Fowlerstefin, a cysteine protease inhibitor of Naegleria fowleri, induces inflammatory responses in BV-2 microglial cells in vitro

• Published in: Parasites & vectors 2020-01, Vol.13 (1), p.41-15, Article 41
• Main Authors: Thái, Thị Lam, Kang, Jung-Mi, Lê, Hương Giang, Lee, Jinyoung, Yoo, Won Gi, Shin, Ho-Joon, Sohn, Woon-Mok, Na, Byoung-Kuk
• Format: Article
• Language: English
• Subjects: Activator protein 1; Amoeba; Analysis; Analysis of Variance; Animals; Antibodies, Protozoan - biosynthesis; Antibodies, Protozoan - immunology; Antibodies, Protozoan - isolation & purification; Antibody Specificity; Cathepsin B; Cathepsin B - antagonists & inhibitors; Cathepsin L - antagonists & inhibitors; Cathepsins; Cell Line; Central Nervous System Protozoal Infections - parasitology; Chemokines; Cloning; Cystatins - chemistry; Cystatins - pharmacology; Cysteine; Cysteine protease inhibitor; Cysteine proteases; Cysteine proteinase; Cysteine Proteinase Inhibitors - chemistry; Cysteine Proteinase Inhibitors - immunology; Cysteine Proteinase Inhibitors - pharmacology; Cystine; Cysts; Cytokines; Cytokines - metabolism; Development and progression; E coli; Electrophoresis, Polyacrylamide Gel; ELISA; Enzyme-Linked Immunosorbent Assay; Enzymes; Escherichia coli; Health aspects; Humans; Hydrogen-Ion Concentration; Immune response; Immunization; Immunoglobulin G - biosynthesis; Immunoglobulin G - immunology; Immunoglobulin G - isolation & purification; Infection; Infections; Inflammation; Inflammatory response; Inhibitors; Interleukin 6; Kinases; MAP kinase; Medical research; Medicine, Experimental; Meningoencephalitis; Mice; Mice, Inbred BALB C; Microglia; Microglia - drug effects; Microglia - immunology; Microglia - pathology; Microglial cells; Mitogens; Naegleria fowleri; Naegleria fowleri - classification; Naegleria fowleri - genetics; Naegleria fowleri - metabolism; NF-κB protein; Nucleotide sequence; Papain; Papain - antagonists & inhibitors; Parasites; Pathogenesis; PCR; Peptides; Phylogeny; Polymerase chain reaction; Primary amebic meningoencephalitis; Protease; Protease inhibitors; Protein kinase; Proteinase inhibitors; Proteins; Recombinant Proteins - chemistry; Recombinant Proteins - pharmacology; Recombinants; Reverse transcription; Roles; Sodium; Thermal cycling; Thiols; Transcription; Transcription factors; Tumor necrosis factor; Vectors (Biology)
• ISSN: 1756-3305; 1756-3305
• DOI: 10.1186/s13071-020-3909-6

Naegleria 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. Recombinant 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. Fowlerstefin 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. Fowlerstefin 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.