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. 2024 Sep 18;16(37):49148-49163.
doi: 10.1021/acsami.4c10426. Epub 2024 Sep 6.

A Targeted and Protease-Activated Genetically Encoded Melittin-Containing Particle for the Treatment of Cutaneous and Visceral Leishmaniasis

Madiha Habib  1 , Jiale Zheng  1 , Chin-Fung Chan  2 , Zaofeng Yang  1 , Iris L K Wong  2 , Larry M C Chow  2 , Marianne M Lee  1 , Michael K Chan  1
Affiliations

A Targeted and Protease-Activated Genetically Encoded Melittin-Containing Particle for the Treatment of Cutaneous and Visceral Leishmaniasis

Madiha Habib et al. ACS Appl Mater Interfaces. .

Abstract

Intracellular infections are difficult to treat, as pathogens can take advantage of intracellular hiding, evade the immune system, and persist and multiply in host cells. One such intracellular parasite, Leishmania, is the causative agent of leishmaniasis, a neglected tropical disease (NTD), which disproportionately affects the world's most economically disadvantaged. Existing treatments have relied mostly on chemotherapeutic compounds that are becoming increasingly ineffective due to drug resistance, while the development of new therapeutics has been challenging due to the variety of clinical manifestations caused by different Leishmania species. The antimicrobial peptide melittin has been shown to be effective in vitro against a broad spectrum of Leishmania, including species that cause the most common form, cutaneous leishmaniasis, and the most deadly, visceral leishmaniasis. However, melittin's high hemolytic and cytotoxic activity toward host cells has limited its potential for clinical translation. Herein, we report a design strategy for producing a melittin-containing antileishmanial agent that not only enhances melittin's leishmanicidal potency but also abrogates its hemolytic and cytotoxic activity. This therapeutic construct can be directly produced in bacteria, significantly reducing its production cost critical for a NTD therapeutic. The designed melittin-containing fusion crystal incorporates a bioresponsive cathepsin linker that enables it to specifically release melittin in the phagolysosome of infected macrophages. Significantly, this targeted approach has been demonstrated to be efficacious in treating macrophages infected with L. amazonensis and L. donovani in cell-based models and in the corresponding cutaneous and visceral mouse models.

