The Jacob2 lectin of the Entamoeba histolytica cyst wall binds chitin and is polymorphic

Abstract

Background: The infectious and diagnostic form of Entamoeba histolytica (Eh), cause of amebic dysentery and liver abscess, is the quadranucleate cyst. The cyst wall of Entamoeba invadens (Ei), a model for Eh, is composed of chitin fibrils and three sets of chitin-binding lectins that cross-link chitin fibrils (multivalent Jacob lectins), self-aggregate (Jessie lectins), and remodel chitin (chitinase). The goal here was to determine how well the Ei model applies to Entamoeba cysts from humans.

Methods/results: An Eh Jacob lectin (EhJacob2) has three predicted chitin-binding domains surrounding a large, Ser-rich spacer. Recombinant EhJacob2 made in transfected Eh trophozoites binds to particulate chitin. Sequences of PCR products using primers flanking the highly polymorphic spacer of EhJacob2 may be used to distinguish Entamoeba isolates. Antibodies to the EhJacob2, EhJessie3, and chitinase each recognize cyst walls of clinical isolates of Entamoeba. While numerous sera from patients with amebic intestinal infections and liver abscess recognize recombinant EhJacob1 and EhJessie3 lectins, few of these sera recognize recombinant EhJacob2.

Conclusions/significance: The EhJacob2 lectin binds chitin and is polymorphic, and Jacob2, Jessie3, and chitinase are present in cyst walls of clinical isolates of Entamoeba. These results suggest there are substantial similarities between cysts of the human pathogen (Eh) and the in vitro model (Ei), even though there are quantitative and qualitative differences in their chitin-binding lectins.

Figure 1. Eh Jacob2 has three chitin-binding domains (CBDs) surrounding a large Ser-rich spacer.

A. EhJacob2 has an N-terminal signal peptide, three CBDs, and a large spacer between the first two CBDs and the last CBD. EhJacob2 has no transmembrane helix or GPI-anchor. B. Sequence of EhJacob2 where signal peptide (grey) and Cys residues (red) within CBDs are highlighted. Also highlighted are short repeats in the spacer, which fall into five families: A (light blue), B (green), C (pink), D (purple), and E (orange). Polymorphisms in these repeat families are further described in Fig. 4. The Ed Jacob2 is shown in Fig. S1.

Figure 2. EhJacob2 is a chitin-binding protein.

A. Coomassie blue-stained SDS-PAGE gel showing a total lysate of Eh trophozoites transfected with c-myc tagged EhJacob2, the fraction that binds chitin beads, and the fraction that does not bind chitin. B. Western blot confirms the chitin binding of EhJacob2, which is detected with anti-c-myc antibodies and chemiluminescence.

Figure 3. Ser-rich domains of EhJacob2 are polymorphic.

Amplification products were generated using PCR primers flanking the Ser-rich region between the second and third chitin-binding domains of Jacob2. A. Jacob2 PCR products from axenized Eh isolates (HM-1:IMSS, HK-9, 200:NIH, and SD157) and Ed isolate (SAW760) have distinct mobilities on agarose gels. B. Jacob2 PCR products from clinical Eh isolates also have distinct mobilities.

Figure 4. EhJacob2 PCR products are distinct for each axenized strain.

A. Coded representations of EhJacob2 repeats from PCR products shown in Fig. 3. Complete sequences were obtained for HM-1:IMSS and SD157. Gaps in the middle of sequences in the HK-9 and 200:NIH products are marked. B. Five EhJacob2 repeats are each assigned a letter (A to E) and a color (as described in Fig. 1). The nucleotide sequences coding for each repeat are numbered in the order of their frequency of occurrence in the sequenced products.

Figure 5. Antibodies to EhJacob2, Jessie3, and chitinase bind to Entamoeba cysts isolated from patient stools.

A to C. Confocal micrographs of stool cysts detected with rabbit antibodies to Eh Jacob2, Jessie3, or chitinase. D. Confocal micrograph of an Eh cyst from xenic culture labeled with antibodies to EhJessie3. Thanks to Upinder Singh for the micrograph shown in D.