Increasing importance of Balamuthia mandrillaris.

Balamuthia mandrillaris is an emerging protozoan parasite, an agent of granulomatous amoebic encephalitis involving the central nervous system, with a case fatality rate of >98%. This review presents our current understanding of Balamuthia infections, their pathogenesis and pathophysiology, and molecular mechanisms associated with the disease, as well as virulence traits of Balamuthia that may be potential targets for therapeutic interventions and/or for the development of preventative measures.

Balamuthia mandrillaris stimulates interleukin-6 release in primary human brain microvascular endothelial cells via a phosphatidylinositol 3-kinase-dependent pathway.

Balamuthia mandrillaris is an emerging protozoan parasite that can cause fatal granulomatous encephalitis. Haematogenous spread is a likely route prior to entry into the central nervous system (CNS), but it is not clear how circulating amoebae cross the blood-brain barrier. Using human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier, we determined HBMEC inflammatory response to B. mandrillaris and the underlying mechanisms associated with this response. We demonstrated that HBMEC incubated with B. mandrillaris released significantly higher levels of interleukin-6 (IL-6) (>400 pg/ml) as compared with less than 50 pg/ml in HBMEC incubated alone. Western blotting assays determined that B. mandrillaris specifically activates phosphatidylinositol 3-kinase (PI3K). By using LY294002, a PI3K inhibitor, as well as by using HBMEC expressing dominant-negative PI3K, we have identified PI3K as an important mediator of B. mandrillaris-mediated IL-6 release. We conclude that B. mandrillaris induces HBMEC signalling pathways, which lead to IL-6 release. This is the first time PI3K has been shown to play a crucial role in B. mandrillaris-mediated IL-6 release in HBMEC.

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.

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 alpha-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.

Acanthamoeba castellanii induces host cell death via a phosphatidylinositol 3-kinase-dependent mechanism.

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.

Ecto-ATPases of clinical and non-clinical isolates of Acanthamoeba.

Acanthamoeba are opportunistic protozoan parasites that can cause fatal granulomatous amoebic encephalitis and eye keratitis, however the pathogenic mechanisms of Acanthamoeba remain unclear. In this study, we described the ability of live Acanthamoeba to hydrolyse extracellular ATP. Both clinical and non-clinical isolates belonging to genotypes, T1, T2, T3, T4 and T7 exhibited ecto-ATPase activities in vitro. Using non-denaturing polyacrylamide gel electrophoresis, ecto-ATPases were further characterized. All Acanthamoeba isolates tested, exhibited a single ecto-ATPase band (approximate molecular weight of 272 kDa). However, clinical isolates exhibited additional bands suggesting that ecto-ATPases may play a role in the pathogenesis of Acanthamoeba. This was supported using suramin (ecto-ATPase inhibitor), which inhibited Acanthamoeba-induced host cell cytotoxicity. Previously, we and others have shown that Acanthamoeba binds to host cells using their mannose-binding protein and binding can be blocked using exogenous alpha-mannose. In this study, we observed that alpha-mannose significantly increased ecto-ATPase activities of pathogenic Acanthamoeba belonging to T1, T2, T3 and T4 genotypes but had no effect on non-pathogenic Acanthamoeba (belonging to T7 genotype). Overall, we have shown, for the first time, that Acanthamoeba exhibit ecto-ATPase activities, which may play a role in the pathogenesis of Acanthamoeba as well as their potential role in the differentiation of pathogenic Acanthamoeba.

Post-mortem culture of Balamuthia mandrillaris from the brain and cerebrospinal fluid of a case of granulomatous amoebic meningoencephalitis, using human brain microvascular endothelial cells.

The first isolation in the UK of Balamuthia mandrillaris amoebae from a fatal case of granulomatous amoebic meningoencephalitis is reported. Using primary cultures of human brain microvascular endothelial cells (HBMECs), amoebae were isolated from the brain and cerebrospinal fluid (CSF). The cultures showed a cytopathic effect at 20-28 days, but morphologically identifiable B. mandrillaris amoebae were seen in cleared plaques in subcultures at 45 days. The identification of the organism was later confirmed using PCR on Chelex-treated extracts. Serum taken while the patient was still alive reacted strongly with slide antigen prepared from cultures of the post-mortem isolate, and also with those from a baboon B. mandrillaris strain at 1:10,000 in indirect immunofluorescence, but with Acanthamoeba castellanii (Neff) at 1:160, supporting B. mandrillaris to be the causative agent. If the presence of amoebae in the post-mortem CSF reflects the condition in life, PCR studies on CSF and on biopsies of cutaneous lesions may also be a valuable tool. The role of HBMECs in understanding the interactions of B. mandrillaris with the blood-brain barrier is discussed.

Acanthamoeba induces cell-cycle arrest in host cells.

Acanthamoeba can cause fatal granulomatous amoebic encephalitis (GAE) and eye keratitis. However, the pathogenesis and pathophysiology of these emerging diseases remain unclear. In this study, the effects of Acanthamoeba on the host cell cycle using human brain microvascular endothelial cells (HBMEC) and human corneal epithelial cells (HCEC) were determined. Two isolates of Acanthamoeba belonging to the T1 genotype (GAE isolate) and T4 genotype (keratitis isolate) were used, which showed severe cytotoxicity on HBMEC and HCEC, respectively. No tissue specificity was observed in their ability to exhibit binding to the host cells. To determine the effects of Acanthamoeba on the host cell cycle, a cell-cycle-specific gene array was used. This screened for 96 genes specific for host cell-cycle regulation. It was observed that Acanthamoeba inhibited expression of genes encoding cyclins F and G1 and cyclin-dependent kinase 6, which are proteins important for cell-cycle progression. Moreover, upregulation was observed of the expression of genes such as GADD45A and p130 Rb, associated with cell-cycle arrest, indicating cell-cycle inhibition. Next, the effect of Acanthamoeba on retinoblastoma protein (pRb) phosphorylation was determined. pRb is a potent inhibitor of G1-to-S cell-cycle progression; however, its function is inhibited upon phosphorylation, allowing progression into S phase. Western blotting revealed that Acanthamoeba abolished pRb phosphorylation leading to cell-cycle arrest at the G1-to-S transition. Taken together, these studies demonstrated for the first time that Acanthamoeba inhibits the host cell cycle at the transcriptional level, as well as by modulating pRb phosphorylation using host cell-signalling mechanisms. A complete understanding of Acanthamoeba-host cell interactions may help in developing novel strategies to treat Acanthamoeba infections.