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Cited by 23Amoebic Infections and TreatmentsAntimicrobial Peptides and ActivitiesLegionella and Acanthamoeba research

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

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Díaz-Godínez, César, González-Galindo, Ximena, Meza-Menchaca, Thuluz, Bobes, Raúl J., de la Garza, Mireya, León-Sicairos, Nidia, Laclette, Juan P., Carrero, Julio C.·2019·Bioscience Reports
DOI 10.1042/bsr20180850PMC PMC6328891
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Abstract

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 μM) showed that Lfampin is far more amoebicidal than Lfcins. Lfampin killed 80% of trophozoites at doses higher than 100 μM 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.

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Figure 1 Lactoferrin (Lf)-derived peptides exhibit varying amoebicidal activity Trophozoites (2 × 104) were cultured in fresh TYI-S-33 medium in the presence of (A) Lfcin-B, (B) Lfcin 17-30, (C) Lfampin, or (D) the three peptides together at crescent concentrations for 24 h at 37°C. After incubation, cells were centrifuged and the supernatant was removed, preserving 100 μl of medium. MTT solution was added and trophozoites were incubated for 1 h at 37°C. Finally, 60°C SDS–HCl was added to lyse the cells and dissolve formazan crystals. Absorbency was measured at 595 nm within 15 min. Values are presented as means ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 1 Lactoferrin (Lf)-derived peptides exhibit varying amoebicidal activity Trophozoites (2 × 104) were cultured in fresh TYI-S-33 medium in the presence of (A) Lfcin-B, (B) Lfcin 17-30, (C) Lfampin, or (D) the three peptides together at crescent concentrations for 24 h at 37°C. After incubation, cells were centrifuged and the supernatant was removed, preserving 100 μl of medium. MTT solution was added and trophozoites were incubated for 1 h at 37°C. Finally, 60°C SDS–HCl was added to lyse the cells and dissolve formazan crystals. Absorbency was measured at 595 nm within 15 min. Values are presented as means ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.CC BY · PubMed Central
Figure 2 Evaluation of apoptosis and necrosis in trophozoites treated with lactoferrin (Lf)-derived peptides Apoptosis assays were performed using the FITC Annexin V Apoptosis Detection Kit I (BD Pharmigen), following the manufacturer’s instructions. (A) In brief, trophozoites (5 × 104) were treated with 250 μM Lfcin-B, Lfcin 17-30, or Lfampin peptides for 24 h. Treated trophozoites were resuspended in binding buffer, to which annexin V/PI stain was added. The samples were analyzed by flow cytometry in a FACScalibur (Beckton Dickinson). (i) Autofluorescence control; (ii) compensation for FITC fluorescence; (iii) compensation for PI fluorescence; (iv) untreated trophozoites; (v) heat-treated trophozoites; (vi) Lfcin-B-treated trophozoites; (vii) Lfcin 17-30-treated trophozoites; and (viii) Lfampin-treated trophozoites. (B) Percentage of apoptotic cells (only annexin V positive). ***P < 0.01.
Figure 2 Evaluation of apoptosis and necrosis in trophozoites treated with lactoferrin (Lf)-derived peptides Apoptosis assays were performed using the FITC Annexin V Apoptosis Detection Kit I (BD Pharmigen), following the manufacturer’s instructions. (A) In brief, trophozoites (5 × 104) were treated with 250 μM Lfcin-B, Lfcin 17-30, or Lfampin peptides for 24 h. Treated trophozoites were resuspended in binding buffer, to which annexin V/PI stain was added. The samples were analyzed by flow cytometry in a FACScalibur (Beckton Dickinson). (i) Autofluorescence control; (ii) compensation for FITC fluorescence; (iii) compensation for PI fluorescence; (iv) untreated trophozoites; (v) heat-treated trophozoites; (vi) Lfcin-B-treated trophozoites; (vii) Lfcin 17-30-treated trophozoites; and (viii) Lfampin-treated trophozoites. (B) Percentage of apoptotic cells (only annexin V positive). ***P < 0.01.CC BY · PubMed Central
