Entamoeba histolytica, which causes amebic dysentery and liver abscesses, is spread via chitin-walled cysts. The most abundant protein in the cyst wall of Entamoeba invadens, a model for amebic encystation, is a lectin called EiJacob1. EiJacob1 has five tandemly arrayed, six-Cys chitin-binding domains separated by lowcomplexity Ser-and Thr-rich spacers. E. histolytica also has numerous predicted Jessie lectins and chitinases, which contain a single, N-terminal eight-Cys chitin-binding domain. We hypothesized that E. invadens cyst walls are composed entirely of proteins with six-Cys or eight-Cys chitin-binding domains and that some of these proteins contain sugars. E. invadens genomic sequences predicted seven Jacob lectins, five Jessie lectins, and three chitinases. Reverse transcription-PCR analysis showed that mRNAs encoding Jacobs, Jessies, and chitinases are increased during E. invadens encystation, while mass spectrometry showed that the cyst wall is composed of an ϳ30:70 mix of Jacob lectins (cross-linking proteins) and Jessie and chitinase lectins (possible enzymes). Three Jacob lectins were cleaved prior to Lys at conserved sites (e.g., TPSVDK) in the Ser-and Thr-rich spacers between chitin-binding domains. A model peptide was cleaved at the same site by papain and E. invadens Cys proteases, suggesting that the latter cleave Jacob lectins in vivo. Some Jacob lectins had O-phosphodiester-linked carbohydrates, which were one to seven hexoses long and had deoxysugars at reducing ends. We concluded that the major protein components of the E. invadens cyst wall all contain chitinbinding domains (chitinases, Jessie lectins, and Jacob lectins) and that the Jacob lectins are differentially modified by site-specific Cys proteases and O-phosphodiester-linked glycans.The infectious form of Entamoeba histolytica, a protozoan parasite that causes amebic dysentery and liver abscesses, is the quadranucleate cyst (10,15,25). Because E. histolytica does not encyst in axenic culture, cyst formation has been studied in Entamoeba invadens, a reptilian pathogen that also forms quadranucleate cysts (7, 31). E. invadens converts to chitinwalled cysts within 2 days when subjected to osmotic shock and/or glucose deprivation (30). The E. invadens cyst wall resembles the E. histolytica cyst wall, and E. invadens cysts are able to excyst readily when placed in full medium. Cyst formation is blocked by the addition of inhibitors of Cys or Ser proteases, although the mechanism of inhibition is unclear (21, 23). E. histolytica has 20 lysosomal Cys protease genes, 8 of which are transcribed by trophozoites in culture, while E. invadens has multiple Cys proteases (4, 23).The most abundant protein in the cyst wall of E. invadens is the Jacob lectin (EiJacob1), a secreted glycoprotein that contains five tandemly arranged chitin-binding domains (CBDs) (9). Each EiJacob1 CBD contains six conserved Cys residues and numerous conserved aromatic amino acids. EiJacob1 was identified by sequencing the largest of Ͼ20 spots on two-dimensional (2-D)...

BackgroundThe 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/ResultsAn 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/SignificanceThe 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.

A bacterial monolayer biofilm is a collection of cells attached to a surface but not to each other. Monolayer formation is initiated when a bacterial cell forms a transient attachment to a surface. While some transient attachments are broken, others transition into the permanent attachments that define a monolayer biofilm. In this work, we describe the results of a large-scale, microscopy-based genetic screen for Vibrio cholerae mutants that are defective in formation of a monolayer biofilm. This screen identified mutations that alter both transient and permanent attachment. Transient attachment was somewhat slower in the absence of flagellar motility. However, flagellar mutants eventually formed a robust monolayer. In contrast, in the absence of the flagellar motor, monolayer formation was severely impaired. A number of proteins that modulate the V. cholerae ion motive force were also found to affect the transition from transient to permanent attachment. Using chemicals that dissipate various components of the ion motive force, we discovered that dissipation of the membrane potential (⌬⌿) completely blocks the transition from transient to permanent attachment. We propose that as a bacterium approaches a surface, the interaction of the flagellum with the surface leads to transient hyperpolarization of the bacterial cell membrane. This, in turn, initiates the transition to permanent attachment.

Chitin in the cyst wall of Entamoeba histolytica is made by two chitin synthases (Chs), one of which is unique (EhCHS-1) and one of which resembles those of insects and nematodes (EhCHS-2). EhCHS-1 is deposited chitin in the lateral wall of transformed Saccharomyces cerevisiae Chs mutants, independent of accessory proteins (Chs4p to Chs7p) required by yeast Chs3p.