Background & Aims

Helicobacter pylori is remarkable for its genetic variation; yet, little is known about its genetic changes during early stages of human infection, as the bacteria adapt to their new environment. We analyzed genome and methylome variations in a fully virulent strain of H pylori during experimental infection.

Methods

We performed a randomized Phase I/II, observer-blind, placebo-controlled study of 12 healthy, H pylori–negative adults in Germany from October 2008 through March 2010. The volunteers were given a prophylactic vaccine candidate (n = 7) or placebo (n = 5) and then challenged with H pylori strain BCM-300. Biopsy samples were collected and H pylori were isolated. Genomes of the challenge strain and 12 reisolates, obtained 12 weeks after (or in 1 case, 62 weeks after) infection were sequenced by single-molecule, real-time technology, which, in parallel, permitted determination of genome-wide methylation patterns for all strains. Functional effects of genetic changes observed in H pylori strains during human infection were assessed by measuring release of interleukin 8 from AGS cells (to detect cag pathogenicity island function), neutral red uptake (to detect vacuolating cytotoxin activity), and adhesion assays.

Results

The observed mutation rate was in agreement with rates previously determined from patients with chronic H pylori infections, without evidence of a mutation burst. A loss of cag pathogenicity island function was observed in 3 reisolates. In addition, 3 reisolates from the vaccine group acquired mutations in the vacuolating cytotoxin gene vacA, resulting in loss of vacuolization activity. We observed interstrain variation in methylomes due to phase variation in genes encoding methyltransferases.

Conclusions

We analyzed adaptation of a fully virulent strain of H pylori to 12 different volunteers to obtain a robust estimate of the frequency of genetic and epigenetic changes in the absence of interstrain recombination. Our findings indicate that the large amount of genetic variation in H pylori poses a challenge to vaccine development. ClinicalTrials.gov no: NCT00736476.

中文翻译：

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幽门螺杆菌与cag致病岛在人类感染的早期阶段的基因组和甲基组变异。

背景与目标

幽门螺杆菌因其遗传变异而著称。然而，由于细菌适应新环境，人们对其在人类感染初期的遗传变化知之甚少。我们分析了幽门螺杆菌的完全有毒力的菌株在实验感染过程中的基因组和甲基化组变异。

方法

我们从2008年10月至2010年3月在德国对12名健康，幽门螺杆菌阴性的健康成年人进行了I / II期，观察员盲目，安慰剂对照的随机研究。为志愿者提供了预防性疫苗候选者（n = 7）或安慰剂（n = 5），然后用幽门螺杆菌BCM-300攻击。收集活检样品并分离幽门螺杆菌。通过单分子实时技术对感染后12周（或在1例中，在62周后）获得的攻击菌株和12个分离株的基因组进行测序，该技术同时可确定全基因组甲基化模式所有菌株。幽门螺杆菌中观察到的遗传变化的功能作用通过测量AGS细胞中白细胞介素8的释放（以检测cag致病岛功能），中性红吸收（以检测空泡细胞毒素活性）和黏附测定，评估了人类感染过程中的毒株。

结果

观察到的突变率与先前从慢性幽门螺杆菌感染患者确定的突变率一致，没有突变爆发的证据。在3个分离株中均观察到cag致病岛功能的丧失。另外，来自疫苗组的3个分离物获得了空泡细胞毒素基因vacA的突变，导致空泡活性的丧失。我们观察到由于编码甲基转移酶的基因的相变，导致甲基化组间的株间变异。

结论

我们分析了一种完全有毒力的幽门螺杆菌菌株对12位不同志愿者的适应性，以在缺乏株间重组的情况下获得对遗传和表观遗传变化频率的可靠估计。我们的发现表明，幽门螺杆菌中大量的遗传变异对疫苗开发提出了挑战。ClinicalTrials.gov编号：NCT00736476。