SUMMARY 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.

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...

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.