ABSTRACT
A novel neutral sphingomyelinase (nSMase) was characterized in Entamoeba histolytica trophozoites. SMase, a sphingomyelin-specific form of phospholipase C, catalyzes the hydrolysis of sphingomyelin to ceramide and phosphorylcholine. Three amebic putative nSMase genes were found to be actively transcribed. Mg(2+)-independent nSMase activity in the soluble fraction of the trophozoites was stimulated by Mn(2+) and partially inhibited by Zn(2+). nSMase activity of the recombinant protein EhnSM1, increased 4.5-fold in the presence of 0.5mM Mn(2+), and abolished by 5mM Zn(2+). A dose-dependent inhibition of rEhnSM1 was observed with scyphostatin, a specific inhibitor of nSMases. The EhnSM1 and EhnSM3 were detected in the soluble fraction of the amebic lysate as 35-37kDa proteins by western blot analysis. Immunofluorescence assay showed that the overexpressed HA-tagged EhnSM1 and EhnSM3 were localized to the cytosol. The biological role of these novel E. histolytica nSMases described in this work remains to be determined.
Subject(s)
Entamoeba histolytica/enzymology , Entamoeba histolytica/genetics , Sphingomyelin Phosphodiesterase/genetics , Sphingomyelin Phosphodiesterase/metabolism , Amides/pharmacology , Amino Acid Sequence , Blotting, Western , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Fluorescent Antibody Technique, Indirect , Molecular Sequence Data , Pyrones/pharmacology , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sequence Alignment , Sphingomyelin Phosphodiesterase/antagonists & inhibitors , Trophozoites/enzymologyABSTRACT
Monoxenic cultivation of pathogenic Entamoeba histolytica trophozoites with Escherichia coli serotype 055 which binds strongly to the Gal/GalNAc amoebic lectin, markedly improved the growth of E. histolytica and produced a significant decrease in cysteine proteinase activity and a lower cytopathic activity on monolayer cells after 3 months of monoxenic culture. However, after long term monoxenic culture (12 months) the proteolytic and cytopathic activities were recovered and the amoebic growth reached the maximum yield. Employing the GeneFishing(R) technology and DNA macroarrays we detected differentially gene expression related to the amoebic interaction with bacteria. A number of differentially expressed genes encoding metabolic enzymes, ribosomal proteins, virulence factors and proteins related with cytoskeletal and vesicle trafficking were found. These results suggest that E. coli 055 has a nutritional role that strongly supports the amoebic growth, and is also able to modulate some biological activities related with amoebic virulence.
Subject(s)
Entamoeba histolytica/growth & development , Entamoeba histolytica/pathogenicity , Escherichia coli/physiology , Animals , Cell Line , Coculture Techniques , Cysteine Endopeptidases/genetics , Cysteine Endopeptidases/metabolism , DNA Primers/chemistry , DNA, Complementary/biosynthesis , DNA, Protozoan/chemistry , Entamoeba histolytica/genetics , Escherichia coli/growth & development , Gene Expression Regulation, Developmental , RNA, Protozoan/genetics , RNA, Protozoan/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction , VirulenceABSTRACT
Serine proteases are one of the biologically most important and widely distributed enzyme families. A protease capable of degrading the substrate Suc-AAF-AMC was isolated from axenically grown trophozoites of Entamoeba histolytica. The enzyme was purified by ion-exchange chromatography and electroelution, and appeared on 2D-PAGE as a spot of 60 kDa and pI of 4.65. Data obtained from zymogram suggest the active protease is present either as homodimer (130 kDa) or homotetramer (250 kDa). The optimal temperature of the enzyme was 37 degrees C, and it exhibited activity over a broad pH range. The protease was strongly inhibited by TPCK and chelating agents. The enzymatic activity was restored upon addition of calcium. BLAST analysis with the sequence of internal peptides of the protein revealed two open reading frames within the genome of E. histolytica, homologous to members of the family S28, clan SC of serine proteases.
