原文链接：Interspecies bacterial competition regulates community assembly in the C. elegans intestine | The ISME Journal (nature.com)

摘要：

从昆虫到哺乳动物，各种动物的肠道中都有复杂的细菌群落，这些细菌群落在健康和疾病中发挥着重要作用。为了进一步了解肠道细菌群落是如何组装和发挥作用的，我们用单一培养的细菌以及2-8种细菌的混合物喂养秀丽隐杆线虫，采用自下而上的方法研究秀丽隐杆线虫的微生物群。我们发现，由于细菌种间的相互作用，在单一培养中定殖良好的细菌在共培养中并不总是表现良好。此外，随着群落多样性的增加，在决定群落组装方面，单一培养物种在线虫肠道的定殖能力变得不如种间相互作用重要。为了探索宿主-微生物适应的作用，我们比较了从秀丽隐杆线虫肠道分离的细菌和非本地分离株，我们发现定殖的成功更多地取决于物种的分类学，而不是分离来源。最后，通过比较两个秀丽隐杆线虫突变体中组装的微生物群，我们发现通过p38 MAPK途径的先天免疫降低了细菌丰度，但对微生物群组成几乎没有影响。这些结果强调，细菌种间相互作用，比宿主-微生物适应或肠道环境过滤更重要，在秀丽隐杆线虫微生物群的组装中发挥着主导作用。

Fig. 1: Different bacterial species reach widely different population sizes in C. elegans gut.图1：不同的细菌种类在秀丽隐杆线虫肠道中达到广泛不同的种群规模。

A Diagram of the C. elegans microbiota assembly and the three biological forces (orange) that might influence this process and that we study in this article. To construct and measure simple microbiotas in C. elegans, a defined number of bacterial species are fed in liquid culture to a same-age adult population of C. elegans previously sterilized with antibiotics. The liquid feeding substrate is restored every day to maintain equal bacterial concentrations during the 4 days of colonization. Afterwards, worms are mechanically disrupted in batches of ~20, and counts of colony forming units (CFU) are used to determine bacterial population sizes in the worm gut. B Phylogenetic tree from full-length 16S rRNA gene sequences of the 11 non-native bacterial species used to colonize the gut of C. elegans. C Bacterial population sizes in monoculture colonization of immunocompromised C. elegans (AU37) span two orders of magnitude. These population sizes reflect the inherent abilities of bacteria to colonize the worm intestine environment. Points are the average of eight or more biological replicates, and error bars are the standard error of the mean (s.e.m.).

A 我们在本文中研究的秀丽隐杆线虫微生物群组装图和可能影响这一过程的三种生物力（橙色）。为了构建和测量秀丽隐杆线虫中的简单微生物群，将一定数量的细菌物种以液体培养物喂养给先前用抗生素灭菌的同龄成年秀丽隐杆虫种群。每天恢复液体喂养基质，以在定殖的4天内保持相等的细菌浓度。然后，以约20个批次的数量对蠕虫进行机械破坏，并使用菌落形成单位（CFU）计数来确定蠕虫肠道中的细菌种群大小。

B 用于在秀丽隐杆线虫肠道定殖的11种非本地细菌的全长16S rRNA基因序列的系统发育树。

C 免疫功能受损的秀丽隐杆线虫（AU37）单一培养定殖中的细菌种群规模跨越两个数量级。这些种群大小反映了细菌在蠕虫肠道环境中定殖的固有能力。点是八个或多个生物重复的平均值，误差条是平均值的标准误差（s.e.m）。

Fig. 2: Monoculture colonization of the worm intestine often fails to predict composition of two-species microbiotas.图2：线虫肠道的单一培养定殖通常无法预测两种微生物群的组成。

A LEFT panels: Fractional abundances of 55 co-culture experiments in C. elegans intestine (AU37); error bars are the s.e.m. of 2–8 biological replicates (Fig. S2). Bacterial species are ordered from left to right by their mean fraction across all co-cultures. RIGHT panels: Null expectation for the fractional abundances based on a noninteracting model where each bacterial species reaches its population size in monoculture; error bars are the s.e.m. from bootstrapping over the monoculture data. * and ** represent a statistically significant difference between the two panels at p values of 0.05 and 0.01, respectively (Welch’s T test). B Coexistence of two species is more common than competitive exclusion in the worm intestine. C Low yields in two species microbiotas—relative to monocultures—are indicative of competitive interactions (Fig. S2); error bars on X-axis are the s.e.m. and on Y-axis the s.e.m. from bootstrapping over monoculture and pairwise data simultaneously. D Competitive ability, defined as the mean fractional abundance in co-culture experiments, relates to monoculture population size, but there are significant deviations; error bars on Y-axis are the propagated error from the s.e.m. of the co-culture experiments.

