Induction of Protease Activity in Vibrio anguillarum by Gastrointestinal Mucus
Induction of Protease Activity in Vibrio anguillarum by Gastrointestinal Mucus
复制标题
DOI：
10.1128/aem.65.8.3555-3560.1999
复制
发表时间：
1999-08
影响因子：
4.4
通讯作者：
Steven M Denkin;D. R. Nelson
中科院分区：
文献类型：
--
作者：
Steven M Denkin;D. R. Nelson
研究了消化道粘液对鳗弧菌蛋白酶活性的影响。通过使用偶氮酪蛋白水解测定来测量蛋白酶活性。在粘液（200 μg粘液蛋白/ml）中生长至稳定期的细胞表现出比在Luria-Bertani肉汤加2%NaCl（LB 20）中生长的细胞高9倍的蛋白酶活性。用在LB 20中生长并重悬于粘液、LB 20、九盐溶液（NSS [无碳、无氮和无磷的盐溶液]）或海洋基本培养基（3 M）（约109 CFU/ml）中的细胞检查蛋白酶诱导。蛋白酶活性的诱导发生在添加粘液后60至90分钟，并且比在LB 20或3 M中孵育的细胞中测量的蛋白酶活性高≥70倍。粘液被分馏成水溶性和氯仿-甲醇可溶性馏分。水性部分支持鳗弧菌细胞的生长，但不诱导蛋白酶活性。氯仿-甲醇可溶性级分不支持生长，也不诱导蛋白酶活性。当两个级分混合时，诱导蛋白酶活性。氯仿-甲醇-可溶性级分没有诱导生长在LB 20中的细胞中的蛋白酶活性。EDTA（50 mM）抑制粘液诱导的蛋白酶。加入二价阳离子后，Mg 2+（100 mM）比等摩尔量的Ca 2+或Zn 2+在恢复活性方面更有效，表明粘液诱导型蛋白酶是镁依赖性金属蛋白酶。鳗弧菌AnempA突变株在暴露于粘液后没有表现出蛋白酶活性，但在粘液中生长。Southern分析和PCR扩增证实鳗弧菌M93含有empA。这些数据表明鳗弧菌的empA金属蛋白酶是由胃肠道粘液特异性诱导的。
ABSTRACT The effect of gastrointestinal mucus on protease activity inVibrio anguillarum was investigated. Protease activity was measured by using an azocasein hydrolysis assay. Cells grown to stationary phase in mucus (200 μg of mucus protein/ml) exhibited ninefold-greater protease activity than cells grown in Luria-Bertani broth plus 2% NaCl (LB20). Protease induction was examined with cells grown in LB20 and resuspended in mucus, LB20, nine-salts solution (NSS [a carbon-, nitrogen-, and phosphorus-free salt solution]), or marine minimal medium (3M) (∼109 CFU/ml). Induction of protease activity occurred 60 to 90 min after addition of mucus and was ≥70-fold greater than protease activity measured in cells incubated in either LB20 or 3M. Mucus was fractionated into aqueous and chloroform-methanol-soluble fractions. The aqueous fraction supported growth of V. anguillarum cells, but did not induce protease activity. The chloroform-methanol-soluble fraction did not support growth, nor did it induce protease activity. When the two fractions were mixed, protease activity was induced. The chloroform-methanol-soluble fraction did not induce protease activity in cells growing in LB20. EDTA (50 mM) inhibited the protease induced by mucus. Upon addition of divalent cations, Mg2+ (100 mM) was more effective than equimolar amounts of either Ca2+ or Zn2+ in restoring activity, suggesting that the mucus-inducible protease was a magnesium-dependent metalloprotease. AnempA mutant strain of V. anguillarum did not exhibit protease activity after exposure to mucus, but did grow in mucus. Southern analysis and PCR amplification confirmed that V. anguillarum M93 contained empA. These data demonstrate that the empA metalloprotease of V. anguillarum is specifically induced by gastrointestinal mucus.