Entamoeba histolytica: Alterations in EhRabB protein in a phagocytosis deficient mutant correlate with the Entamoeba dispar RabB sequence

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Abstract

We analyzed the expression and location of EhRabB in clone L-6, a phagocytosis-deficient mutant of Entamoeba histolytica, in comparison with the wild-type clone A. Intriguingly, trophozoites of clone L-6 express more EhRabB than those of clone A. However, the majority of EhRabB-containing vesicles remained in the cytoplasm of clone L-6 during phagocytosis. To investigate molecular alterations in EhRabB of clone L-6 we compared the EhrabB gene sequences from clones L-6 and A. We also isolated, sequenced and compared the RabB protein of Entamoeba dispar. Results showed that EhrabB gene of clone L-6 is 98.2 and 94.1% identical to rabB genes of E. dispar and clone A, respectively. The rabB genes from clone A and E. dispar have 92.2% identity. Four out of five amino acids changes in RabB proteins of clone L-6 and E. dispar are shared. These changes may alter the binding of effector proteins and the specific subcellular location of EhRabB.

Introduction

Phagocytosis is an event that participates in the pathogenic mechanism of Entamoeba histolytica, the causative protozoan of human amoebiasis. In this event are involved, among others molecules, adhesins (Garcia-Rivera et al., 1999), cytoskeleton proteins, regulator proteins of the cytoskeleton structure (Godbold and Mann, 1998, Labruyere et al., 2003, Voigt and Guillen, 1999), hydrolytic enzymes (Ankri et al., 1998, Que et al., 2002), and regulator proteins of the vesicle transport as the Rab GTPases (Rodriguez et al., 2000, Saito-Nakano et al., 2004).

Rab proteins are conserved through evolution of eukaryotic organisms and they are central regulators of the vesicular transport, acting as molecular switches by cycling between active GTP-bound and inactive GDP-bound protein forms (Zerial and McBride, 2001). In addition, accessory proteins modulate the Rab membrane association, the nucleotide binding and hydrolysis, and the interaction of Rab with effector proteins that help to provide vesicle motility, or that bring appropriate membranes into close contact (Zerial and McBride, 2001).

EhRabB is an E. histolytica Rab protein containing the four domains involved in the guanine nucleotide binding and the motif called “effector region,” involved in interactions with particular effector molecules. It also has two cysteine residues in its carboxy-terminus that are subjected to isoprenyl modification for its association with vesicle membranes (Rodriguez et al., 2000). The EhRabB recombinant protein bound specifically GTP and GDP (Rodriguez et al., 2000, Rodriguez and Orozco, 2000). EhRabB is located in small cytoplasmic vesicles that are translocated to plasma membrane and to phagocytic mouths when trophozoites are incubated with red blood cells (RBCs) (Rodriguez et al., 2000), suggesting that EhRabB could be involved in phagocytosis.

On the other hand, the phagocytosis-deficient clone L-6 was obtained from strain HM1:IMSS (Orozco et al., 1983). This clone is also virulence-deficient, but displays normal adherence to RBCs, indicating that a subsequent event to adherence is responsible of its phagocytosis deficience (Orozco et al., 1983). This mutant is an excellent biological tool to investigate the participation of different molecules in E. histolytica pathogenicity. In fact, L-6 shows low proteinase activity (Carpeniseanu et al., 2000) and low expression of the EhCPADH complex (Ocadiz et al., 2005), which participates in the E. histolytica pathogenicity and is formed by a cysteine protease (EhCP112) and an adhesin (EhADH112) (Garcia-Rivera et al., 1999). Here, we analyzed the EhRabB expression and its cellular location during phagocytosis in trophozoites of clone L-6. We also compared the RabB nucleotide and amino acid sequences of clones A and L-6 and of the non-pathogenic Entamoeba dispar.

Section snippets

Entamoeba histolytica cultures

Trophozoites of E. histolytica, clones A (wild-type) and L-6 (phagocytosis-deficient mutant) (Orozco et al., 1983), were axenically cultured in TYI-S-33 medium and harvested as described (Diamond et al., 1978).

SDS–PAGE and Western blot

Trophozoites were lysed in the presence of 2 mM phenylmethylsulphonyl fluoride (PMSF) and 20 mM p-hydroxymercuribenzoate (PHMB). Then, 30 μg of trophozoites extracts were electrophoresed on 12% SDS–PAGE and transferred to nitrocellulose membranes. Detection of EhRabB was performed using

Clone L-6 shows a higher EhRabB expression than the wild-type clone A

We analyzed the expression of EhRabB protein in the phagocytosis-deficient mutant trophozoites by Western blot assays using antibodies against a divergent peptide of EhRabB comprising from 177 to 190 amino acid residues (anti-EhRabB). The antibodies recognized the expected 21 kDa EhRabB protein in extracts of both, the wild-type and the mutant trophozoites (Fig. 1A, upper panel). Interestingly, the antibodies revealed a stronger band in clone L-6 than in the wild-type clone A (Fig. 1A, upper

Acknowledgments

This work was supported by the European Community and CONACyT (México). We are also grateful for the excellent technical assistance of Alfredo Padilla-Barberi and Blanca Estela Reyes-Marquez. We thank Dr. Nancy Guillen at the Pasteur Institute, France, who kindly provided the DNA of E. dispar.

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    The sequence data reported herein have been submitted to GenBank and assigned Accession No. AY883996 for the rabB gene of E. histolytica clone L-6 and Accession No. AY882575 for the rabB gene of Entamoeba dispar.

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