A 左图：秀丽隐杆线虫肠道55个共培养实验的部分丰度（AU37）；误差条是2–8个生物重复的s.e.m（图S2）。细菌种类按其在所有共培养物中的平均比例从左到右排列。

   右图：基于非相互作用模型，即每个细菌物种在单一培养中达到的其种群规模，对部分丰度的零期望值；误差条是在单一培养数据上引导的s.e.m。*和**分别表示p值为0.05和0.01时两个面板之间的统计学显著差异（Welch’s T检验）。

B 在线虫肠道中，两种物种共存比竞争排斥更常见。

C相对于单一培养，两物种的微生物群产量较低，这表明存在竞争性相互作用（图S2）；X轴上的误差条是s.e.m，Y轴上的误差条是同时在单一培养和成对数据上引导的s.e.m。

D竞争能力，定义为共培养实验中的平均部分丰度，与单一培养的种群规模有关，但存在显著偏差；Y轴上的误差条是来自共培养实验的s.e.m的传播误差。

Fig. 3: Fractional abundances in three-species microbiotas are well predicted by pairwise outcomes.图3：三物种微生物群的部分丰度可以通过成对结果很好地预测。

A Outcome of trio Ea-Pf-Sm in C. elegans (AU37) intestine, together with predictions based on monoculture population sizes, two-species microbiotas, or pairwise outcomes in vitro liquid media (normalized arithmetic mean). B Simplex representation of trio outcome and predictions in (A), with the edges of the triangle depicting the two-species microbiotas in C. elegans. The error bars on measurement are the s.e.m. of four biological replicates, and the clouds of points around predictions are 400 bootstrap replicates (“N”s sampling the monoculture data, and “W”s and “M”s sampling the pairwise data in worm and media, respectively). C Twenty trio outcomes represented in one sixth of a simplex. D 3, 8, and 9 out of the 20 trios show full competitive exclusion, two- and three-species coexistence, respectively. E Assembly rules help the quantitative prediction of the trio outcomes based on pairwise outcomes when one of the pairs is competitive exclusion. F Cumulative distribution of error of predictions. Error calculated as the linear distance between prediction and measurement in the simplex. The distances are normalized by the maximal distance, √2. The dashed line is the mean distance between the measured mean and the four biological replicates of each trio, and serves as a lower bound for the error of the predictions.

A. 三物种群落Ea-Pf-Sm在秀丽隐杆线虫（AU37）肠道中的结果，以及基于单一培养种群规模、两物种微生物群或体外液体培养基中成对结果的预测（归一化算术平均值）。

B（A）中三物种群落结果和预测的简单表示，三角形的边描绘了秀丽隐杆线虫中的两物种微生物群。测量的误差条是四个生物复制的s.e.m.，预测周围的点云是400个自举复制（“N”s对单一培养数据进行采样，“W”s和“M”s分别对线虫和培养基中的成对数据进行采样）。

C 20个三物种群落结果代表了一个简单的六分之一。

D 20个三物种群落中的3个、8个和9个分别表现出完全竞争排斥、两物种和三物种共存。

E 当其中一对是竞争排斥时，组装规则有助于基于成对结果对三物种群落结果进行定量预测。

F  预测误差的累积分布。误差计算为简单中预测和测量之间的线性距离。距离通过最大距离√2进行归一化。虚线是测量的平均值和每个三物种群落四个生物重复之间的平均距离，并作为预测误差的下限。

Fig. 4: Experimental colonization of C. elegans by a wide range of native and non-native bacteria reveals that phylogeny rather than isolation origin determines abundance in the gut microbiota.图4：秀丽隐杆线虫被各种本地和非本地细菌定殖的实验表明，系统发育而非分离源决定了肠道微生物群的丰度。

A Phylogenetic classification of previously shown laboratory species (non-native) and bacterial strains isolated from C. elegans intestines (native; dark and light blue from MYb and CR collections, respectively; Methods). Phylogenetic tree built with maximum likelihood estimate utilizing alignment of full-length 16S gene sequences. The phylogenetic tree is sorted at each internal node to have the higher monoculture colonizers at the bottom. High level phylogenetic classification is given on the left side of the tree for ease of interpretation. Stars indicate bacteria used in follow-up two-species microbiotas. B Bacterial population sizes in monoculture colonization of wild-type C. elegans (N2); error bars are s.e.m. of two to three replicates. C Left panels: Fractional abundances in two-species microbiotas with native and non-native bacteria in C. elegans intestine (AU37). Right panels: Null expectation for the fractional abundances based on monoculture population sizes. “*” and “**” represent a statistically significant difference between measurement and null expectation at p values of 0.05 and 0.01, respectively (Welch’s T test). D Although two native strains can reach substantial colonization of the worm intestine in monoculture, these strains reach low fractional abundances in two-species microbiotas. E Differences in competitive ability correlate with phylogenetic distances regardless of the isolation origin of the bacteria. Phylogenetic distances are the horizontal distances in the phylogenetic tree. Differences in competitive ability are normalized by the maximum competitive ability of the pair (i.e., competitive abilities 0.8 and 0.4 are as different as 0.2 and 0.1).

