Extracellular proteases of Acanthamoeba castellanii (encephalitis isolate belonging to T1 genotype) contribute to increased permeability in an in vitro model of the human blood–brain barrier

Abstract

Objectives Granulomatous amoebic encephalitis (GAE) is a serious human infection with fatal consequences, however, the pathogenic mechanisms associated with this disease remain unclear. Several lines of evidence suggest that haematogenous spread is a prerequisite for Acanthamoeba encephalitis but it is not clear how circulating amoebae cross the blood–brain barrier to gain entry into the central nervous system. Objectives of this study were to determine the effects of Acanthamoeba on the permeability of an in vitro blood–brain barrier model and factors contributing to these changes. Methods Using human brain microvascular endothelial cells, an in vitro blood–brain barrier model was constructed in 24-well Transwell plates. Acanthamoeba (GAE isolate belonging to T1 genotype) or its conditioned media were used to determine permeability changes. Zymography assays were performed to characterise Acanthamoeba proteases. In addition, the ability of Acanthamoeba to bind brain microvascular endothelial cells was determined using adhesion assays. Results We observed that Acanthamoeba produced an increase of more than 45% in the blood–brain barrier permeability. Acanthamoeba-conditioned media exhibited similar effects indicating Acanthamoeba-mediated blood–brain barrier permeability is contact-independent. Prior treatment of conditioned media with phenylmethyl sulfonyl fluoride (PMSF, serine protease inhibitor), abolished permeability changes indicating the role of serine proteases. Of interest, methyl α- d-mannopyranoside inhibited Acanthamoeba binding to human brain microvascular endothelial cells but had no effect on Acanthamoeba-mediated blood–brain barrier permeability. Zymography assays revealed that Acanthamoeba produced two major proteases, one of which was inhibited by PMSF (serine protease inhibitor) and the second with 1,10-phenanthroline (metalloprotease inhibitor). Conclusions We have for the first time shown that Acanthamoeba produces human brain microvascular endothelial cells permeability, which can be blocked by PMSF. A metalloprotease of approx. molecular weight of 150 kDa is produced by A. castellanii (GAE isolate belonging to T1 genotype) and its role in the disease is suggested.