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首页> 美国卫生研究院文献>Infection and Immunity >Sequence Analysis of the mip Gene of the Soilborne Pathogen Legionella longbeachae
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Sequence Analysis of the mip Gene of the Soilborne Pathogen Legionella longbeachae

机译：土传病原菌军团菌mip基因的序列分析

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摘要

To understand the basis of pathogenesis by Legionella longbeachae serogroup 1, the importance of the Mip protein in this species was examined. Amino-terminal analysis of the purified, cloned L. longbeachae serogroup 1 ATCC 33462 Mip protein confirmed that the cloned gene protein was expressed and processed in an Escherichia coli background. DNA sequence analysis of plasmid pIMVS27, containing the entire L. longbeachae serogroup 1 mip gene, revealed a high degree of homology to the mip gene of Legionella pneumophila serogroup 1, 76% homology at the DNA level and 87% identity at the amino acid level. Primer extension analysis determined that the start site of transcription was the same for both species, with some differences observed for the −10 and −35 promoter regions. Primers designed from the mip gene sequence obtained for L. longbeachae serogroup 1 ATCC 33462 were used to amplify the mip genes from L. longbeachae serogroup 2 ATCC 33484 and an Australian clinical isolate of L. longbeachae serogroup 1 A5H5. The mip gene from A5H5 was 100% identical to the type strain sequence. The serogroup 2 strain of L. longbeachae differed by 2 base pairs in third-codon positions. Allelic exchange mutagenesis was used to generate an isogenic mip mutant in ATCC 33462 and strain A5H5. The ATCC mip mutant was unable to infect a strain of Acanthamoebae sp. both in liquid and in a potting mix coculture system, while the A5H5 mip mutant behaved in a manner siilar to that of L. pneumophila serogroup 1, i.e., it displayed a reduced capacity to infect and multiply within Acanthamoebae. To determine if this mutation resulted in reduced virulence in the guinea pig animal model, the A5H5 mip mutant and its parent strain were assessed for their abilities to establish an infection after aerosol exposure. Unlike the virulent parent strain, the mutant strain did not kill any animals under two different dose regimes. The data indicate that the Mip protein plays an important role in the intracellular life cycle of L. longbeachae serogroup 1 species and is required for full virulence.
机译：为了了解长军团菌血清群1发病机理的基础，检查了该物种中Mip蛋白的重要性。纯化，克隆的长滩乳杆菌血清群1 ATCC 33462 Mip蛋白的氨基末端分析证实，克隆的基因蛋白在大肠杆菌背景中表达和加工。质粒pIMVS27的DNA序列分析包含完整的长滩乳杆菌血清群1 mip基因，显示与军团菌嗜肺杆菌血清群1的mip基因高度同源，在DNA水平上具有76％的同源性，在氨基酸水平上具有87％的同一性。引物延伸分析确定两种物种的转录起始位点相同，但在-10和-35启动子区域观察到一些差异。从为长滩头球菌血清群1 ATCC 33462获得的mip基因序列设计的引物用于扩增来自长滩头球菌血清群2 ATCC 33484的mip基因和澳大利亚临床上的L. longbeachae血清群1 A5H5分离株。来自A5H5的mip基因与类型菌株序列100％相同。 L. longbeachae的血清群2菌株在第三密码子位置相差2个碱基对。等位基因交换诱变用于在ATCC 33462和菌株A5H5中产生等基因mip突变体。 ATCC mip突变体无法感染 Acanthamoebae sp。菌株。在液体和盆栽混合共培养系统中，A5H5 mip 突变体的行为与 L相似。肺炎血清群1，即在Acanthamoebae 中的感染和繁殖能力降低。为了确定这种突变是否会导致豚鼠动物模型的毒力降低，评估了A5H5 mip 突变体及其亲本菌株在暴露于气溶胶​​后感染的能力。与有毒的亲本菌株不同，突变菌株在两种不同剂量方案下均未杀死任何动物。数据表明，Mip蛋白在 L的细胞内生命周期中起着重要作用。 longbeachae 血清群1种，是充分毒力所必需的。

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