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Synthetic bovine lactoferrin peptide Lfampin kills Entamoeba histolytica trophozoites by necrosis and resolves amoebic intracecal infection in mice.

ABSTRACT: Amoebiasis caused by the protozoan parasite Entamoeba histolytica remains a public health problem in developing countries, making the identification of new anti-amoebic compounds a continuing priority. Previously, we have shown that lactoferrin (Lf) and several Lf-derived peptides exhibit in vitro anti-amoebic activity independently of their iron-binding activity. Here, we evaluated the amoebicidal effect of synthetic Lf-derived peptides Lfcin-B, Lfcin 17-30, and Lfampin, analyzed the mechanism of death induced by the peptides and determined their therapeutic effects on murine intestinal amoebiasis. MTT assays in trophozoite cultures of E. histolytica exposed to each peptide (1-1000 ?M) showed that Lfampin is far more amoebicidal than Lfcins. Lfampin killed 80% of trophozoites at doses higher than 100 ?M in 24 h, and FACs analysis using Annexin V/propidium iodide showed that death occurred mainly by necrosis. In contrast, Lfcin-B and Lfcin 17-30 appeared to have no significant effect on amoebic viability. FACs and confocal microscopy analysis using FITC-labeled peptides showed that all three peptides are internalized by the amoeba mainly using receptor (PI3K signaling) and actin-dependent pathways but independent of clathrin. Docking studies identified cholesterol in the amoeba's plasma membrane as a possible target of Lfampin. Oral treatment of intracecally infected mice with the abovementioned peptides at 10 mg/kg for 4 days showed that Lfampin resolved 100% of the cases of intestinal amoebiasis, whereas Lfcin 17-30 and Lfcin-B were effective in resolving infection in 80 and 70% of cases, respectively. These data show that although synthetic bovine Lf-derived peptides exhibit varying amoebicidal potentials in vitro, they do resolve murine intestinal amoebiasis efficiently, suggesting that they may be useful as a therapeutic treatment.

SUBMITTER: Diaz-Godinez C

PROVIDER: S-EPMC6328891 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Synthetic bovine lactoferrin peptide Lfampin kills Entamoeba histolytica trophozoites by necrosis and resolves amoebic intracecal infection in mice.

Díaz-Godínez César C González-Galindo Ximena X Meza-Menchaca Thuluz T Bobes Raúl J RJ de la Garza Mireya M León-Sicairos Nidia N Laclette Juan P JP Carrero Julio C JC

Bioscience reports 20190108 1

Amoebiasis caused by the protozoan parasite Entamoeba histolytica remains a public health problem in developing countries, making the identification of new anti-amoebic compounds a continuing priority. Previously, we have shown that lactoferrin (Lf) and several Lf-derived peptides exhibit in vitro anti-amoebic activity independently of their iron-binding activity. Here, we evaluated the amoebicidal effect of synthetic Lf-derived peptides Lfcin-B, Lfcin 17-30, and Lfampin, analyzed ...[more]

PMID: 30429239

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Project description:In a previous work we described the transcriptional silencing of the amoebapore A (AP-A) gene (Ehap-a) of Entamoeba histolytica strain HM-1:IMSS. The silencing occurred following transfection with a plasmid containing a 5' upstream region (473 bp) of Ehap-a that included a truncated segment (140 bp) of a short interspersed nuclear element (SINE1). Silencing remained in effect even after removal of the plasmid (clone G3). Neither short interfering RNA nor methylated DNA were detected, but the chromatin domain of Ehap-a in the gene-silenced trophozoites was modified. Two other similar genes (Ehap-b and one encoding a Saposin-like protein, SAPLIP 1) also became silenced. In the present work we demonstrate the silencing of a second gene of choice, one that encodes the light subunit of the Gal/GalNAc inhibitable lectin (Ehlgl1) and the other, the cysteine proteinase 5 (EhCP-5). This silencing occurred in G3 trophozoites transfected with a plasmid in which the 473 bp 5' upstream Ehap-a fragment was directly ligated to the second gene. Transcriptional silencing occurred in both the transgene and the chromosomal gene. SINE1 sequences were essential, as was a direct connection between the Ehap-a upstream region and the beginning of the open reading frame of the second gene. Gene silencing did not occur in strain HM-1:IMSS with any of these plasmid constructs. The trophozoites with two silenced genes were virulence-attenuated as were those of clone G3. In addition, trophozoites not expressing Lgl1 and AP-A proteins had a significantly reduced ability to cap the Gal/GalNAc-lectin to the uroid region when incubated with antibodies against the heavy (170 kDa) subunit of the lectin. Lysates of trophozoites lacking cysteine proteinase 5 and AP-A proteins had 30% less cysteine proteinase activity than those of HM-1:IMSS strain or the G3 clone. Silencing of other genes in G3 amoebae could provide a model to study their various functions. In addition, double gene-silenced, virulence-attenuated trophozoites may be an important tool in vaccine development.

Project description:The cyst stage of Entamoeba histolytica is a promising therapeutic target against human amoebiasis. Our research team previously reported the production in vitro of Cyst-Like Structures (CLS) sharing structural features with cysts, including rounded shape, size reduction, multinucleation, and the formation of a chitin wall coupled to the overexpression of glucosamine 6-phosphate isomerase, the rate-limiting enzyme of the chitin synthesis pathway. A proteomic study of E. histolytica trophozoites, cysts, and in vitro-produced CLS is reported herein to determine the nature of CLS, widen our knowledge on the cyst stage, and identify possible proteins and pathways involved in the encystment process. Total protein extracts were obtained from E. histolytica trophozoites, CLS, and partially purified cysts recovered from the feces of amoebic human patients; extracts were trypsin-digested and analyzed by LC-MS/MS. In total, 1029 proteins were identified in trophozoites, 550 in CLS, and 411 in cysts, with 539, 299, and 84 proteins unique to each sample, respectively, and only 74 proteins shared by all three stages. About 70% of CLS proteins were shared with trophozoites, even though differences were observed in the relative protein abundance. While trophozoites showed a greater abundance of proteins associated to a metabolically active cell, CLS showed higher expression of proteins related to proteolysis, redox homeostasis, and stress response. In addition, the expression of genes encoding for the cyst wall proteins Jessie and Jacob was detected by RT-PCR and the Jacob protein identified by Western blotting and immunofluorescence in CLS. However, the proteomic profile of cysts as determined by LC-MS/MS was very dissimilar to that of trophozoites and CLS, with almost 40% of hypothetical proteins. Our global results suggest that CLS are more alike to trophozoites than to cysts, and they could be generated as a rapid survival response of trophozoites to a stressful condition, which allows the parasite to survive temporarily inside a chitin-like resistant cover containing Jacob protein. Our findings lead us to suggest that encystment and CLS formation could be distinct stress responses. In addition, we show that cysts express a high number of genes with unknown function, including four new, highly antigenic, possibly membrane-located proteins that could be targets of therapeutic and diagnostic usefulness.

Dataset Information

Synthetic bovine lactoferrin peptide Lfampin kills Entamoeba histolytica trophozoites by necrosis and resolves amoebic intracecal infection in mice.

Publications

Synthetic bovine lactoferrin peptide Lfampin kills <i>Entamoeba histolytica</i> trophozoites by necrosis and resolves amoebic intracecal infection in mice.

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