Reduced Parasite Motility and Micronemal Protein Secretion by a p38 MAPK Inhibitor Leads to a Severe Impairment of Cell Invasion by the Apicomplexan Parasite Eimeria tenella

E. tenella infection is associated with a severe intestinal disease leading to high economic losses in poultry industry. Mitogen activated protein kinases (MAPKs) are implicated in early response to infection and are divided in three pathways: p38, extracellular signal-regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK). Our objective was to determine the importance of these kinases on cell invasion by E. tenella. We evaluated the effect of specific inhibitors (ERK: PD98059, JNKII: SP600125, p38 MAPK: SB203580) on the invasion of epithelial cells. Incubation of SP600125 and SB203580 with epithelial cells and parasites significantly inhibited cell invasion with the highest degree of inhibition (90%) for SB203580. Silencing of the host p38α MAPK expression by siRNA led to only 20% decrease in cell invasion. In addition, when mammalian epithelial cells were pre-treated with SB203580, and washed prior infection, a 30% decrease in cell invasion was observed. This decrease was overcome when a p38 MAPK activator, anisomycin was added during infection. This suggests an active but limited role of the host p38 MAPK in this process. We next determined whether SB203580 has a direct effect on the parasite. Indeed, parasite motility and secretion of micronemal proteins (EtMIC1, 2, 3 and 5) that are involved in cell invasion were both decreased in the presence of the inhibitor. After chasing the inhibitor, parasite motility and secretion of micronemal proteins were restored and subsequently cell invasion. SB203580 inhibits cell invasion by acting partly on the host cell and mainly on the parasite.

柔嫩艾美耳球虫（Eimeria tenella，E. tenella）感染可引发严重肠道疾病，给家禽养殖业造成高额经济损失。丝裂原活化蛋白激酶（mitogen-activated protein kinases，MAPKs）参与感染早期应答，可分为p38、细胞外调节蛋白激酶（extracellular signal-regulated protein kinase，ERK）与c-Jun氨基末端激酶（c-Jun N-terminal kinase，JNK）三条信号通路。本研究旨在明确上述激酶在柔嫩艾美耳球虫侵袭宿主细胞过程中的关键作用。我们评估了三类特异性抑制剂（ERK抑制剂：PD98059、JNKII抑制剂：SP600125、p38 MAPK抑制剂：SB203580）对上皮细胞侵袭过程的影响。将SP600125与SB203580分别与上皮细胞及寄生虫共孵育，可显著抑制细胞侵袭，其中SB203580的抑制效果最为显著，抑制率高达90%。通过小干扰RNA（small interfering RNA，siRNA）沉默宿主p38α MAPK的表达，仅能使细胞侵袭率降低20%。此外，若先用SB203580预处理哺乳动物上皮细胞，洗去抑制剂后再进行感染，可观察到细胞侵袭率下降30%；若在感染过程中加入p38 MAPK激活剂茴香霉素（anisomycin），则可逆转该侵袭率下降趋势。上述结果提示宿主p38 MAPK在该过程中发挥活跃但有限的调控作用。我们进一步探究了SB203580是否对寄生虫具有直接作用，实验发现，在抑制剂存在的条件下，寄生虫的运动能力以及参与细胞侵袭的微线体蛋白（EtMIC1、2、3及5）的分泌水平均显著下降。移除抑制剂后，寄生虫的运动能力与微线体蛋白的分泌可恢复至正常水平，细胞侵袭能力亦随之恢复。综上，SB203580主要通过直接作用于寄生虫，同时部分作用于宿主细胞，从而抑制柔嫩艾美耳球虫对上皮细胞的侵袭过程。