[Role of protein palmitoylation modification in the proliferation and gametogenesis of Plasmodium falciparum].

OBJECTIVE: To investigate the palmitoyltransferase (PATs) activity during different developmental stages of Plasmodium falciparum and to explore the impact of PATs activity on intra-erythrocytic replication and gametocytogenesis, so as to provide insights into development of novel antimalarial targets.

METHODS: The PATs activity was measured using the click chemistry method during different developmental stages of P. falciparum, including rings, trophozoites, schizonts, and gametocytes. P. falciparum-infected erythrocytes were divided into three groups, including a control group, a dimethyl sulfoxide (DMSO) group, and a 2-bromopalmitate (2-BP) group. Erythrocytes in the control group were incubated in normal culture media, and cells in DMSO and 2-BP groups were exposed to DMSO or 2-BP at a final concentration of 10 μmol/L to examine the inhibitory effect of 2BP on the PATs activity. The growth curve analysis and gametocyte production assay were employed to investigate changes in asexual proliferation and gametocyte production of P. falciparum following inhibition of the PATs activity with 2-BP, and transcriptomics sequencing was performed to examine the impact of inhibition of the PATs activity with 2-BP on transcriptional levels of P. falciparum and possible mechanisms.

RESULTS: The green fluorescence intensity of PATs varied across developmental stages of P. falciparum (F = 38.120, P < 0.001), with a higher fluorescence intensity seen in trophozoites (35.680 ± 8.439), merozoites (33.380 ± 9.030) and gametocytes (21.540 ± 8.654), and a lower intensity in ring bodies (10.720 ± 3.183) (all P values < 0.05). The green fluorescence intensities were 8.738 ± 1.576, 8.633 ± 1.827 and 4.911 ± 0.318 in the control group, DMSO group, and 2-BP group 4 days post-culture (schizont stage), respectively (F = 91.490, P < 0.001), and the PATs activity was significantly inhibited post-treatment with 2-BP (P < 0.05). The areas under the time curve for the parasitemias were 25.700 ± 0.696, 28.630 ± 3.062 and 8.370 ± 1.751 in the control group, DMSO group, and 2-BP group following inhibition of the PATs activity during the asexual stage of P. falciparum (F = 83.440, P < 0.001), and the parasitemia was lower in the 2-BP group than in the control group and the DMSO group (both P values < 0.001). In addition, the asexual stage development was delayed in the 2BP group, with abnormal morphology seen. The numbers of merozoites were 18.050 ± 4.362, 18.200 ± 3.517 and 14.020 ± 4.320 in each schizont in the control group, DMSO group and 2-BP group, respectively (H = 39.100, P < 0.001), and the merozoite number was significantly lower in the 2-BP group than in the control group and the DMSO group (both Padjusted values < 0.001). The areas under the time curve for P. falciparum gametocyte production were 18.900 ± 0.384, 18.240 ± 0.177 and 7.507 ± 0.201 in the control group, the DMSO group, and the 2-BP group following inhibition of the PATs activity, respectively (F = 1 677.000, P < 0.001), and the proportion of gametocyte production was statistically lower in the 2-BP group than in the control group and DMSO group (both P values < 0.001). The formation and maturation of gametophytes were blocked in the 2-BP group, and most of them were arrested in the middle and late stages. Following 2-BP treatment, significantly down-regulated genes during the asexual stage of P. falciparum were significantly enriched in cell cycle regulation, mitosis, DNA damage/response and structural organization, and significantly down-regulated genes during the gametocyte stage were significantly enriched in biological processes of cell cycle (mitosis and G1/S transition), RNA regulation and metabolism (such as carbon catabolite repression 4-negative on TATA-less) and cell development and differentiation.

CONCLUSIONS: The palmitoylation modification plays an important role in the asexual reproduction and gametocyte generation and development of P. falciparum.