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

Abstract

Background: 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. Methods: 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. Fowlerstefininduced pro-inflammatory response in BV-2 microglial cells was anayzed by cytokine array assay, reverse transcription polymerase chain reaction, and enzyme-linked immunosorbent assay. Results: 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. Conclusions: 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.

Host-Parasite Interaction of Atlantic salmon (Salmo salar) and the Ectoparasite Neoparamoeba perurans in Amoebic Gill Disease

“…This includes proteins to facilitate the destruction of the mucus barrier, apoptosis of epithelial cells and re-arrangement of the host actin cytoskeleton to gain access to host tissue. In protozoan and sporozoite parasites cysteine proteases, such as cathepsin B identified in our study are critical for contact dependent host invasion through proteolysis of host extracellular matrix proteins and degradation of host immune proteins, including the amoeba genus Naegleria and E. histolytica (98,150,151). Cathepsin B expression in the parasite Giardia duodenalis, is induced by soluble host factors that deter attachment (105).…”

A Novel Cysteine Protease Inhibitor of Naegleria fowleri That Is Specifically Expressed during Encystation and at Mature Cysts

“…Studies have reported the essential role of parasitic protozoan cysteine protease inhibitors in the survival and infectivity of parasites in hosts by regulating endogenous or exogenous proteases [27][28][29][30]. Previously, we identified and characterized a novel stefin-family cysteine protease inhibitor of N. fowleri, known as fowlerstefin [13]. In this study, we characterized the second cysteine protease inhibitor of N. fowleri, NfCPI.…”

“…Inhibitory activity of NfCPI against cysteine proteases was determined by measuring the residual enzyme activity after incubation of each enzyme with NfCPI [12,13]. Cysteine proteases used in this study were papain (Sigma, St. Louis, MO, USA) and two recombinant cathepsin-B and cathepsin-B-like cysteine proteases of N. fowleri (NfCB and NfCBL).…”

“…In order to analyze the structural properties of NfCPI, electrophoretic analysis of the NfCPI was performed in the presence and absence of SDS or β-mercaptoethanol [β-ME, 5% (vol/vol)] with or without prior heating at 100 • C for 5 min [12,13]. The native molecular size of NfCPI was also analyzed by gel filtration chromatography using a Superdex 200 HR 10/30 column with an Äcta FPLC system (GE Biosciences, Pittsburgh, PA, USA).…”

“…The expression profiles of nfcb, nfcbl, cathepsin Z (nfcz) and calpain-like (nfcalpain-like) were also analyzed with the following primer sets: nfcb (5 -GAGC-TCATGATGATGAGATGCGTGAAT-3 and 5 -AAGCTTTTAACAACATGCTCCATCA-ATGAAAT-3 ), nfcbl (5 -GTGAGTAG-GAGGTAGCCTTCCA-3 and 5 -CCTCTTGTTCA-TCACCTTGTTG-3 ), nfcz (5 -ATGAA-AAATCTCTGGCTTGTCTTGCTG-3 and 5 -TTAA-ACAATTGGAACAGCAAAGGCACA-3 ), and nfcalpain-like (5 -ATGAACATTCCAGACAAAATTCCG-3 and 5 -TTACACAA-CTTTTCTAACTTC-3 ). Glyceraldehyde 3-phosphate dehydrogenase of N. fowleri (nfgapdh) was included as an internal control for relative quantitation of other genes [13].…”