Acanthamoeba culbertsoni의 단백분해효소와 병원성과의 상관성

Abstract

[한글]

자유생활아메바의 병원성과 단백분해효소의 연관성을 밝히기 위해 병원성인 Acanthamoeba culbertsoni와 비병원성 인 Acanthamoeba sp. 한국 분리주 YM-3에서 단백분해효소의 활성을 비교 관찰하였다.

각 아메바의 영양형을 ICR 마우스에 접종시켜 사망률을 관찰하였다. A.culbertsoni 와 Acanthamoeba sp., YM-3의 영양형 1x10**5개가 함유된 부유액 5μl를 secobarbital로 마취한 마우스의 비강에 떨어뜨려 감염시켰다. A.culbertsoni에 감염된 마우스의 사망률이

60.0%였고 YM-3를 감염시켰을 때 사망률이 0%였다.

Gelatin을 기질로 한 Sodium dodesyl sulfate polyacrylamide gel electrophoresis의 결과 A.culbertsoni는 6개, YM-3는 5개의 분획을 보였다. Chinese hamster ovary 세포에 대한 세포독성을 Cytotox 96 assay kit(Promega, Madison, Wl, U.S.A.)을 사용하여 측정한 결과, A.culbertsoni와 YM-3는 차이를 보이지 않았다. A.culbertsoni 와 YM-3의 단백분해효소원은 azocasein을 기질로 하여 활성잔기 억제 실험을 한 결과 차이를 보였다. A.culbertsoni와 YM-3의 용해물자 24시간 배양액에 여러 단백분해 활성 억제제를 처리하였다. A.culbertsoni에 cysteine계 단백분효소와 serine 및 histidine계 단백분해효소가 존재함을 알수 있었고 YM-3에는 serine 및 histidine계 단백분해효소가 있음이 관찰되었다 Sephacryl S-300-HR column을 사용하여 부분정제한 A.culbertsoni의 용해물을 임의로 6개의 분획으로 나누고 단백분해 활성 억제제를 처리하여 Ⅲ번 분획이 cysteine계 단백분해효소원이고, V번 분획이 serine 및 histidine계 단백분해효소원임을 추정할 수 있었으며, 그 분자량을 측정한 결과 Ⅲ번 분획이 63,100 Da, V분획은 33,900 Da였다.

[영문]

The pathogenicity of free-living amoeba has been 7own to be related to the proteinase activity. This study aimed to verify the proteinase acclivity of Acanthamoeba sp. Korean isolate YM-3 and /acanthamoeba culbertsoni with reference to the pathogenicity. Mortality of ICR mice inoculated intranasally with 1×10**5 Acanthamoeba app. trophozoites was observed. Forty of the mice infected with A.culbertsoni were survived after 17 days. None of the mice which was infected with YM-3 was dead. This demonstrated that A.culbertsoni was pathogenic strain and YM-3 was non-pathogenic strain, To determine the type of proteinase, various modification reagents were added to Acanthamoeba app. Iysate and media. Various reagents used were as follows; Aspartic type proteinase inhibitors, serine type proteinase inhibitors, Phenylglyoxal, partial serine type proteinase inhibitor and cysteine type proteinase inhibitor(leupeptin), some serine type proteinase inhibitor and histidine type proteinase inhibitor. As a result, cysteine type, serine type, histidine type proteinases were detected in A.culbertsoni and serine type, histidine type poteinases were detected in YM-3. Proteinase activity of A.culbertsoni and YM-3 was tested by SDS-PAGE containing 0.4% gelatin. A.culbertsoni and YM-3 revealed different band patterns, 6 bands for A.culbertsoni and 5 bands for YM-3.

The cytotoxicty was observed against Chinese hamster ovarian cell using the Cytotox 96 assay kit(Promega, Madison Wl, U.S.A.) This was not different between A.culbertsoni and YM-3.

The Gel filtratifnl of A.culbertsoni Iysate was performed using Sephacryl S-300-HR. Six fractions were demonstrated and tested by various proteinase inhibitors. Ⅲ fiaction was inhibited by cysteine type proteinase inhibitor, and V flaction was inhibited by serine and histidine type proteinase inhibitor. Molecular weight of Ⅲ fraction was 63,100 Da'and V fraction 33,900Da.