Abstract
Focal adhesion kinase (FAK), a cytoplasmic protein tyrosine kinase (PTK), is implicated in diverse cellular processes, including the regulation of F-actin dynamics. Host cell F-actin rearrangement is critical for invasion of Trypanosoma cruzi, the protozoan parasite that causes Chagas disease. It is unknown whether FAK is involved in the internalization process of metacyclic trypomastigote (MT), the parasite form that is important for vectorial transmission. MT can enter the mammalian host through the ocular mucosa, lesion in the skin, or by the oral route. Oral infection by MT is currently a mode of transmission responsible for outbreaks of acute Chagas disease. Here we addressed the question by generating HeLa cell lines deficient in FAK. Host cell invasion assays showed that, as compared to control wild type (WT) cells, FAK-deficient cells were significantly more susceptible to parasite invasion. Lysosome spreading and a disarranged actin cytoskeleton, two features associated with susceptibility to MT invasion, were detected in FAK-deficient cells, as opposed to WT cells that exhibited a more organized F-actin arrangement, and lysosomes concentrated in the perinuclear area. As compared to WT cells, the capacity of FAK-deficient cells to bind a recombinant protein based on gp82, the MT surface molecule that mediates invasion, was higher. On the other hand, when treated with FAK-specific inhibitor PF573228, WT cells exhibited a dense meshwork of actin filaments, lysosome accumulation around the nucleus, and had increased resistance to MT invasion. In cells treated with PF573228, the phosphorylation levels of FAK were reduced and, as a consequence of FAK inactivation, diminished phosphorylation of extracellular signal-regulated protein kinases (ERK1/2) was observed. Fibronectin, known to impair MT invasion, induced the formation of thick bundles of F-actin and ERK1/2 dephosphorylation.
Highlights
Focal adhesion kinase (FAK), a cytoplasmic protein tyrosine kinase (PTK) involved in the control of several biological processes, regulates the flow of signals from the extracellular matrix (ECM) to the actin cytoskeleton (Parsons et al, 2000)
Our study has shown that cells depleted in FAK are more susceptible to T. cruzi metacyclic trypomastigote (MT) invasion
As the MT entry into cells is not impaired by FAK deficiency, it is clear that this tyrosine kinase is not required for the invasion process
Summary
Focal adhesion kinase (FAK), a cytoplasmic protein tyrosine kinase (PTK) involved in the control of several biological processes, regulates the flow of signals from the extracellular matrix (ECM) to the actin cytoskeleton (Parsons et al, 2000). The formation of focal adhesion-like complexes induced at sites of S. typhimurium attachment, and the dramatic impairment of bacterial uptake by FAK-depleted cells, demonstrated that FAK is required (Shi and Casanova, 2006). S. aureus, which triggered the recruitment of focal contact associated proteins, including FAK, to sites of bacterial attachment, had its internalization severely impaired in FAK-deficient cells (Agerer et al, 2005). Invasive E. coli K1 induced tyrosine phosphorylation of human brain microvascular endothelial cells FAK, which was recruited to focal plaques at the site of bacterial entry (Reddy et al, 2000). Treatment of target cells with specific FAK inhibitor reduced N. meningitidis internalization by more than 90% (Slanina et al, 2012)
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