Novel cathepsin B and cathepsin B-like cysteine protease of Naegleria fowleri excretory–secretory proteins and their biochemical properties

Abstract

Naegleria fowleri causes a lethal primary amoebic meningoencephalitis (PAM) in humans and experimental animals, which leads to death within 7-14 days. Cysteine proteases of parasites play key roles in nutrient uptake, excystment/encystment, host tissue invasion, and immune evasion. In this study, we cloned N. fowleri cathepsin B (nfcpb) and cathepsin B-like (nfcpb-L) genes from our cDNA library of N. fowleri. The full-length sequences of genes were 1,038 and 939 bp (encoded 345 and 313 amino acids), and molecular weights were 38.4 and 34 kDa, respectively. Also, nfcpb and nfcpb-L showed a 56 and 46 % identity to Naegleria gruberi cathepsin B and cathepsin B-like enzyme, respectively. Recombinant NfCPB (rNfCPB) and NfCPB-L (rNfCPB-L) proteins were expressed by the pEX5-NT/TOPO vector that was transformed into Escherichia coli BL21, and they showed 38.4 and 34 kDa bands on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis using their respective antibodies. Proteolytic activity of refolded rNfCPB and rNfCPB-L was maximum at a pH of 4.5, and the most effective substrate was Z-LR-MCA. rNfCPB and rNfCPB-L showed proteolytic activity for several proteins such as IgA, IgG, IgM, collagen, fibronectin, hemoglobin, and albumin. These results suggested that NfCPB and NfCPB-L cysteine protease are important components of the N. fowleri ESP, and they may play important roles in host tissue invasion and immune evasion as pathogens that cause N. fowleri PAM.

Naegleria fowleri: Sources of infection, pathophysiology, diagnosis, and management; a review

“…The important components of the N. fowleri ESP are the cysteine protease named N. fowleri cathepsin B‐like (nfcpb‐L) and cathepsin B (nfcpb), the study indicated that these refolding proteins have a principal role in host immune evasion, tissue invasion, nutrient uptake, and encystment/excystment. Both rNfCP‐B and rNfCPB‐L have shown proteolytic activity for several proteins including IgA, IgG, IgM, fibronectin, haemoglobin, albumin and collagen . Further, it was recently evaluated that N. fowleri excretory and secretory proteins (NfESP) were involved in producing inflammatory responses in BV‐2 microglial cells.…”

Cathepsins: Proteases that are vital for survival but can also be fatal

“…RAAS activation underlies numerous pathologies [20]. Cathepsin B from amoeba can cleave several human proteins including immunoglobulins (IgA, IgG, IgM), hemoglobin, collagen, fibronectin, and albumin [21]. Cathepsin D cleaves fibronectin and laminin.…”

Naegleria fowleri after 50 years: is it a neglected pathogen?

“…Finally, the resistant form of N. fowleri, the cyst, is usually spherical, smooth, double walled and refractive, and measures approximately 20 µm in diameter (Fig. 2c); the wall is composed mainly of polysaccharides (Ch avez- Munguía et al, 2009;Lee et al, 2014). The cysts contain pores that are sealed by a thin mucoid layer.…”

“…Recently, cathepsin B and cathepsin-B-like cysteine proteases have been cloned and purified. These proteases can degrade a variety of human substrates, such as IgA, IgG, IgM, collagen, fibronectin, haemoglobin and albumin (Lee et al, 2014). Considering all of these findings, the proteases of N. fowleri might be excellent targets of chemotherapeutic agents directed against this pathogen (Klemba & Goldberg, 2002;Sajid & McKerrow, 2002;McKerrow et al, 2008).…”