Pathogenesis and molecular genetics of expression of iron acquisition systems in Vibro parahaemolyticus

1.纯化了与氯化血红素结合的83 kDa铁阻抑外膜蛋白，并制备了兔抗该蛋白的抗血清。用该抗血清进行免疫印迹分析表明，该蛋白的分子质量和抗原性在物种间高度保守。对109株来自不同来源的副溶血弧菌在花期培养中产生弧铁蛋白的活性进行了检测，并建立了一种新的高效液相色谱方法。大多数受检菌株(95.2%)检出弧菌铁蛋白。44株临床分离株弧菌铁蛋白的平均值为742.7±46.7um，与食品分离株(386.5±lt；+-gt；43.6，n=35)和环境分离株(378，8~lt；+-gt；37.2，n=25)的平均值有显著差异。这些结果表明，临床分离株的较高生产力可能为cond…的生长提供了显著的选择优势。更多的铁限制，如在IN3中存在。对副溶血性弧菌WP1的耐锰突变株进行了分离和鉴定。突变体表达的78 kDa和83 kDa外膜蛋白不依赖于铁，表明存在毛皮调节子。编码铁调节蛋白的毛皮基因已经被克隆和测序。其产物与已知毛皮序列具有高度的同源性。在铁限制条件下生长的副溶血弧菌的胞外蛋白水解酶活性显著提高。一些耐锰突变株即使在铁充足的条件下也表现出比野生型菌株Wp1更高的蛋白酶活性，这表明编码一种蛋白酶的基因(S)的表达可能受到Fur的调控。由于已经报道了四种蛋白酶的存在，所以对于Fur调控的是哪种蛋白酶基因还需要进一步的研究。对这些基因的进一步核苷酸序列分析可能会导致铁反应基因的鉴定和整个相关基因的克隆。较少

1. The 83-kDa iron-repressible outer membrane protein which binds hemin was purified and the antiserum against this protein was raised in rabbits. Immunoblot analysis using this antiserum indicated that the molecular mass and antigenic properties of the protein were highly conserved among this species.2. A total of 109 V.parahaemolyticus strains isolated from different sources were examined for their activity to produced vibrioferrin in spend cultures, which was determined by a new developed HPLC method. Vibrioferrin was detected in most of the strains tested (95.2%). The mean value <plus-minus> SD of vibrioferrin in the clinical isolates (n=44) was 742.7 <plus-minus> 46.7 muM in sup./OD660, signifcantly different with p<0.01 from the mean values in food isolate (386.5 <plus-minus> 43.6, n=35) and environmental isolates (378,8 <plus-minus> 37.2, n=25). These results suggest that the higher productivity of clinical isolates may afford a significant selective advantage for growth in cond … More itions of iron-limitation such as exist in the in3. Manganese resistant mutants of V.parahaemolyticus WP1 were isolated and characterized. The mutants expressed the 78- and 83-kDa outer membrane proteins independently of iron, suggesting the existence of an Fur-regulon. The fur gene, encoding an iron-regulatory protein has been cloned and sequenced. Its product showed a high degree of homology to known Fur sequence.4. Exocellular protease activity of V.parahaemolyticus strains was significantly enhanced when they were grown under iron-limited conditions. Some manganese resistant mutants exhibited higher protease activity than the wild type strain WP1 even when grown under iron sufficient conditions, suggesting that expression of the gene encoding a protease (s) may be regulated by the Fur.Further study is required to clarify as to what kind of protease gene is regulated by Fur, since the presence of four proteases has been reported for this species.Many clones which may have genes regulated by Fur were isolated by Fur titration assay method. Further nucleotide sequence analysis of these genes may lead to identification of iron-responsive genes and to cloning of the whole genes responsible. Less