Figure 3 Interaction of lactoferrin (Lf)-derived peptides with E. histolytica trophozoites (A) Trophozoites (1 × 105) were cultured in 200 μl of fresh TYI-S-33 medium and treated with 50 μM FITC-labeled Lfcin-B, Lfcin 17-30, or Lfampin for 5 min on ice chilling (blue histogram) or 2 h at 37°C (red histogram). After incubation, the trophozoites were washed with PBS and then fixed with formaldehyde. The samples were analyzed by flow cytometry in a FACScalibur. Non-stained trophozoites were used as the autofluorescence control (dotted line histogram). R1 positive region was determined selecting all values higher than the autofluorescence control. R2 region (binding peptide) was determined selecting 90% of values at 5 min of incubation on ice chilling. R3 region (bound and internalized peptide) was defined as all values higher than R2. (B) Interacting peptide established as mean intensity fluorescence values at 5 min and 2 h (FRU, fluorescence relative unities). Values are presented as means ± SD of three independent experiments.
Figure 3 Interaction of lactoferrin (Lf)-derived peptides with E. histolytica trophozoites (A) Trophozoites (1 × 105) were cultured in 200 μl of fresh TYI-S-33 medium and treated with 50 μM FITC-labeled Lfcin-B, Lfcin 17-30, or Lfampin for 5 min on ice chilling (blue histogram) or 2 h at 37°C (red histogram). After incubation, the trophozoites were washed with PBS and then fixed with formaldehyde. The samples were analyzed by flow cytometry in a FACScalibur. Non-stained trophozoites were used as the autofluorescence control (dotted line histogram). R1 positive region was determined selecting all values higher than the autofluorescence control. R2 region (binding peptide) was determined selecting 90% of values at 5 min of incubation on ice chilling. R3 region (bound and internalized peptide) was defined as all values higher than R2. (B) Interacting peptide established as mean intensity fluorescence values at 5 min and 2 h (FRU, fluorescence relative unities). Values are presented as means ± SD of three independent experiments.CC BY · PubMed Central
Figure 4 E. histolytica trophozoites internalize lactoferrin (Lf)-derived peptides Trophozoites (1 × 105) were cultured in 200 μl of fresh TYI-S-33 medium with Hoechst and treated with 50 μM FITC-labeled peptides for 2 h at 37°C. After incubation, the trophozoites were washed with PBS and fixed with formaldehyde. Amoebas were visualized by fluorescence microscopy using a Nikon A1R+ confocal microscope. Scale bar = 20 μm.
Figure 4 E. histolytica trophozoites internalize lactoferrin (Lf)-derived peptides Trophozoites (1 × 105) were cultured in 200 μl of fresh TYI-S-33 medium with Hoechst and treated with 50 μM FITC-labeled peptides for 2 h at 37°C. After incubation, the trophozoites were washed with PBS and fixed with formaldehyde. Amoebas were visualized by fluorescence microscopy using a Nikon A1R+ confocal microscope. Scale bar = 20 μm.CC BY · PubMed Central
Figure 5 Internalization pathway of FITC-coupled lactoferrin-derived Lfampin into E. histolytica trophozoites Amoebas (1 × 105) were cultured in fresh TYI-S-33 medium in the presence of 50 μM FITC-labeled Lfampin for 2 h at 37°C. Cells were fixed and immunofluorescence was performing using anti-clathrin HC, anti-EEA1, anti-MPR, and anti-LAMP-1 antibodies, followed by anti-mouse IgG conjugated to rhodamine (for clathrin HC and MPR), anti-rat IgG conjugated to Alexa Fluor 594 (for LAMP-1), or anti-rabbit conjugated to TRITC (for EEA1). White arrows indicate vesicles that appear to interact with vacuoles and asterisks indicate tubular-like structures. Scale bar = 5 μm.
Figure 5 Internalization pathway of FITC-coupled lactoferrin-derived Lfampin into E. histolytica trophozoites Amoebas (1 × 105) were cultured in fresh TYI-S-33 medium in the presence of 50 μM FITC-labeled Lfampin for 2 h at 37°C. Cells were fixed and immunofluorescence was performing using anti-clathrin HC, anti-EEA1, anti-MPR, and anti-LAMP-1 antibodies, followed by anti-mouse IgG conjugated to rhodamine (for clathrin HC and MPR), anti-rat IgG conjugated to Alexa Fluor 594 (for LAMP-1), or anti-rabbit conjugated to TRITC (for EEA1). White arrows indicate vesicles that appear to interact with vacuoles and asterisks indicate tubular-like structures. Scale bar = 5 μm.CC BY · PubMed Central
