Campbell Danielle E, Wu Xiaofen, Hall Lindsey R, Lawrence Dylan, Molleston Jerome M, Schriefer Lawrence A, Droit Lindsay, Weagley James S, Olson Benjamin S, Rimmer Joanna, Parkes Miles, Handley Scott A, Baldridge Megan T
Department of Medicine, Division of Infectious Diseases, Washington University School of Medicine, St. Louis, MO, USA.
Edison Family Center for Genome Sciences & Systems Biology, Washington University School of Medicine, St. Louis, MO, USA.
Gut Microbes. 2025 Dec;17(1):2526719. doi: 10.1080/19490976.2025.2526719. Epub 2025 Aug 3.
The associations of the gut microbiome and virome with human health and disease are increasingly numerous and clear. The mechanistic roles of bacteriophages (phages) in the microbiome, however, are especially unclear, as their cultivation is exceedingly difficult and their diversity so immense. We use viral tagging (VT), a technique wherein fluorescently stained uncultivated viruses are allowed to adsorb to host cells and then host cells are singly sorted. This method identifies interacting phage-bacteria pairs to better sample and characterize the phages in human stool samples from healthy and inflammatory bowel disease (IBD)-affected patients. First, we apply VT to uncultivated bacteria from a healthy human sample, demonstrating far-reaching ability to observe diverse bacteria and phages alike. We also use VT with a cultured isolate, a bacterial host of interest due to its anti-inflammatory effects and strong negative correlation with IBD. Comparing VT with virome sequencing and phage identification from single amplified genomes shows that it is a practical technique for phage discovery, especially when it is used to focus on individual bacterial cultivars for which genomes have been sequenced. VT can detect phages so rare as to be undetectable in standard virome sequencing, which is biased toward the most abundant phage species even at high sequencing depth. Remarkably, VT also identified novel prophage integration events in , demonstrating that VT interactions can extend beyond the level of surface attachment and constitute active infection events. In total, VT identified at least 328 unique and highly diverse phage-host pairs, almost all of which are entirely uncharacterized, and several phages that are differentially abundant in IBD patients compared to healthy controls. Taken together, we show that VT is an extremely powerful tool to move beyond the cultivation and abundance biases inherent to current techniques and suggest that the phage-host pairs identified by VT here are crucial first step to enable future mechanistic studies of phage-bacteria-human interactions.
肠道微生物群和病毒组与人类健康和疾病的关联越来越多且日益清晰。然而，噬菌体在微生物群中的作用机制尤其不清楚，因为它们的培养极其困难，且多样性极大。我们使用病毒标记（VT）技术，即让荧光染色的未培养病毒吸附到宿主细胞上，然后对宿主细胞进行单细胞分选。这种方法可识别相互作用的噬菌体-细菌对，以便更好地对健康和炎症性肠病（IBD）患者的人类粪便样本中的噬菌体进行采样和表征。首先，我们将VT应用于来自健康人类样本的未培养细菌，证明其具有观察各种细菌和噬菌体的广泛能力。我们还将VT与一种培养的分离株一起使用，该分离株因其抗炎作用以及与IBD的强负相关性而成为感兴趣的细菌宿主。将VT与病毒组测序以及从单扩增基因组中鉴定噬菌体进行比较表明，它是一种用于噬菌体发现的实用技术，特别是当用于关注已测序基因组的单个细菌培养品种时。VT可以检测到极为罕见的噬菌体，这些噬菌体在标准病毒组测序中即使在高测序深度下也因偏向于最丰富的噬菌体种类而无法检测到。值得注意的是，VT还在[具体对象]中鉴定出了新的原噬菌体整合事件，表明VT相互作用可以超越表面附着水平，构成活跃的感染事件。总体而言，VT鉴定出至少328个独特且高度多样的噬菌体-宿主对，几乎所有这些对都完全未被表征，并且还鉴定出了几种在IBD患者中与健康对照相比丰度有差异的噬菌体。综上所述，我们表明VT是一种极其强大的工具，可以超越当前技术固有的培养和丰度偏差，并表明此处由VT鉴定出的噬菌体-宿主对是实现未来噬菌体-细菌-人类相互作用机制研究的关键第一步。
