Purification and characterization of a 33 kDa serine protease from Acanthamoeba lugdunensis KA/E2 isolated from a Korean keratitis patient

Kim, Hyo-Kyung; Ha, Young-Ran; Yu, Hak Sun; Kong, Hyun-Hee; Chung, Dong‐Il

doi:10.3347/kjp.2003.41.4.189

Cited by 24 publications

(26 citation statements)

References 18 publications

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“…more, by adding 0.1 mM PMSF, the CPE of 3 Acanthamoeba strains with different degree of virulence came to be equal level. This indicates that the difference in virulence and cytopathy have may originated mainly from PMSF susceptible serine proteinases as suggested by previous studies (Leher et al, 1998;Alfrei et al, 2000;Kong et al, 2000;Kim et al, 2003).…”

Section: Discussionsupporting

confidence: 56%

“…Northern blot analysis revealed that AhSUB was expressed at higher levels in the high-virulent strain than low-or avirulent strains. Recently, we confirmed the secretion of a 33 kDa serine protease from ocular and environmental isolates of Acanthamoeba (Kim et al, 2003).…”

Section: Introductionmentioning

confidence: 57%

“…Purification of 33 kDa serine proteases from culture media of 3 strains were performed as described in previous papers Kim et al, 2003). The amoeba culture supernatant was precipitated with 65% ammonium sulfate and centrifuged at 10,000 rpm for 15 min.…”

Section: Purification Of Subtilasementioning

confidence: 99%

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Comparison of specific activity and cytopathic effects of purified 33 kDa serine proteinase from Acanthamoeba strains with different degree of virulence

Kim

Kong

et al. 2006

Korean J Parasitol

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Section: Discussionsupporting

confidence: 56%

Section: Introductionmentioning

confidence: 57%

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Comparison of specific activity and cytopathic effects of purified 33 kDa serine proteinase from Acanthamoeba strains with different degree of virulence

Kim

Kong

et al. 2006

Korean J Parasitol

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“…Considering the high phagocytic activity of Acanthamoeba as a free-living organism and tissue-invasive behavior as a parasite, proteases would be involved in various processes of Acanthamoeba, such as pathogenesis, nutrient obtainment, and host tissue destruction. To date, several reports on the serine, cysteine, and metalloproteinases of Acanthamoeba were published (9,12,18,19). Serine proteinase purified from culture supernatant was reported to play roles in host tissue invasion because of its strong activity against a broad spectrum of extracellular matrix and serum proteins of humans (12).…”

mentioning

confidence: 99%

Intracellular Localization and Trafficking of Serine Proteinase AhSub and Cysteine Proteinase AhCP of Acanthamoeba healyi

et al. 2006

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Proteinases have been proposed to play important roles in pathogenesis and various biologic actions in Acanthamoeba. Although genetic characteristics of several proteases of Acanthamoeba have been reported, the intracellular localization and trafficking of these enzymes has yet to be studied. In the present study, we analyzed the intracellular localization and trafficking of two proteinases, AhSub and AhCP, of Acanthamoeba healyi by transient transfection. Full-length AhSub-enhanced green fluorescent protein (EGFP) fusion protein was found in intracellular vesicle-like structures of transfected amoebae. Time-lapse photographs confirmed the secretion of the fluorescent material of the vesicle toward the extracellular space. The mutated AhSub, of which the pre or prepro region was deleted, was found to localize diffusely throughout the cytoplasm of the amoeba rather than concentrated in the secretory vesicle. Transfection of the construct containing the pre region only showed the same localization and trafficking of the full-length AhSub. A cysteine proteinase AhCP-EGFP fusion protein showed similar localization in the vesicle-like structure in the amoeba. However, using Lyso Tracker analysis, these vesicular structures of AhCP were confirmed to be lysosomes rather than secretory vesicles. The AhCP construct with a deletion of the prepro region showed a dispersed distribution of fluorescence in the cytoplasm of the cells. These results indicated that AhSub and AhCP would play different roles in Acanthameoba biology and that the pre region of AhSub and pro region of AhCP are important for proper intracellular localization and trafficking of each proteinase.The genus Acanthamoeba, one of the amphizoic amoebae with ubiquitous distribution in human environments, has been known to cause granulomatous amoebic encephalitis in immune-compromised hosts and sight-threatening amebic keratitis, especially in contact lens users (15).Proteinases have been known to play important roles in the metabolism, development, or survival of protozoa (16). Considering the high phagocytic activity of Acanthamoeba as a free-living organism and tissue-invasive behavior as a parasite, proteases would be involved in various processes of Acanthamoeba, such as pathogenesis, nutrient obtainment, and host tissue destruction. To date, several reports on the serine, cysteine, and metalloproteinases of Acanthamoeba were published (9,12,18,19). Serine proteinase purified from culture supernatant was reported to play roles in host tissue invasion because of its strong activity against a broad spectrum of extracellular matrix and serum proteins of humans (12). The gene of the purified serine proteinase was characterized and named AhSub based on high homology with bacterial subtilisins (3). Cathepsin L-like cysteine proteinase genes were cloned from Acanthamoeba healyi and Acanthamoeba culbertsoni (3, 29). However, the specific function of these cysteine proteinases in Acanthamoeba is still unclear. The intracellular localization and trafficking of the e...