Figure 6 Internalization of lactoferrin-derived peptides depends on receptor-mediated endocytosis and actin cytoskeleton dynamics Trophozoites (5 × 105) were incubated for 1 h in TYI-S-33 medium added with Wortmannin (100 µM), cytochalasin B (10 µg/ml), or 1% DMSO (vehicle). Amoebas were then incubated with 50 µM FITC-labeled peptides for 2 h at 37°C. After incubation, cells were washed with cold PBS. Trophozoites were resuspended in cold PBS and 2 mg/ml Trypan blue was added. The samples were analyzed by flow cytometry in a FACScalibur.
Figure 6 Internalization of lactoferrin-derived peptides depends on receptor-mediated endocytosis and actin cytoskeleton dynamics Trophozoites (5 × 105) were incubated for 1 h in TYI-S-33 medium added with Wortmannin (100 µM), cytochalasin B (10 µg/ml), or 1% DMSO (vehicle). Amoebas were then incubated with 50 µM FITC-labeled peptides for 2 h at 37°C. After incubation, cells were washed with cold PBS. Trophozoites were resuspended in cold PBS and 2 mg/ml Trypan blue was added. The samples were analyzed by flow cytometry in a FACScalibur.CC BY · PubMed Central
Figure 7 Clustering of free energy of binding vs calculated Ki for lactoferrin (Lf)-derived peptides docked to E. histolytica molecules and cholesterol Molecular dataset of hierarchical clustering plot from two different docking calculation datasets against the peptides. One subset of molecules (red dots) displayed belong to Lfcin 17-30, the second subset (blue dots) to Lfcin-B, and the third subset (green dots) to Lfampin. Numbers on the plot correspond to the following molecules in order of best docking scores: (1) deoxyuridine 5-triphosphate nucleotidohydrolase (3LQW), (2) Protein G (4FID), (3) cholesterol (5997), (4) triplet AAA, (5) clathrin B (10588140), (6) EhCBP1 (5XOP), (7) phenylkainic acid (10933893), (8) Dbr1 (5K73), (9) HP1 (2M2L), and (10) α-actinin-1 (2M7L).
Figure 7 Clustering of free energy of binding vs calculated Ki for lactoferrin (Lf)-derived peptides docked to E. histolytica molecules and cholesterol Molecular dataset of hierarchical clustering plot from two different docking calculation datasets against the peptides. One subset of molecules (red dots) displayed belong to Lfcin 17-30, the second subset (blue dots) to Lfcin-B, and the third subset (green dots) to Lfampin. Numbers on the plot correspond to the following molecules in order of best docking scores: (1) deoxyuridine 5-triphosphate nucleotidohydrolase (3LQW), (2) Protein G (4FID), (3) cholesterol (5997), (4) triplet AAA, (5) clathrin B (10588140), (6) EhCBP1 (5XOP), (7) phenylkainic acid (10933893), (8) Dbr1 (5K73), (9) HP1 (2M2L), and (10) α-actinin-1 (2M7L).CC BY · PubMed Central
Figure 8 Representative images of cecal tissue sections from infected mice treated with lactoferrin (Lf)-derived peptides C3H/HeJ mice intracecally infected with E. histolytica trophozoites were treated at 15 days post-infection via the oral route with 10 mg/kg of each peptide for 4 days. Animals were killed 2 days later and ceca were processed for histopathological analysis. (A and B) Tissue sections from Sham mice showing abundant amoebas in the intestinal lumen, with erosion of the enteric epithelia. (C and D) Tissue sections from Lfcin 17-30- and Lfcin-B-treated mice, respectively, showing reduction in the load of amoebas contained in the intestinal lumen. (E and F) Tissue sections from Lfampin-treated mice showing complete absence of amoebas in the intestinal lumen, indicating eradication of the infection. H&E staining. Arrows show amoebas in intestinal lumen. Scale bar = 100 μm.
Figure 8 Representative images of cecal tissue sections from infected mice treated with lactoferrin (Lf)-derived peptides C3H/HeJ mice intracecally infected with E. histolytica trophozoites were treated at 15 days post-infection via the oral route with 10 mg/kg of each peptide for 4 days. Animals were killed 2 days later and ceca were processed for histopathological analysis. (A and B) Tissue sections from Sham mice showing abundant amoebas in the intestinal lumen, with erosion of the enteric epithelia. (C and D) Tissue sections from Lfcin 17-30- and Lfcin-B-treated mice, respectively, showing reduction in the load of amoebas contained in the intestinal lumen. (E and F) Tissue sections from Lfampin-treated mice showing complete absence of amoebas in the intestinal lumen, indicating eradication of the infection. H&E staining. Arrows show amoebas in intestinal lumen. Scale bar = 100 μm.CC BY · PubMed Central

