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 Tyr(397)) 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.