Pumilacidins from the Octocoral-Associated Bacillus sp. DT001 Display Anti-Proliferative Effects in Plasmodium falciparum

Abstract

Chemical examination of the octocoral-associated Bacillus species (sp.) DT001 led to the isolation of pumilacidins A (1) and C (2). We investigated the effect of these compounds on the viability of Plasmodium falciparum and the mechanism of pumilacidin-induced death. The use of inhibitors of protein kinase C (PKC) and phosphoinositide 3-kinase (PI3K) was able to prevent the effects of pumilacidins A and C. The results indicated also that pumilacidins inhibit parasite growth via mitochondrial dysfunction and decreased cytosolic Ca²⁺.

Keywords: Bacillus; P. falciparum; apoptosis; coral-associated bacteria; pumilacidins; surfactins.

Figure 1

Structures of pumilacidins A and C.

Figure 2

Effect of pumilacidins on the proliferation of Plasmodium falciparum. (A) Ring stage; (B) schizont stage; (C) chloroquine (CQ) control in ring stage. Plasmodium parasites were incubated together with pumilacidins A and C at 10, 2, 0.4, and 0.08 μM, and CQ at 1000, 10, and 0.1 nM for 48 h. The proliferation of pumilacidin-treated parasites was measured by PicoGreen DNA fluorometric measurement. Data represent the means ± standard error of the mean (SEM) of one representative observation out of three experiments performed in duplicate. ** p < 0.01, *** p < 0.001.

Figure 3

Effect of pumilacidins on the proliferation of P. falciparum. Plasmodium parasites were cultured in the presence of pumilacidins A and C at 10 μM for 24 h. The proliferation of pumilacidin-treated parasites was measured by flow cytometry quantifying their DNA content with Hoechst 33342 staining. Data represent the means ± SEM of two independent observations performed in triplicate. * p < 0.05.

Figure 4

Effect of pumilacidins on hemolytic activity in red blood cells (RBCs). Red blood cells were incubated with pumilacidins at a concentration of 10 μM for 24 h. Data represent the percentage of hemolysis relative to 0.1% Triton as a positive control (100%) of one representative experiment run in triplicate. *** p < 0.001.

Figure 5

Effect of pumilacidins on cell-membrane integrity. Infected and uninfected red blood cells were cultured with pumilacidins A and C at 10 μM for 3 h. As a positive control, heating at 80 °C was used. Integrity of the membranes was determined by the incorporation of propidium iodide (PI) measured by flow cytometry and plotted as mean fluorescence intensity (MFI). Data represent the means ± SEM of one representative experiment performed in triplicate. *** p < 0.001.

Figure 6

Morphological changes induced by pumilacidins A and C. Panel A1 and A2: Untreated parasites. Panel B1 and B2: P. falciparum-infected erythrocytes in the schizont stage were treated with 10 μM pumilacidins A and C, and their morphology was analyzed 48 h after treatment through Giemsa-stained smears.

Figure 7

Mitochondrial membrane potentials. Changes in the mitochondrial membrane potentials relative to their untreated controls were measured with the mitochondrial potential-dependent dye DiO6 using flow cytometry after 3 h (A), 6 h (B), and 9 h (C) post treatment with pumilacidins A and C at 10 μM in the schizont stage of the parasites. * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 8

Reactive oxygen species (ROS) generation after surfactin treatment. After incubating P. falciparum parasites in the schizont stage with pumilacidins A and C at 10 μM for different time points, the levels of ROS were measured at 3 h (A), 6 h (B), and 9 h (C) post treatment with surfactins.

Figure 9

Kinetic measurement of cytosolic Ca²⁺. Saponin-isolated parasites stained with fura-2-acetoxymethyl ester (FURA 2AM) were treated with pumilacidins A and C at a concentration of 10 μM. Untreated parasites were used as controls. Readings were taken at 10, 30, 60, and 90 min after pumilacidin introduction. Samples were tested in duplicate. A representative result is shown.

Figure 10

Erythrocytes infected with P. falciparum were either incubated without (w/o inhibitor) or pre-incubated with inositol triphosphate 3-kinase (IP3K; 5 μM) and protein kinase C (PKC; 4 μg/mL) inhibitors and then treated with pumilacidins A and C at 10 μM (A) or not (B). The growth of the parasites was analyzed by flow cytometric detection of DNA 24 h afterward, and compared to untreated culture controls incubated with each respective inhibitor. The bars represent the mean of three individual experiments in duplicate (A) or the mean of two individual experiments in duplicate (B). * p < 0.05; *** p < 0.001.

Figure 11

Morphology changes induced by treatment with PKC and phosphoinositide 3-kinase (PI3K) inhibitors. Erythrocytes infected with P. falciparum in the schizont stage were either incubated without (w/o inhibitor) or pre-incubated with (A) no inhibitor, (B) 3-methyladenine (3MA), (C) LY29400, and (D) bisindolylmaleimide I, and then treated with pumilacidins A and C at 10 μM for 24 h. The parasite morphology was analyzed by Giemsa smear under light microscopy.