A 先前显示的实验室物种（非本地）和从秀丽隐杆线虫肠道分离的细菌菌株（本地；深蓝色和浅蓝色分别来自MYb和CR收集的；方法）的系统发育分类。利用全长16S基因序列比对建立具有最大似然估计的系统发育树。系统发育树在每个内部节点进行排序，以在底部具有较高的单一培养定殖体。为了便于解释，在树的左侧给出了高级系统发育分类。星星表示在后续两物种的微生物群中使用的细菌。

B 单一培养定殖野生型秀丽隐杆线虫（N2）中的细菌种群大小；误差条是两到三个重复的s.e.m。

C 左图：秀丽隐杆线虫肠道（AU37）中本地和非本地细菌组成的两物种微生物群的部分丰度。右图：基于单一培养种群规模的部分丰度的零期望值。“*”和“**”分别代表p值为0.05和0.01时测量值和零期望值之间的统计学显著差异（Welch’s T检验）。

D 尽管两种本地菌株在单一培养中可以在线虫肠道大量定殖，但这些菌株在两物种微生物群中的丰度很低。

E 无论细菌的分离来源如何，竞争能力的差异都与系统发育距离相关。系统发育距离是系统发育树中的水平距离。竞争能力的差异通过成对的最大竞争能力来归一化（即，竞争能力0.8和0.4与0.2和0.1一样不同）。

Fig. 5: Bacterial interspecies interactions are similar between the in vitro and in vivo environments, with some differences caused by the acidity of the worm gut.图5：细菌种间相互作用在体外和体内环境中相似，但一些差异是由线虫肠道的酸性引起的。

A Black points are the mean fractional abundance in co-culture experiments in C. elegans intestine and liquid media (1% AXN); error bars are the propagated error from the s.e.m. of the underlying co-culture experiments. Blue points are the outcomes of individual co-culture experiments in worms and media. B S. marcescens and P. putida reach different fractional abundances in vivo worm gut and in vitro liquid media on a coupled experiment, where worms and liquid media from the same test tube are tested. C An acidic version of the media resembling the average pH of the worm intestine (4.5) shifts back the pairwise outcome to a worm-like state; error bars are the s.e.m. of at least four replicates. 

A 黑点是秀丽隐杆线虫肠道和液体培养基（1%AXN）共培养实验中的平均部分丰度；误差条是来自基础共培养实验的s.e.m的传播误差。蓝点是在线虫和培养基中单独共培养实验的结果。

B 在一项耦合实验中，粘质链霉菌和恶臭假单胞菌在体内蠕虫肠道和体外液体培养基中达到不同的部分丰度，其中测试来自同一试管的线虫和液体培养基。

C类似于线虫肠道平均pH（4.5）的酸性介质将成对结果转变回线虫样状态；误差条是至少四个重复的s.e.m。

Fig. 6: Innate immunity of C. elegans via the p38 MAPK pathway reduces bacterial population sizes, but has little influence on the composition of the two- and eight-species microbiotas. 图6：秀丽隐杆线虫通过p38 MAPK途径的先天免疫减少了细菌种群规模，但对两物种和八物种微生物群的组成几乎没有影响。

A Immune system of C. elegans reduces bacterial monoculture population sizes unevenly for different bacteria. Immunocompromised C. elegans (AU37) has larger bacterial population sizes in its intestine than immunocompetent C. elegans (GLP4). B The mean fractional abundances in co-cultures are similar between the two worm strains with different immune activity. C Composition of an intestinal microbiota in immunocompromised C. elegans AU37 and immunocompetent SS104, together with predictions based on monoculture colonization and pairwise outcomes in the same worm strains. Three or more batches of ~20 worms digested for each measurement. D Errors are the L1 norm (Manhattan distance) between measurement replicates and predictions. The variability across different batches of digested worms generates a measurement error of 9.3% and 14.2% for AU37 and SS104, respectively. The errors are normalized by 2, the maximum error. Confidence intervals of the prediction errors were calculated by bootstrapping over the corresponding data.

A 秀丽隐杆线虫的免疫系统不均衡地减少了不同细菌的单一培养种群规模。免疫受损的秀丽隐杆线虫（AU37）肠道中的细菌数量比免疫活性的秀丽隐杆线虫（GLP4）大。

B 具有不同免疫活性的两种线虫菌株在共培养物中的平均部分丰度相似。

C 免疫功能受损的秀丽隐杆线虫AU37和免疫活性秀丽隐杆线虫SS104中肠道微生物群的组成，以及基于相同线虫菌株的单一培养定殖和成对结果的预测。每次消化测量三批或三批以上约~20只线虫。

D 误差是测量重复和预测之间的L1范数（曼哈顿距离）。不同批次消化蠕虫的变异性分别导致AU37和SS104的测量误差为9.3%和14.2%。误差通过最大误差2进行归一化。通过对相应数据进行自举来计算预测误差的置信区间。