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Research works printed but not linked: 2

  1. Leon-Sicairos, 2006, Microbicidal action of lactoferrin and lactoferricin and their synergistic effect with metronidazole in Entamoeba histolytica, Biochem. Cell Biol., 84, 327
  2. Samarawickrema, 1997, Involvement of superoxide dismutase and pyruvate:ferredoxin oxidoreductase in mechanisms of metronidazole resistance in Entamoeba histolytica, J. Antimicrob. Chemother., 40, 833, 10.1093/jac/40.6.833 · DOI

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Authors
Díaz-Godínez, C., González-Galindo, X., Meza-Menchaca, T., Bobes, R. J., de la Garza, M., León-Sicairos, N., Laclette, J. P., & Carrero, J. C.
Affiliations
  • Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México (MX)
  • Laboratorio de Genómica Humana, Facultad de Medicina, Universidad Veracruzana, Calle Médicos y Odontólogos S/N., Col. Unidad del Bosque, 91010, Xalapa, Veracruz, México
  • Department of Cellular Biology, Centro de Investigación y de Estudios Avanzados del IPN, Av. IPN 2508, San Pedro Zacatenco, 07360 México, D.F., México
  • Universidad Autónoma de Sinaloa (MX)
Corresponding author
Carrero, Julio C.
ORCID
Venue
Bioscience Reports
Publisher
Portland Press Ltd.
Published
2019-01
Volume
39
Issue
1
ISSN
  • 0144-8463 (ISSN-L)
  • 1573-4935
DOI
10.1042/bsr20180850
PMC
PMC6328891
OpenAlex
W2900943109
Cited by
23
References
38
Citations by year
2026: 1 · 2025: 3 · 2024: 4 · 2023: 6 · 2022: 3 · 2020: 6
FWCI
1.13
Type
journal article
Language
en
Open access
Licence
Locations
  • Bioscience Reports (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, other-oa)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Greater South Information System
Funders
Consejo Nacional de Ciencia y Tecnología (284830)
Topics
  • Health Sciences › Medicine › Infectious Diseases › Amoebic Infections and Treatments
  • Life Sciences › Immunology and Microbiology › Microbiology › Antimicrobial Peptides and Activities
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Endocrinology › Legionella and Acanthamoeba research
Keywords
Entamoeba histolytica, amoebiasis, lactoferrin-derived peptides, amoebicidal activity, necrosis, peptide internalization, PI3K signaling, cholesterol, plasma membrane
MeSH terms
Animals, Antiprotozoal Agents, Cattle, Entamoeba histolytica, Entamoebiasis, Lactoferrin, Male, Mice, Inbred C3H, Necrosis, Peptides, Phosphatidylinositol 3-Kinases, Mice, Trophozoites

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