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

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.

Free-Living Amoebas in Extreme Environments: The True Survival in our Planet

“…The cellulose precursor is glucose incorporated into the cell wall as β(1 → 4)-glucans (i.e., cellulose). However, in N. fowleri expression studies, an increase in the BioMed Research International expression of the cysteine protease inhibitor genes (belonging to the cystatin family) was evidenced in the encystment process and mature amoeba cysts in N. fowleri (NfCPI) [96]. These findings collectively suggest that NfCPI may play a critical role in N. fowleri cyst formation by regulating cysteine proteases that may be actively involved in mediating cyst formation or else in the encystment process of amoebae [97].…”

“…fowleri expression studies, an increase in the expression of the cysteine protease inhibitor genes (belonging to the cystatin family) was evidenced in the encystment process and mature amoeba cysts in N . fowleri (NfCPI) [ 96 ]. These findings collectively suggest that NfCPI may play a critical role in N .…”

Naegleria genus pangenome reveals new structural and functional insights into the versatility of these free-living amoebae

“…The information in differential expression of genes between the different forms of Naegleria is very limited. Lê and co-workers found evidence that a Cystatin-B like protein (encoded by the gene mRNA1_NF0117700-p1 in NF_ATCC 30863) may play a critical role in N. fowleri encystation ( Lê et al, 2021 ). We recovered Cystatin-B orthologs in N. gruberi , N. fowleri (7 out the 8 strains analyzed) and in only N. lovaniensis strain ( Supplementary Table S5 ).…”

Inhibitor of Cysteine Protease of Plasmodium malariae Regulates Malapains, Endogenous Cysteine Proteases of the Parasite

“…Inhibitory activity of recombinant PmICP against cysteine proteases was analyzed by measuring the residual enzyme activity after incubation of each enzyme with PmICP [ 21 , 25 ]. Cysteine proteases used in this study were papain (Sigma, St. Louis, MO, USA), human cathepsin B (HCB; Sigma), human cathepsin L (HCL; Sigma), malapain-2 (MP-2), malapain-4 (MP-4), vivapain-3 (VX-3), vivpapain-4 (VX-4), and falcipain-3 (FP-3).…”