Abstract
Brucellosis is a zoonotic disease caused by Brucella, with extensive implications in both economic and public health. To date, no human vaccine has been developed and the current treatment with antibiotics can cause severe side effects and relapses. Due to the need for new treatments to fight brucellosis, one possibility is to target host cells mechanisms to block entry and/or intracellular replication of bacteria. In addition to such host-directed therapy (HDT), we used a drug repositioning strategy, which consists in evaluating drugs already on the market. Previous findings from our lab showed that Gefitinib, a drug currently used for the treatment of a type of lung cancer, strongly inhibits the proliferation of B. melitensis, B. abortus and B. suis inside macrophages and trophoblasts in vitro, even proving a curative effect. In vivo evaluation of the drug is currently ongoing in a murine model of brucellosis in collaboration with the group of Prof. Renee Tsolis (UC Davis, CA, USA). The aim of this study is to understand the molecular mechanisms by which Gefitinib achieves the control of Brucella infection. BeWo cells (human cytotrophoblasts) were treated with Gefitinib. The activation of several intracellular signaling pathways was analysed using Western Blot (WB). In parallel, cells were infected with fluorescent B. melitensis using gentamicin protection assays and cells were analyzed using confocal microcopy. WB analyses revealed a decreased phosphorylation of EGFR upon stimulation with Gefitinib and alteration of downstream signaling pathways was detected, including FAK and MAPKs. Gefitinib treatment increased both the number and size of Lysotracker-positive vesicles (LPV). Upon infection, the number of bacteria per infected cell is significantly decreased and Brucella highly colocalized with LPV in Gefitinib treated cells. Gefitinib modifies intracellular molecular pathways and bacteria are rapidly eliminated from infected cells by an apparent increase in lysosomal activity. These results suggest Gefitinib as a promising therapeutic alternative for the treatment of human brucellosis.
