Abstract
Naegleria fowleri is a free-living amoeba that is ubiquitous in diverse natural environments. It causes a fatal brain infection in humans known as primary amoebic meningoencephalitis. Despite the medical importance of the parasitic disease, there is a great lack of knowledge about the biology and pathogenicity of N. fowleri. In this study, we identified and characterized a novel cysteine protease inhibitor of N. fowleri (NfCPI). NfCPI is a typical cysteine protease inhibitor belonging to the cystatin family with a Gln-Val-Val-Ala-Gly (QVVAG) motif, a characteristic motif conserved in the cystatin family of proteins. Bacterially expressed recombinant NfCPI has a dimeric structure and exhibits inhibitory activity against several cysteine proteases including cathespin Bs of N. fowleri at a broad range of pH values. Expression profiles of nfcpi revealed that the gene was highly expressed during encystation and cyst of the amoeba. Western blot and immunofluorescence assays also support its high level of expression in cysts. These findings collectively suggest that NfCPI may play a critical role in encystation or cyst formation of N. fowleri by regulating cysteine proteases that may mediate encystation or mature cyst formation of the amoeba. More comprehensive studies to investigate the roles of NfCPI in encystation and its target proteases are necessary to elucidate the regulatory mechanism and the biological significance of NfCPI.
Highlights
Naegleria fowleri is a free-living amoeba that is ubiquitously distributed in diverse natural environments including freshwaters and soil [1]
The clinical manifestations of N. fowleri infection normally appear within 2–8 days after infection, which include symptoms of fever, severe headache, nausea, chills, neck stiffness, seizures, and coma and eventually result in fatal primary amoebic meningoencephalitis (PAM) characterized by necrotizing, fulminant, and hemorrhagic meningoencephalitis [2,3]
Naegleria fowleri trophozoites (Carter NF69 strain, ATCC 30215) were cultured in Nelson medium supplemented with 2% heat-inactivated fetal bovine serum (FBS; Gibco, Rockville, MA, USA) and 1% penicillin-streptomycin (Gibco) at 37 ◦C [11]
Summary
Naegleria fowleri is a free-living amoeba that is ubiquitously distributed in diverse natural environments including freshwaters and soil [1]. It can infect humans and cause a rare but fatal brain infection known as primary amoebic meningoencephalitis (PAM) [2]. The clinical manifestations of N. fowleri infection normally appear within 2–8 days after infection, which include symptoms of fever, severe headache, nausea, chills, neck stiffness, seizures, and coma and eventually result in fatal PAM characterized by necrotizing, fulminant, and hemorrhagic meningoencephalitis [2,3]. Cases of PAM induced by N. fowleri infection have been reported in diverse global areas, including Asia, Africa, Europe, and South America [6]
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