A Combination of Metagenomic and Cultivation Approaches Reveals Hypermutator Phenotypes within Vibrio cholerae-Infected Patients.
A Combination of Metagenomic and Cultivation Approaches Reveals Hypermutator Phenotypes within Vibrio cholerae-Infected Patients.
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DOI：
10.1128/msystems.00889-21
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发表时间：
2021-08-31
期刊：
影响因子：
6.4
通讯作者：
Shapiro BJ
中科院分区：
文献类型：
--
作者：
Levade I;Khan AI;Chowdhury F;Calderwood SB;Ryan ET;Harris JB;LaRocque RC;Bhuiyan TR;Qadri F;Weil AA;Shapiro BJ
霍乱弧菌可引起一系列症状，从严重腹泻到无症状感染。先前使用每个患者的多种细菌分离株的全基因组测序（WGS）的研究表明，霍乱弧菌在症状感染期间可以进化适度的遗传多样性。为了进一步探索宿主内霍乱弧菌多样性的程度，我们将基于培养的WGS和宏基因组学应用于来自孟加拉国的有症状和无症状霍乱患者的队列。虽然宏基因组学允许我们在有症状的患者中检测到更多的突变，但培养分离株的WGS对于检测无症状携带者中的霍乱弧菌多样性是必要的，这可能是由于他们的霍乱弧菌负荷低。使用宏基因组学和分离的WGS，我们报告了三条证据，表明患者体内的霍乱弧菌超变子进化。首先，我们在11名患者宏基因组中的5名中鉴定出霍乱弧菌DNA修复基因的非同义突变，其中霍乱弧菌基因组的覆盖率足够高，并且在3名患者中的1名中鉴定出霍乱弧菌DNA修复基因的非同义突变。其次，DNA修复基因中的这些突变往往伴随着过量的宿主内单核苷酸变异（iSNV）。第三，这些iSNV富含颠换突变，这是超变基因表型的已知标志。虽然高变基因似乎产生大多数选择性中性突变，但非变基因在多个患者中显示出会聚突变的迹象，表明宿主内的霍乱弧菌适应。我们的研究结果强调了宏基因组学与分离测序相结合的能力和局限性，以表征急性霍乱弧菌感染的患者内多样性，同时为霍乱患者内的超变表型提供证据。重要性患者体内的病原体演变可影响表型，如耐药性和毒力，可能影响临床结局。霍乱弧菌感染可导致危及生命的肠炎或无症状感染。在这里，我们描述了霍乱弧菌分离株的全基因组测序和来自有症状的霍乱患者和无症状携带者粪便的无培养宏基因组测序。尽管霍乱的持续时间通常很短，但我们发现了霍乱弧菌基因组中适应性突变的证据，这些突变在多个有症状的患者中独立和重复发生。我们还在几名患者中鉴定了霍乱弧菌超变子表型，这些表型似乎主要产生中性或有害突变。我们的工作为今后研究hypermutators的作用和患者内的演变，解释从无症状携带症状霍乱的变化奠定了基础。
Vibrio cholerae can cause a range of symptoms, from severe diarrhea to asymptomatic infection. Previous studies using whole-genome sequencing (WGS) of multiple bacterial isolates per patient showed that V. cholerae can evolve modest genetic diversity during symptomatic infection. To further explore the extent of V. cholerae within-host diversity, we applied culture-based WGS and metagenomics to a cohort of both symptomatic and asymptomatic cholera patients from Bangladesh. While metagenomics allowed us to detect more mutations in symptomatic patients, WGS of cultured isolates was necessary to detect V. cholerae diversity in asymptomatic carriers, likely due to their low V. cholerae load. Using both metagenomics and isolate WGS, we report three lines of evidence that V. cholerae hypermutators evolve within patients. First, we identified nonsynonymous mutations in V. cholerae DNA repair genes in 5 out of 11 patient metagenomes sequenced with sufficient coverage of the V. cholerae genome and in 1 of 3 patients with isolate genomes sequenced. Second, these mutations in DNA repair genes tended to be accompanied by an excess of intrahost single nucleotide variants (iSNVs). Third, these iSNVs were enriched in transversion mutations, a known hallmark of hypermutator phenotypes. While hypermutators appeared to generate mostly selectively neutral mutations, nonmutators showed signs of convergent mutation across multiple patients, suggesting V. cholerae adaptation within hosts. Our results highlight the power and limitations of metagenomics combined with isolate sequencing to characterize within-patient diversity in acute V. cholerae infections, while providing evidence for hypermutator phenotypes within cholera patients. IMPORTANCE Pathogen evolution within patients can impact phenotypes such as drug resistance and virulence, potentially affecting clinical outcomes. V. cholerae infection can result in life-threatening diarrheal disease or asymptomatic infection. Here, we describe whole-genome sequencing of V. cholerae isolates and culture-free metagenomic sequencing from stool of symptomatic cholera patients and asymptomatic carriers. Despite the typically short duration of cholera, we found evidence for adaptive mutations in the V. cholerae genome that occur independently and repeatedly within multiple symptomatic patients. We also identified V. cholerae hypermutator phenotypes within several patients, which appear to generate mainly neutral or deleterious mutations. Our work sets the stage for future studies of the role of hypermutators and within-patient evolution in explaining the variation from asymptomatic carriage to symptomatic cholera.