High-Throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants during Acute Infection.
High-Throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants during Acute Infection.
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DOI：
10.1371/journal.pntd.0005117
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发表时间：
2016-11
影响因子：
3.8
通讯作者：
Haake DA
中科院分区：
文献类型：
--
作者：
Lourdault K;Matsunaga J;Haake DA
钩端螺旋体的致病种是钩端螺旋体病的病原体，钩端螺旋体病是一种在世界范围内引起死亡和发病的人畜共患疾病。由于可用于这种微生物的遗传工具数量有限，因此对钩端螺旋体属的毒力机制的理解仍处于早期阶段。十年前在致病菌株中随机转座子诱变的发展有助于鉴定几种毒力因子。在这项研究中，我们使用转座子测序（Tn-Seq）技术，它结合了转座子诱变与大规模的平行测序，研究在体内健身池的钩端螺旋体问号突变体。我们用42个突变体（输入池）感染仓鼠，其中包括先前通过毒力测试分析的4个基因（loa 22，ligB，flaA 1和lic 20111）中插入的对照突变体和23个信号转导基因被破坏的突变体。我们通过高通量测序在攻击后4天定量不同组织（血液、肾脏和肝脏）中的突变体，并将从组织中回收的突变体的频率与它们在输入池中的频率进行比较。当在致死性钩端螺旋体病的仓鼠模型中单独测试时，在Tn-Seq实验中不太适合的对照突变体的毒力被减毒。适应性未改变的对照突变体与野生型菌株的毒性相同。我们发现了两个突变体的转座子插入在同一个假定的腺苷酸/鸟苷酸环化酶基因（lic 12327），降低了在血液，肾脏和肝脏的体内健身。当在仓鼠中单独测试时，两种lic 12327突变体的毒力都减弱。对照突变体和lic 12327突变体在培养基中的生长与野生型菌株相似。这些结果证明了筛选大库L.通过转座子测序，对具有改变的适应性和潜在减弱的毒力的那些问号线虫转座子突变体进行测序。钩端螺旋体病是一种被忽视的传染病，在世界各地使许多人和动物患病。它是由致病性钩端螺旋体引起的。很少有钩端螺旋体的基因，有助于疾病已被确定。我们产生了800 L的文库。在不同基因中插入转座子的问号突变体。在仓鼠模型中单独筛选每种突变体的致病能力将是费力的，需要数千只动物。在一个试点实验中，我们用42个突变体的池感染仓鼠，以确定在感染期间被破坏的基因对细菌适应性的作用。在这三种组织中适应性减弱的12种突变体中，有两种在编码可能产生小信号分子cAMP的酶的基因中插入了转座子。当分别测试时，两个突变体不能使仓鼠窒息，表明L.怀疑者可能在致病中起作用。这些发现表明，大池的转座子插入突变体可以筛选在有限数量的动物，以确定钩体基因，可能是至关重要的疾病过程。
Pathogenic species of Leptospira are the causative agents of leptospirosis, a zoonotic disease that causes mortality and morbidity worldwide. The understanding of the virulence mechanisms of Leptospira spp is still at an early stage due to the limited number of genetic tools available for this microorganism. The development of random transposon mutagenesis in pathogenic strains a decade ago has contributed to the identification of several virulence factors. In this study, we used the transposon sequencing (Tn-Seq) technique, which combines transposon mutagenesis with massive parallel sequencing, to study the in vivo fitness of a pool of Leptospira interrogans mutants. We infected hamsters with a pool of 42 mutants (input pool), which included control mutants with insertions in four genes previously analyzed by virulence testing (loa22, ligB, flaA1, and lic20111) and 23 mutants with disrupted signal transduction genes. We quantified the mutants in different tissues (blood, kidney and liver) at 4 days post-challenge by high-throughput sequencing and compared the frequencies of mutants recovered from tissues to their frequencies in the input pool. Control mutants that were less fit in the Tn-Seq experiment were attenuated for virulence when tested separately in the hamster model of lethal leptospirosis. Control mutants with unaltered fitness were as virulent as the wild-type strain. We identified two mutants with the transposon inserted in the same putative adenylate/guanylate cyclase gene (lic12327) that had reduced in vivo fitness in blood, kidney and liver. Both lic12327 mutants were attenuated for virulence when tested individually in hamsters. Growth of the control mutants and lic12327 mutants in culture medium were similar to that of the wild-type strain. These results demonstrate the feasibility of screening large pools of L. interrogans transposon mutants for those with altered fitness, and potentially attenuated virulence, by transposon sequencing. Leptospirosis is a neglected infectious disease that sickens many humans and animals throughout the world. It is caused by pathogenic Leptospira spp. Few leptospiral genes that contribute to the disease have been identified. We generated a library of 800 L. interrogans mutants with transposon insertions in different genes. Screening each mutant individually for the ability to cause disease in the hamster model would be laborious and requires thousands of animals. In a pilot experiment, we infected hamsters with a pool of 42 mutants to determine the role of the disrupted genes on fitness of the bacterium during infection. Out of the 12 mutants with diminished fitness in the three tissues, two had transposon insertions in the gene encoding an enzyme that may generate the small signaling molecule cAMP. When tested separately, the two mutants failed to sicken hamsters, indicating that intracellular cAMP signaling within L. interrogans could have a role in causing disease. These findings indicate that large pools of transposon insertion mutants can be screened in a limited number of animals to identify leptospiral genes that may be critical for the disease process.
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影响因子：
6.7
作者：
Caimano MJ;Sivasankaran SK;Allard A;Hurley D;Hokamp K;Grassmann AA;Hinton JC;Nally JE
通讯作者：
Nally JE
影响因子：
3.2
作者：
JOHNSON, RC;HARRIS, VG
通讯作者：
HARRIS, VG
影响因子：
3.1
作者：
Eshghi, Azad;Becam, Jerome;Picardeau, Mathieu
通讯作者：
Picardeau, Mathieu
影响因子：
3.8
作者：
Lehmann JS;Fouts DE;Haft DH;Cannella AP;Ricaldi JN;Brinkac L;Harkins D;Durkin S;Sanka R;Sutton G;Moreno A;Vinetz JM;Matthias MA
通讯作者：
Matthias MA
影响因子：
7
作者：
Giardine, B;Riemer, C;Nekrutenko, A
通讯作者：
Nekrutenko, A