细菌泛基因组关联研究揭示多胺代谢和碱性耐受在中耳感染中的作用。

文献信息

中文摘要

背景： 中耳感染，即中耳炎（OM），是一种常见于儿童的疾病，而流感嗜血杆菌是最常见的OM相关细菌。我们试图通过识别与OM疾病相关的细菌基因来获得对发病机制的新见解。方法：我们对随时间从9名健康和12名易患OM儿童中收集的>200株临床分离株进行了全基因组测序。基因组分析首先注释并聚类了来自各装配体的同源基因，推断了系统发育，并识别了密切相关的菌株集合，即克隆谱系，发现不同谱系的短暂和持续定植。结果：基于系统发育信息的关联测试随后识别出7个基因/操纵子与健康或疾病的强关联。值得注意的是，5个关联涉及基因缺失与OM，并且所有5个关联的预测功能均与pH稳态和氮代谢中的作用一致，具体涉及多胺的转运和修饰。使用实验室菌株，测试了候选“抗OM”操纵子potE-speF的敲除。结论：改变pH和补充多胺的体外生长实验表明，在鸟氨酸存在下，potE-speF的存在赋予细胞碱性敏感性。对人类中耳液pH的测量发现，在收集到流感嗜血杆菌的标本中碱性高于其他OM病原体的标本，支持了OM中碱性中耳微环境源于或选择流感嗜血杆菌感染的假设。使用小鼠OM模型的实验发现，potE-speF的缺失导致中耳洗液中细菌疾病负担增加约63倍。相比之下，小鼠肺部感染模型显示无显著差异。我们得出结论：缺乏potE-speF的流感嗜血杆菌在OM疾病发病机制中具有选择优势，因为这些菌株在儿童中耳中出现的原本不利的碱性环境中茁壮成长。

英文摘要

Infection of the middle ear, or otitis media (OM), is a disease that commonly afflicts children, and Haemophilus influenzae is the most prevalent OM-associated bacterium. We sought new insights into mechanisms of pathogenesis by identifying bacterial genes associated with OM disease. We whole-genome sequenced >200 clinical isolates collected over time from 9 healthy and 12 OM-prone children. Genome analysis first annotated and clustered homologous genes from across assemblies, inferred the phylogeny, and identified closely related set of strains, or clonal lineages, finding both transient and persistent colonization by distinct lineages. Phylogeny-informed association tests then identified 7 strong associations of genes/operons with health or disease. Remarkably, 5 associations were with gene absences and OM, and all 5 had predicted functions consistent with roles in pH homeostasis and nitrogen metabolism, specifically in transport and modification of polyamines. Using a laboratory strain, knockouts of candidate "anti-OM" operon potE-speF were tested. In vitro growth assays varying pH and supplementation with polyamines showed that the presence of potE-speF conferred alkaline sensitivity to cells in the presence of ornithine. Measurements of human middle ear fluid pH found higher alkalinity in specimens where H. influenzae was collected over those with other OM pathogens, supporting the hypothesis that the alkaline middle ear microenvironment in OM arises from or selects for H. influenzae infection. Experiments using a mouse OM model found that absence of potE-speF caused a ~ 63-fold increase in bacterial disease burden in middle ear washes. By contrast, a mouse lung infection model showed no significant difference. We conclude that H. influenzae lacking potE-speF have a selective advantage in OM disease pathogenesis, because these strains thrive in the otherwise hostile alkaline environment that occurs in the middle ears of children.