Keywords: Cry3Aa crystal protein; Leishmania; antileishmanial peptides; melittin; parasites.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Scheme 1
Scheme 1. Schematic Illustration of Cry3Aa-AMP Fusion Crystals and Their Use in Treating Leishmaniasis Created with Biorender.com
(A) The design and production of Cry3Aa-AMP fusion crystals. (B) Leishmania-infected mouse injected with Cry3Aa-AMP fusion crystals. Preferential uptake of the Cry3Aa-AMP fusion crystals by infected macrophages and the stimuli-responsive release of the AMP from the Cry3Aa therapeutic particle mediated by cathepsin D/E enzymes whose presence is elevated in the acidic environment of infected macrophages.
Figure 1
Figure 1
Antileishmanial activity of melittin and miltefosine. (A) Antipromastigote activity of melittin peptide and miltefosine drug against L. amazonensis LV78 at different concentrations. (B) Development of resistance in L. amazonensis LV78 against melittin peptide and miltefosine. Values represent the fold change in IC50 (log2) from the starting IC50 at passage 0.
Figure 2
Figure 2
Construction of Cry3Aa-AMP fusion crystals and their antileishmanial activity. (A) Amino acid sequences of promelittin, melittin peptide, and its variants used in the construction of the Cry3Aa-AMP fusion crystals. (B) SDS-PAGE analysis of Cry3Aa-AMP fusion crystals. Cry3Aa-mPMLT, Cry3Aa-mMLT, Cry3Aa-MLT, and Cry3Aa-PMLT were successfully expressed in Bt cells and purified by sucrose gradient centrifugation. The molecular weight of Cry3Aa is ∼73 kDa, Cry3Aa-MLT and Cry3Aa-mMLT are ∼75 kDa, and Cry3Aa-PMLT and Cry3Aa-mPMLT are ∼78 kDa. M = molecular weight marker. The bands in each lane slightly greater than 15 kDa are residual lysozyme added during purification. (C–F) Cry3Aa-mediated delivery of melittin to Leishmania parasite-infected peritoneal elicited macrophages (PEMs). Quantification of (C) L. donovani LU3 amastigotes and (D) L. amazonensis LV78 amastigotes in at least 100 randomly selected PEMs after treatment with 0.8 μM of Cry3Aa-AMP fusion crystals or Cry3Aa crystals or free AMP peptides for 72 h. Representative light microscope images of (E) L. donovani LU3-infected PEMs and (F) L. amazonensis LV78-infected PEMs. The cells were fixed and stained with Giemsa for parasite enumeration. The control groups were untreated parasite-infected-PEMs. Red arrows indicate the presence of parasites in macrophages. ****P < 0.0001 and ***P < 0.001. ns, not significant.
Figure 3
Figure 3
Hemolytic activity and cytotoxicity of Cry3Aa-AMP fusion crystals. (A,B) RBCs were treated with different concentrations (0.1–1.6 μM) of Cry3Aa-mMLT and Cry3Aa-mPMLT fusion crystals and their corresponding free peptide counterparts for 4 h at 37 °C. (A) Note that the free melittin peptides readily lysed the RBCs, while no RBC lysis was observed for the Cry3Aa-mMLT fusion crystals. (B) Release of hemoglobin after RBC lysis could be easily observed due to the color of the supernatant turning red. Triton X-100 was used as a positive control, while PBS solvent was employed as a negative control. (C) Percentage of viable PEMs after treatment with Cry3Aa control, fusion crystals (Cry3Aa-mPMLT and Cry3Aa-mMLT), and their corresponding peptides is shown. ****P < 0.0001. ns, not significant.
Figure 4
Figure 4
Cellular internalization and colocalization of Cry3Aa-AMP fusion crystals into the lysosomes of macrophages. (A–B) Representative confocal images of RAW 264.7 cells treated with Alexa 647-labeled (red) (A) Cry3Aa-mPMLT fusion crystal and (B) Cry3Aa-mMLT fusion crystal taken at different time points. Efficient internalization of the Cry3Aa-AMP crystals into macrophages could be observed as soon as at the 4 h time point based on the strong red fluorescent signals observed in most cells. Their colocalization with lysosomes is indicated as yellow punctate dots (white arrow) in the merged images. Nuclei of macrophages were stained with Hoechst 33342 (blue), and lysosomes were stained with lysotracker (green). Scale bars: 10 μm. (C,D) Corresponding colocalization intensity profiles of (A) and (B). Macrophages boxed in white in the merged images in (A–B) were used for determining the fluorescence intensity profile generated using ImageJ software. (E–G) Flow cytometric analysis of fusion crystals internalized by macrophages upon incubation with different lengths of time (4, 12, and 24 h). (E) Staggered histograms of the macrophages internalized with labeled fusion crystals and negative controls at different time points. (F) Percentage of macrophages loaded with Alexa 647 labeled fusion crystals and their (G) mean fluorescent intensities (MFI).
Figure 5
Figure 5
Characterization of Cry3Aa-mPMLT fusion crystals. (A) SEM micrograph and (B) size distribution and (C) zeta potential of the Cry3Aa-mPMLT fusion crystal. (D) LU3-infected PEMs were treated with either pepstatin A (20 μg/mL) or Cry3Aa-mPMLT fusion crystals (0.8 μM) or a combination of pepstatin A and Cry3Aa-mPMLT for 72 h at 37 °C. The control groups were untreated parasite-infected-PEMs. At least 100 macrophages were randomly selected across three coverslips of each experimental group for parasite counting. (E–F) Serum stability of Cry3Aa-mPMLT fusion crystals. In vitro antileishmanial activity of Cry3Aa-mPMLT fusion crystals was evaluated by preincubation in serum. (E) Corresponding quantification of the number of L. amazonensis LV78 amastigotes in 100 macrophages randomly selected for parasite enumeration. (F) Representative images of LV78-infected PEMs treated with Cry3Aa-mPMLT fusion crystals that were preincubated in human serum for different periods of time (0, 0.5, 2, 4, 6, 8, and 12 h). The control groups were untreated parasite-infected-PEMs. The presence of parasites in macrophages was indicated by the red arrows. ****P < 0.0001 and ***P < 0.001. ns, not significant.
Figure 6
Figure 6
In vivo cytotoxicity of Cry3Aa-mMPLT fusion crystals. (A) Body weight of mice before and after treatment with different dose levels of Cry3Aa-mMPLT fusion crystals (n = 5) and the PBS control (n = 8). (B) Organ indices of the mice treated with a single dose of Cry3Aa-mPMLT fusion crystals for 24 h. (C–E) Serum levels of the (C) ALT, (D) AST, and (E) creatinine of the blood samples of the fusion crystal-treated mice collected 24 h postadministration. (F) Representative images of the H & E-stained liver, spleen, heart, lungs, and kidney tissues of the differentially treated groups. No obvious injuries or anomalies were observed. ns, not significant.
Figure 7
Figure 7
In vivo antileishmanial activity of Cry3Aa-mPMLT and Cry3Aa-mMLT fusion crystals in a mouse model of cutaneous leishmaniasis. (A) Study protocol used for developing footpad Leishmania mouse model and treatment schedule. Schematic created with Biorender.com. (B) Thickness of lesions in the left footpads of Balb/c mice infected with L. amazonensis LV78 and treated with either PBS, the Cry3Aa crystal group, or the fusion crystal group (Cry3Aa-mMLT and Cry3Aa-mPMLT) over time. Black arrows indicate the days of injections. (C) Representative images showing the morphologic appearance of the differentially treated footpads at day 46 post infection. Significant inflammation and footpad swelling could be observed for the PBS, Cry3Aa, and Cry3Aa-mMLT groups. (D) Parasite burden after the in vitro culturing of the L. amazonensis LV8 parasites recovered from the lesions of the differentially treated mice at day 62 post infection. ***P < 0.001 and **P < 0.01. ns, not significant.
Figure 8
Figure 8
Visceral Leishmania mouse model. (A) Schematic of study protocol used for developing visceral Leishmania mouse model and treatment schedule created with Biorender.com. (B) Body weight of Balb/c mice infected with L. donovani LU3 remained within a normal range during the entire treatment period (n = 11). The black arrows represent the days of injection subsequent to the onset of infection. (C) Number of amastigotes per 1000 nucleated cells was counted as Leishman–Donovan units (LDU) in an individual mouse liver smear. (D) LU3-infected liver tissue smear stained with Giemsa. Black arrows indicate amastigotes inside parasitized macrophages. Data were from two independent trials. **P < 0.01. ns, not significant.
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