleton in parasite pathogenicity.

Figure 1

Cloning and expression of light meromyosin in E. histolytica. (A) The 3′ end of the gene encoding the myosin heavy chain was amplified by PCR. Oligonucleotides were designed to incorporate an epitope tag at the amino terminus of the expressed LMM. The generated DNA fragment was cloned into the ExEhNeo vector and introduced into the amoebae by electroporation. (B) Immunoblot of proteins obtained from the transfectant cells revealed by the anti-myosin II and by the anti-VSV-G antibodies. Myosin is at 250 kDa, and VSV-LMM is at 71 kDa. Lane 1, LMM20; lane 2, ExEh20, lane 3, LMM30; lane 4, ExEh30. Analysis shows that the LMM protein is expressed and correctly tagged.

Figure 2

Analysis of mean speed of wild-type, ExEh20, and LMM transfectant amoebae. The histogram shows the comparison of the means speed of the two subpopulations of LMM20: slower () and LMM20 faster (). The same type of representation was chosen for LMM30: slower (▥) and LMM30 faster (▨). Finally, the mean speeds for ExEh20 (□) and wild type (▪) are represented.

Figure 3

Computer-assisted cellular movement analysis of wild-type, ExEh20, and LMM cells. Images of amoebae were recorded and computer analyzed using conditions described in MATERIALS AND METHODS. Micrographs of analyzed amoebae are shown in the upper panel, and their trajectories are represented in the lower panel. The wild-type strain moves in random directions; some of the amoebae return on their trajectories. ExEh20 cells move at the same rate as the wild-type strain but show more direct trajectories. LMM20 and LMM30 show reduced trajectories. The majority of the amoebae wobble in place around stationary points of contact with the substratum. In addition, LMM transfectants are round, and some of them seem to extend small pseudopods.

Figure 4

TEM of transfectant amoebae induced for capping with ConA. ExEh30 and LMM30 strains were induced for capping and fixed, and redistribution of ConA–receptor complexes was observed by the labeling of ConA binding sites by peroxidase. The membrane region in which capped complexes are concentrated is stained in black by oxidized diaminobenzidine. Micrograph A (4500× magnification) shows the cellular localization of ConA–receptor complexes in the LMM30 strain (arrows). The membrane appears poorly folded when compared with membrane in the uroid region of the ExEh30 transfectant used as a control (micrograph B, 8000× magnification).

Figure 5

Distribution of myosin II, LMM, and Gal-GalNAc lectin in E. histolytica transfected with LMM. Transfectants growing in 30 μg/ml G418 amoebae were fixed and stained for myosin II, VSV-LMM, and Gal-GalNAc lectin. Confocal laser scanning microscopy was performed to detect immunofluorescence. Micrograph A shows rounded cells with myosin II labeling the plasma membrane (panel 1); some discrete myosin II spots are also observed in the cytoplasm. In the same cell, LMM is essentially diffuse throughout the cytoplasm (panel 2). The focal plane shown in this micrograph represents the middle of the cell. Micrograph B shows myosin II distribution in rounded cells (panel 3) and Gal-GalNAc in the same cell (panel 4). Myosin II is located under the plasma membrane as described previously. The Gal-GalNAc lectin is present at the membrane level; some patches of receptor can be seen (arrows). Micrograph C shows transfected amoeba that display a uroid (arrowhead). Myosin II is recruited to and concentrates in the uroid (panel 5); VSV-LMM is also observed in the uroid (panel 6). These results indicate the recruitment of both proteins during capping. Bar, 5 μm.

Figure 6

Viability of Caco-2 cells in the presence of LMM transfectants. A ⁵¹Cr-labeled, postconfluent monolayer of Caco-2 cells, cultivated in 24-well plates, was infected with growing E. histolytica (1 amoeba for 5 cells) in a 1-ml vol medium and incubated for the indicated times. After incubation, medium was removed and centrifuged. The cytolysis is reflected by the ⁵¹Cr molecules liberated in the medium. Percentage of lysis corresponds to the fraction of radioactivity in the supernatant compared with the total radioactivity. Each incubation was condition tested in triplicate, and the experiment was repeated three times. ▴—▴, HM1 wild-type strain (40% ± 3% of lysis; n = 3); ▪—▪, NEO-transfected strain: (40% ± 1% of lysis; n = 3); •—•, LMM transfected strain (5% ± 1% of lysis; n = 3).