Fig 1.
In vitro antimalarial activity of LL-37 and its truncated peptides.
(a) Helical wheel analysis of antimicrobial peptide LL-37. The helical wheel diagram was generated using HeliQuest to visualize the spatial distribution of residues. Residues are color-coded by physicochemical properties: yellow (hydrophobic: I, L,V,F), blue (positively charged: K,R), purple (uncharged polar: S,T), red (negatively charged: D,E), pink (uncharged polar amide: Q), green (non-polar: P), gray (aliphatic: G,A). The projection angle is rotated to maximize amphipathicity with hydrophobic residues (yellow) clustered on one face and polar/charged residues (blue/red/purple) on the opposing face. (b) Hydrophobicity profile of antimicrobial peptide LL-37. Residue numbers are indicated on the x-axis. Hydrophobicity index was calculated using the Kyte-Doolittle scale (window size = 5). Positive values indicate hydrophobic regions, negative values indicate hydrophilic regions. (c) IC50s of LL-37 and its truncated peptide against P. falciparum 3D7. (d) IC50s of LL-37 against sensitive and resistant strains of Plasmodium falciparum. Data in c and d are presented as means ± SEM from three biological replicates.
Fig 2.
Stage-specific and time-dependent antiplasmodial activity of LL-37 in the blood stage.
(a) Schematic of the experimental design. Highly synchronized P. falciparum 3D7 parasites were treated with LL-37 at its IC50 for 1, 3, 6, 9 and 12 h across three consecutive periods of the intraerythrocytic life cycle. Subsequently, the parasitemia of re-invasion was analyzed at 50 hpi, and another analysis was conducted after a 1:40 dilution and 4 days of culture. (b) Viability of parasites re-invasion at 50 hpi was measured. (c) Parasites were washed, followed by 1:40 dilution, and cultured for 4 days while parasitemia was measured. Data in b and c are presented as means ± SEM of 10,000 RBCs from three biological replicates.
Fig 3.
LL-37 selectively targets infected erythrocytes by exploiting synergistic phosphatidylserine externalization and cholesterol depletion.
(a) Comparative hemolysis of uninfected erythrocytes (uRBC) and schizont-infected erythrocytes (iRBC) exposed to LL-37 at its IC50 and 10 × IC50 concentrations. (b) Stage-specific hemolysis of P. falciparum-infected erythrocytes treated with LL-37 (IC50). R: Ring stage; T: Trophozoite stage; S: Schizont stage. (c) Hemolysis of LL-37 under increasing A23187 concentrations with or without 1.5 mM MβCD. (d) Hemolysis of LL-37 under increasing concentrations of MβCD with or without 0.75 μM calcium ionophore A23187. (e) Parasite parasitemia under treatment with different proportions of MβCD-cholesterol complexes with or without LL-37 present. (f) Parasite parasitemia under treatment with different concentrations of DOTAP with or without LL-37 present. (g) Parasite parasitemia of LL-37 treatment with MβCD-cholesterol complexes and/or DOTAP. All data are presented as means ± SEM from three biological replicates. ns: not significant; *p < 0.05, *p < 0.01, ***p < 0.001 indicate statistically significant differences.
Fig 4.
Elevated plasma levels of LL-37 and CRAMP in malaria-infected individuals and Plasmodium- infected mice.
(a) Plasma levels of LL-37 in Plasmodium falciparum (P.f) - infected patients and healthy donors. (b) Plasma levels of CRAMP in Plasmodium berghei (P.b) - infected mice and non-infected control mice. Each dot represents an individual sample. Horizontal lines indicate the median values. ***p < 0.001 indicate statistically significant differences.
Fig 5.
Exogenous LL-37 and CRAMP treatment confers antiplasmodial and anti-pathological effects.
(a) Representative images of blood smears (day 4 post-infection) following treatments with different concentrations of LL-37 and CRAMP (n = 6 mice per group). (b) Dynamic changes in parasitemia from day 0 to day 4 post-P. berghei ANKA infection in untreated control mice and mice treated with LL-37/CRAMP (1, 4, 16 mg/kg/day, intravenous administration for 4 consecutive days). Parasitemia was determined by microscopic examination of Giemsa-stained thin blood smears at 24 h intervals. Data are presented as mean ± SEM (n = 6 mice per group). (c) Quantification of parasitemia (day 4, n = 6 mice/group) in mice treated with different concentrations of LL-37 and CRAMP. Data are presented as mean ± SEM (n = 6 mice per group). (d) Survival curves of mice administered different concentrations of LL-37 and CRAMP (day 4, n = 6 mice per group). Statistical analysis: Log-rank (Mantel-Cox) test, ns: not significant; *p < 0.05, **p < 0.01 vs. NS group. (e–h) Quantification of various inflammatory cytokines in mice treated with different concentrations of LL-37 and CRAMP (day 4, n = 6 mice per group). (i) Representative H&E-stained sections of liver, spleen, and brain tissues (day 4, n = 3 mice per group) from mice under distinct conditions: normal control, infected control, CQ positive control, and treatment with different concentrations of LL-37 or CRAMP. Black arrows point to malarial pigment; red arrows indicate the sequestration of RBCs; yellow arrows denote inflammatory cells; yellow boxes show fat vacuoles; red boxes indicate RePu or WhPu. Scale bar = 10 μm. ns: not significant; **p < 0.01, ***p < 0.001 indicate statistically significant differences. In all panels, NS (normal saline) served as a negative control, treated with normal saline (0.9% NaCl); NC (normal control) denotes the normal uninfected control group, serving as a baseline for normal physiological status and CQ (chloroquine) as a positive control.
Fig 6.
Endogenous cathelicidin (Cramp) is essential for parasite control.
(a) Representative images of blood smears from C57BL/6 mice (day 4 post-infection), Cramp⁻/⁻ mice. Scale bar = 5 μm. (b) Quantification of parasitemia (day 4, n = 6 mice/group) in C57BL/6 mice, Cramp⁻/⁻ mice. (c) Survival curves (day 21, n = 6 mice/group) of C57BL/6 mice, Cramp⁻/⁻ mice after infection. (d–g) Quantification of plasma levels of IL-1β (d), IL-6 (e), TNF-α (f), and IFN-γ (g) (day 4, n = 6 mice/group) in C57BL/6 mice, Cramp⁻/⁻ mice. (h) Representative H&E-stained sections of liver, spleen, and brain tissues (day 4, n = 3 mice/group) from C57BL/6 mice, Cramp⁻/⁻ mice. Black arrows point to malarial pigment; red arrows indicate the sequestration of RBCs; yellow arrows denote inflammatory cells; red boxes indicate RePu or WhPu. Scale bar = 10 μm. ns: not significant; **p < 0.01, ***p < 0.001 indicate statistically significant differences.