Heparan sulfate proteoglycan triggers focal adhesion kinase signaling during Trypanosoma cruzi invasion

BACKGROUND Trypanosoma cruzi, the etiologic agent of Chagas disease, is capable of triggering different signaling pathways that modulate its internalisation in mammalian cells. Focal adhesion kinase (FAK), a non-receptor tyrosine kinase protein, has been demonstrated as a mechanism of T. cruzi invasion in cardiomyocytes. Since the involved cell surface receptors are not yet known, we evaluated whether heparan sulfate proteoglycans (HSPG), a molecule involved in T. cruzi recognition and in the regulation of multiple signaling pathways, are able to trigger the FAK signaling pathway during T. cruzi invasion. METHODS To investigate the role of HSPG in the regulation of the FAK signaling pathway during trypomastigote entry, we performed heparan sulfate (HS) depletion from the cardiomyocyte surface by treatment with heparinase I or p-nitrophenyl-β-D-xylopyranoside (p-n-xyloside), which abolishes glycosaminoglycan (GAG) attachment to the proteoglycan core protein. Wild-type (CHO-k1) and GAG-deficient Chinese hamster ovary cells (CHO-745) were also used as an approach to evaluate the participation of the HSPG-FAK signaling pathway. FAK activation (FAK Tyr397) and spatial distribution were analysed by immunoblotting and indirect immunofluorescence, respectively. FINDINGS HS depletion from the cardiomyocyte surface inhibited FAK activation by T. cruzi. Cardiomyocyte treatment with heparinase I or p-n-xyloside resulted in 34% and 28% FAK phosphorylation level decreases, respectively. The experiments with the CHO cells corroborated the role of HSPG as a FAK activation mediator. T. cruzi infection did not stimulate FAK phosphorylation in CHO-745 cells, leading to a 36% reduction in parasite invasion. FAK inhibition due to the PF573228 treatment also impaired T. cruzi entry in CHO-k1 cells. MAIN CONCLUSION Jointly, our data demonstrate that HSPG is a key molecule in the FAK signaling pathway activation, regulating T. cruzi entry.

背景 克氏锥虫（Trypanosoma cruzi）是恰加斯病（Chagas disease）的病原体，可激活多条信号通路以调控其在哺乳动物细胞中的内化过程。黏着斑激酶（focal adhesion kinase, FAK）作为一种非受体酪氨酸激酶蛋白，已被证实参与克氏锥虫入侵心肌细胞的过程。由于目前尚未明确其所涉及的细胞表面受体，本研究评估了硫酸乙酰肝素蛋白聚糖（heparan sulfate proteoglycans, HSPG）——一种参与克氏锥虫识别并调控多条信号通路的分子——在克氏锥虫入侵过程中是否能够激活FAK信号通路。方法 为探究锥鞭毛体入侵阶段HSPG对FAK信号通路的调控作用，本研究通过肝素酶I（heparinase I）或对硝基苯基-β-D-吡喃木糖苷（p-nitrophenyl-β-D-xylopyranoside, p-n-xyloside）处理，去除心肌细胞表面的硫酸乙酰肝素（heparan sulfate, HS），该处理可阻断糖胺聚糖（glycosaminoglycan, GAG）与蛋白聚糖核心蛋白的结合。此外，本研究还采用野生型中国仓鼠卵巢细胞（Chinese hamster ovary, CHO）CHO-k1及糖胺聚糖缺陷型CHO-745细胞，以验证HSPG-FAK信号通路的参与情况。分别通过免疫印迹法与间接免疫荧光法，检测FAK的活化状态（FAK Tyr397）及其空间分布。结果 去除心肌细胞表面的HS可抑制克氏锥虫诱导的FAK活化。经肝素酶I或p-n-xyloside处理的心肌细胞，其FAK磷酸化水平分别下降34%与28%。CHO细胞实验进一步证实了HSPG作为FAK活化介导因子的作用。克氏锥虫感染无法诱导CHO-745细胞的FAK磷酸化，导致寄生虫入侵率降低36%。经PF573228处理以抑制FAK活性后，CHO-k1细胞的克氏锥虫入侵能力也受到损伤。主要结论 综上，本研究数据证实HSPG是FAK信号通路激活的关键分子，可调控克氏锥虫的细胞入侵过程。