Respiratory hydrogen use by Salmonella enterica serovar typhimurium is essential for virulence
Respiratory hydrogen use by Salmonella enterica serovar typhimurium is essential for virulence
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DOI：
10.1128/iai.72.11.6294-6299.2004
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发表时间：
2004-11-01
影响因子：
3.1
通讯作者：
Gunn, J
中科院分区：
文献类型：
--
作者：
Maier, RJ;Olczak, A;Gunn, J
基于现有的带注释的基因序列信息，肠道病原体沙门氏菌与其他肠道细菌一样，含有三种假定的与膜相关的利用H - 2的氢化酶。这些酶分解分子氢（H₂），释放出低电位电子，用于还原呼吸链中含醌或含血红素的成分。在此我们表明，鼠伤寒沙门氏菌的三种不同的与膜相关的氢化酶中的每一种都与一个以氧作为末端电子受体的呼吸途径相偶联。在基于血液的培养基中生长的细胞所表达的氢化酶（H₂氧化）活性是在卢里亚 - 贝尔塔尼培养基上生长的细胞的四倍。悬浮在磷酸盐缓冲盐水中的细胞在H₂ - O₂呼吸途径中每消耗1摩尔O₂会消耗2摩尔H₂，并且该活性受到呼吸抑制剂氰化物的抑制。在活小鼠的器官（即肝脏和脾脏）中测得的分子氢水平平均超过40μM，而肠道内（假定为气体的来源）的水平是这个数值的四倍。鼠伤寒沙门氏菌对H₂的半饱和亲和力仅为2.1μM，因此预计在体内利用H₂的氢化酶会被还原底物所饱和。基于三种氢化酶基因中每种基因都有突变的菌株的结果，所有三种氢化酶都对伤寒热 - 小鼠模型中的细菌毒力有贡献。所引入的突变是非极性的，突变菌株的生长情况与亲本菌株相似。去除所有三种氢化酶后得到的菌株是无毒的，并且（与亲本菌株相反）无法侵入肝脏或脾脏组织。将其中一种氢化酶基因以低拷贝数质粒的形式导入三重突变菌株中，得到的菌株能够氧化H₂，并在接种后11天内使小鼠发病；因此，三重突变体的无毒表型不是由于未知的假突变引起的。我们得出结论，以呼吸方式利用H₂对于能量产生是必需的，这使得沙门氏菌在感染过程中能够生长并具有后续的毒力。
Based on available annotated gene sequence information, the enteric pathogen salmonella, like other enteric bacteria, contains three putative membrane-associated H-2-using hydrogenase enzymes. These enzymes split molecular H-2, releasing low-potential electrons that are used to reduce quinone or heme-containing components of the respiratory chain. Here we show that each of the three distinct membrane-associated hydrogenases of Salmonella enterica serovar Typhimurium is coupled to a respiratory pathway that uses oxygen as the terminal electron acceptor. Cells grown in a blood-based medium expressed four times the amount of hydrogenase (H, oxidation) activity that cells grown on Luria Bertani medium did. Cells suspended in phosphate-buffered saline consumed 2 mol of H-2 per mol of O-2 used in the H-2-O-2 respiratory pathway, and the activity was inhibited by the respiration inhibitor cyanide. Molecular hydrogen levels averaging over 40 muM were measured in organs (i.e., livers and spleens) of live mice, and levels within the intestinal tract (the presumed origin of the gas) were four times greater than this. The half-saturation affinity of S. enterica serovar Typhimurium for H-2 is only 2.1 muM, so it is expected that H-2-utilizing hydrogenase enzymes are saturated with the reducing substrate in vivo. All three hydrogenase enzymes contribute to the virulence of the bacterium in a typhoid fever-mouse model, based on results from strains with mutations in each of the three hydrogenase genes. The introduced mutations are nonpolar, and growth of the mutant strains was like that of the parent strain. The combined removal of all three hydrogenases resulted in a strain that is avirulent and (in contrast to the parent strain) one that is unable to invade liver or spleen tissue. The introduction of one of the hydrogenase genes into the triple mutant strain on a low-copy-number plasmid resulted in a strain that was able to both oxidize H-2 and cause morbidity in mice within 11 days of inoculation; therefore, the avirulent phenotype of the triple mutant is not due to an unknown spurious mutation. We conclude that H-2 utilization in a respiratory fashion is required for energy production to permit salmonella growth and subsequent virulence during infection.