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“…This is achieved using a specific pseudopodial projection, the "amoebastome" (16). Another mechanism proposed is the use of cytotoxic toxins and cytolytic proteinases that destroy target cells (5,11,12,15). At the molecular level, a 17-kDa unique membrane protein (MP2CL5) was found to be expressed by Mp2CL5 cloned from a cDNA expression library of N. fowleri (20).…”

Section: Discussionmentioning

confidence: 99%

Expression of the nfa1 Gene Cloned from Pathogenic Naegleria fowleri in Nonpathogenic N. gruberi Enhances Cytotoxicity against CHO Target Cells In Vitro

et al. 2005

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The pathogenic amoeba Naegleria fowleri has a 360-bp nfa1 gene that encodes the Nfa1 protein (13.1 kDa), which is located in the pseudopodia of the amoeba, and an anti-Nfa1 antibody reduces N. fowleri-induced mammalian-cell cytotoxicity in vitro. In contrast, an anti-Nfa1 antibody cannot detect Nfa1 protein expression in the nonpathogenic amoeba Naegleria gruberi, which also possesses the nfa1 gene. In the present study, the nfa1 gene cloned from pathogenic N. fowleri was transfected into nonpathogenic N. gruberi to determine whether it was related to pathogenicity. The nfa1 gene was initially inserted into a eukaryotic transfection vector, pEGFP-C2, containing a cytomegalovirus promoter and the green fluorescent protein (GFP) gene, and was designed as pEGFP-C2/nfa1UTR (nfa1UTR contains 5 upstream regions, the nfa1 open reading frame, and 3 downstream regions). After transfection, the green fluorescence was observed in the cytoplasm of N. gruberi trophozoites. These transfectants were preserved for more than 9 months after selection. The transfected nfa1 gene was observed by PCR using nfa1-and vector-specific primers in the genomic DNA of N. gruberi transfected with the pEGFP-C2/nfa1UTR vector. In addition, the nfa1 and GFP genes were identified by reverse transcription-PCR in transgenic N. gruberi. The Nfa1 protein expressed in transgenic N. gruberi was identified as a 13.1-kDa band by Western blotting using an anti-Nfa1 antibody. Finally, N. gruberi transfected with the pEGFP-C2/nfa1UTR vector was found to have enhanced cytotoxicity against CHO cells compared with naïve N. gruberi.

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Purification and characterization of a 33 kDa serine protease from Acanthamoeba lugdunensis KA/E2 isolated from a Korean keratitis patient

Cited by 24 publications

References 18 publications

Comparison of specific activity and cytopathic effects of purified 33 kDa serine proteinase from Acanthamoeba strains with different degree of virulence

Comparison of specific activity and cytopathic effects of purified 33 kDa serine proteinase from Acanthamoeba strains with different degree of virulence

Intracellular Localization and Trafficking of Serine Proteinase AhSub and Cysteine Proteinase AhCP of Acanthamoeba healyi

Expression of the nfa1 Gene Cloned from Pathogenic Naegleria fowleri in Nonpathogenic N. gruberi Enhances Cytotoxicity against CHO Target Cells In Vitro

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