Abstract: 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 2⁃BP 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 2⁃BP 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. Conclusion　The palmitoylation modification plays an important role in the asexual reproduction and gametocyte generation and development of P. falciparum.

Key words: Plasmodium falciparum, Palmitoyltransferase, Palmitoylation, Gametocyte, 2?bromopalmitic acid

摘要： 目的　观察恶性疟原虫不同发育阶段棕榈酰转移酶（palmitoyltransferases，PATs）活性变化及其对红内期增殖和配子体生成的影响，为开发抗疟药物新靶点提供科学依据。方法　采用点击化学技术检测疟原虫环状体、滋养体、裂殖体和配子体等不同发育阶段PATs的活性特征。将感染红细胞分成对照组、二甲基亚砜（dimethyl sulfoxide，DMSO）组和2⁃溴棕榈酸（2⁃bromopalmitate，2⁃BP）组，其中对照组使用普通培养基，DMSO组和2⁃BP组分别在培养基中加入DMSO和终浓度为10 μmol/L 的2⁃BP，以验证2⁃BP对PATs活性的抑制情况；通过绘制生长曲线和配子体诱导实验，观察2⁃BP抑制PATs活性后恶性疟原虫无性增殖及配子体生成的变化，并结合转录组测序进一步分析其对疟原虫转录水平的影响及可能机制。结果　PATs绿色荧光强度在恶性疟原虫不同发育阶段存在差异（F = 38.120，P < 0.001）；其中滋养体（35.680 ± 8.439）、裂殖体（33.380 ± 9.030）及配子体阶段（21.540 ± 8.654）荧光强度较强，环状体阶段（10.720 ± 3.183）较弱（P均< 0.05）；培养4 d后（裂殖体期）对照组、DMSO组、2⁃BP组绿色荧光强度分别为8.738 ± 1.576、8.633 ± 1.827、4.911 ± 0.318（F = 91.490，P < 0.001），2⁃BP处理后PATs活性受到显著抑制（P < 0.05）。抑制PATs活性后，对照组、DMSO组、2⁃BP组疟原虫无性期原虫率⁃时间曲线下面积分别为25.700 ± 0.696、28.630 ± 3.062、8.370 ± 1.751，差异有统计学意义（F = 83.440，P < 0.001）；两两比较结果显示，2⁃BP组无性期原虫率低于对照组和DMSO组（P均< 0.001）；且无性期原虫发育延迟、形态异常。对照组、DMSO组和2⁃BP组每个裂殖体中裂殖子数量分别为（18.050 ± 4.362）、（18.200 ± 3.517）、（14.020 ± 4.320）个/裂殖体，差异有统计学意义（H = 39.100，P < 0.001）；两两比较结果显示，2⁃BP组较对照组和DMSO组均显著下降（P校正均< 0.001）。抑制PATs活性后，对照组、DMSO组和2⁃BP组恶性疟原虫配子体生成率⁃时间曲线下面积分别为18.900 ± 0.384、18.240 ± 0.177、7.507 ± 0.201，差异有统计学意义（F = 1 677.000，P < 0.001）；其中2⁃BP组配子体生成率低于对照组和DMSO组（P均< 0.001），配子体生成及成熟受阻，且多数停滞在中晚期。添加2⁃BP后，恶性疟原虫无性期下调显著的差异基因主要富集于细胞周期、有丝分裂、DNA损伤响应与细胞结构；在配子体阶段下调显著的差异基因主要富集于细胞周期（如有丝分裂、G1/S期转换）、RNA代谢与转录（如碳代谢抑制4⁃无TATA负调控复合物）以及细胞发育与分化相关的生物学过程。结论　棕榈酰化修饰在恶性疟原虫无性增殖、配子体生成和发育中发挥重要作用。

关键词: 恶性疟原虫, 棕榈酰转移酶, 棕榈酰化, 配子体, 2?溴棕榈酸