Acanthamoeba castellanii Induces Host Cell Death via a Phosphatidylinositol 3-Kinase-Dependent Mechanism

Abstract

Granulomatous amoebic encephalitis due to Acanthamoeba castellanii is a serious human infection with fatal consequences, but it is not clear how the circulating amoebae interact with the blood-brain barrier and transmigrate into the central nervous system. We studied the effects of an Acanthamoeba encephalitis isolate belonging to the T1 genotype on human brain microvascular endothelial cells, which constitute the blood-brain barrier. Using an apoptosis-specific enzyme-linked immunosorbent assay, we showed that Acanthamoeba induces programmed cell death in brain microvascular endothelial cells. Next, we observed that Acanthamoeba specifically activates phosphatidylinositol 3-kinase. Acanthamoeba-mediated brain endothelial cell death was abolished using LY294002, a phosphatidylinositol 3-kinase inhibitor. These results were further confirmed using brain microvascular endothelial cells expressing dominant negative forms of phosphatidylinositol 3-kinase. This is the first demonstration that Acanthamoeba-mediated brain microvascular endothelial cell death is dependent on phosphatidylinositol 3-kinase.Acanthamoeba spp. are opportunistic protozoan parasites that are widely distributed throughout the environment (12, 18). The genus Acanthamoeba consists of both pathogenic and nonpathogenic species. Given the host susceptibility and correct environmental conditions, Acanthamoeba can cause granulomatous amoebic encephalitis (GAE), a fatal central nervous system (CNS) infection that occurs in immunocompromised patients (7-10, 11, 19). Several lines of evidence suggest that hematogenous spread is a prerequisite in Acanthamoeba encephalitis (19-21), but it is not clear how circulating amoebae cross the blood-brain barrier and gain access to the CNS to produce disease. We have demonstrated that pathogenic Acanthamoeba exhibits more than 60% binding to human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier (2). Acanthamoeba binding to HBMEC is mediated by a mannose-binding protein expressed on the surface of Acanthamoeba cells (2). Moreover, we showed that Acanthamoeba produces severe HBMEC cytotoxicity by secreting extracellular proteases, as well as using contact-dependent mechanisms such as phagocytosis (12), which may play an important role in blood-brain barrier perturbations. However, the host intracellular signaling pathways and the molecular mechanisms associated with Acanthamoeba-mediated HBMEC cytotoxicity have not been determined.Lipid second messengers, such as those derived from the polyphosphoinositide cycle, play a central role in many signaling networks. The majority of inositol lipids reside in membranes and serve as substrates for kinases, phosphatases, and phospholipases. Phosphatidylinositol 3-kinases (PI3Ks) are important signaling molecules that phosphorylate the 3Ј OH position of the inositol ring of phosphoinositides (PIs), generating the second messengers PI(3)P, PI(3,4)P 2 , and PI(3,4,5)P 3 (4, 17). These second messengers recruit the downstream effector molecu...

An epitope from Acanthamoeba castellanii that cross-react with proteolipid protein 139-151-reactive T cells induces autoimmune encephalomyelitis in SJL mice

“…AGE is a chronic progressive disease of the CNS commonly seen in chronically ill, debilitated, or immunocompromised individuals such as AIDS patients (Marciano-Cabral and Cabral, 2003). The amoebae activate phosphatidylinositol 3-kinase to induce apoptosis of brain microvascular endothelial cells and successfully traverse the blood-brain barrier to induce AGE (Sissons et al, 2005). IL-1ß has been implicated in the formation of granuloma in the CNS tissues, but it is unclear whether the hemorrhagic necrosis caused by Acanthamoeba infections results from destruction of brain tissue or inflammatory cytokines (Marciano-Cabral et al, 2004).…”

Use of In Vitro Assays To Determine Effects of Human Serum on Biological Characteristics of Acanthamoeba castellanii

“…genotype was originally isolated from an AGE patient and routinely grown in PYG medium (0.75% Proteose Peptone, 0.75% yeast extract, 1.5% glucose) at 30°C, and the medium was refreshed 17 to 20 h prior to experiments as previously described (16). This resulted in more than 95% acanthamoebae in the trophozoite form.…”

“…This resulted in more than 95% acanthamoebae in the trophozoite form. For in vitro assays, primary HB-MEC were isolated from human brain tissue as described previously (16,18) and routinely grown in RPMI 1640 medium containing 10% fetal bovine serum (heat inactivated), 10% NuSerum, 2 mM glutamine, 1 mM pyruvate, penicillin and streptomycin, nonessential amino acids, and vitamins (16,18).…”

Status of the effectiveness of contact lens disinfectants in Malaysia against keratitis-causing pathogens

“…It is originally isolated from a patient suffering from Acanthamoeba keratitis. Acanthamoeba castellanii was cultured in a T-75 tissue culture flask in 10 mL Proteose peptone-Yeast extract-Glucose (PYG) medium [0.75% (w/v) proteose peptone, 0.75% (w/v) yeast extract, and 1.5% (w/v) glucose] at 37°C as previously described (Sissons et al, 2005). Before performing assays, Fresh PYG media was added in each flask approximately 17-20 h before, to obtain amoebae in the trophozoite form.…”