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

1. 纯化了与血红蛋白结合的83-kDa铁抑制外膜蛋白，并在家兔中制备了该蛋白的抗血清。免疫印迹分析表明，该蛋白的分子量和抗原性在该物种中高度保守。采用高效液相色谱法测定了从不同来源分离的109株副溶血性弧菌在体外培养中产生弧菌铁蛋白的活性。大多数菌株（95.2%）检出铁弧菌蛋白。临床分离株（n=44）的弧菌铁蛋白<正负> SD平均值为742.7 <正负> 46.7 muM，与食物分离株（386.5 <正负> 43.6,n=35）和环境分离株（378,8 <正负> 37.2,n=25）的平均值差异有统计学意义，p<0.01。这些结果表明，临床分离株的高生产力可能为在低温条件下的生长提供了显着的选择优势。分离并鉴定了副溶血性弧菌WP1的耐锰突变体。突变体独立于铁表达78-和83-kDa外膜蛋白，表明存在fur调控子。编码铁调节蛋白的毛皮基因已被克隆并测序。其产物与已知的Fur序列具有高度的同源性。在限铁条件下培养的副溶血性弧菌胞外蛋白酶活性显著增强。即使在铁充足的条件下生长，一些抗锰突变体也表现出比野生型菌株WP1更高的蛋白酶活性，这表明编码蛋白酶的基因的表达可能受到皮毛的调节。由于该物种中存在四种蛋白酶，因此需要进一步研究以明确哪种蛋白酶基因受Fur调控。用Fur滴定法分离了许多可能含有Fur调控基因的克隆。进一步分析这些基因的核苷酸序列可能导致铁反应基因的鉴定和整个基因的克隆。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