Abstract
BackgroundNaegleria fowleri is a free-living amoeba that causes an opportunistic fatal infection known as primary amoebic meningoencephalitis (PAM) in humans. Cysteine proteases produced by the amoeba may play critical roles in the pathogenesis of infection. In this study, a novel cysteine protease inhibitor of N. fowleri (fowlerstefin) was characterized to elucidate its biological function as an endogenous cysteine protease inhibitor of the parasite as well as a pathogenic molecule that induces immune responses in microglial cells.MethodsRecombinant fowlerstefin was expressed in Escherichia coli. The inhibitory activity of fowlerstefin against several cysteine proteases, including human cathepsins B and L, papain and NfCPB-L, was analyzed. Fowlerstefin-induced pro-inflammatory response in BV-2 microglial cells was anayzed by cytokine array assay, reverse transcription polymerase chain reaction, and enzyme-linked immunosorbent assay.ResultsFowlerstefin is a cysteine protease inhibitor with a monomeric structure, and belongs to the stefin family. Recombinant fowlerstefin effectively inhibited diverse cysteine proteases including cathepsin B-like cysteine proteases of N. fowleri (NfCPB-L), human cathepsins B and L, and papain. Expression of fowlerstefin in the amoeba was optimal during the trophozoite stage and gradually decreased in cysts. Fowlerstefin induced an inflammatory response in BV-2 microglial cells. Fowlerstefin induced the expression of several pro-inflammatory cytokines and chemokines including IL-6 and TNF in BV-2 microglial cells. Fowlerstefin-induced expression of IL-6 and TNF in BV-2 microglial cells was regulated by mitogen-activated protein kinase (MAPKs). The inflammatory response induced by fowlerstefin in BV-2 microglial cells was downregulated via inhibition of NF-κB and AP-1.ConclusionsFowlerstefin is a pathogenic molecule that stimulates BV-2 microglial cells to produce pro-inflammatory cytokines through NF-κB- and AP-1-dependent MAPK signaling pathways. Fowlerstefin-induced inflammatory cytokines exacerbate the inflammatory response in N. fowleri-infected areas and contribute to the pathogenesis of PAM.
Highlights
Naegleria fowleri is a free-living amoeba that causes an opportunistic fatal infection known as primary amoebic meningoencephalitis (PAM) in humans
The expression of C5, IL-5, TIMP-1, G-CSF, IL-6, MCP-3, MIG, IFN-γ and IL-1β was increased in BV-2 cells upon treatment with fowlerstefin. These results suggested that fowlerstefin induced the expression of diverse cytokines and chemokines in BV-2 microglial cells, which are associated with the pro-inflammatory response
< 0.0001) and IL-6 (ANOVA: F(8, 18) = 1826, P < 0.0001) (Fig. 7b, Additional file 3: Text S1). These results collectively suggested that the fowlerstefin-induced proinflammatory response in BV-2 cells was mediated via Mitogen‐activated protein kinase (MAPK) signaling pathway
Summary
Naegleria fowleri is a free-living amoeba that causes an opportunistic fatal infection known as primary amoebic meningoencephalitis (PAM) in humans. A novel cysteine protease inhibitor of N. fowleri (fowlerstefin) was char‐ acterized to elucidate its biological function as an endogenous cysteine protease inhibitor of the parasite as well as a pathogenic molecule that induces immune responses in microglial cells. Naegleria fowleri is a free-living amoeba that causes a lethal brain infection known as primary amoebic meningoencephalitis (PAM) in humans [1,2,3]. Naegleria fowleri infection is initiated by inhaling water containing amoebae into the host nasal cavity. The inhaled amoebae pass the respiratory epithelium and olfactory mucosa and migrate through the cribriform plate into the brain [9]. The amoebae trigger extensive tissue damage along with acute inflammation. The acute hemorrhagic meningoencephalitis that follows invasion of the central nervous system (CNS) generally results in death within 7–10 days of infection [10]
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