Deciphering molecular mechanisms underlying vertical transmission of Listeria monocytogenes

摘要 与普通人群相比，孕妇更有可能感染细胞内病原体单核细胞增多性李斯特菌(Listeria monchrontogen，简称Lm)，这种细菌可以垂直传播，导致胎儿感染和妊娠丢失。尽管它与公共/健康/相关，但母婴传播的决定因素仍然不确定。对临床分离的肺炎链球菌的检测表明，菌株的亚群具有增强的垂直传播能力。其中一种菌株07PF0776首次被发现具有靶向心脏的能力，这种向心功能需要InlB，这是一种已知对哺乳动物细胞入侵很重要的细菌表面蛋白。实验证据表明，垂直传播同样需要In1B，仅高In1B表达就足以显著增强无关菌株的垂直传播。该项目的目标是阐明In1B增强超强毒力Lm株和Lm的垂直传播的机制。该项目旨在将特定菌株的基因变异与改变的细菌行为、宿主病理和免疫反应联系起来。目前的工作假设是，在LM超强毒力亚群中，in1B的翻译增强是由于in1B转录本5‘非翻译区(UTR)上促进核糖体结合的三核苷酸替换的结果。它还假设，In1B内的氨基酸变化增强了In1B与细菌细胞壁的结合，从而增加了In1B表面的丰度。目标1将通过观察In1B的合成、降解和细胞壁亲和力来确定07PF0776中InlB水平是如何变化的。这一目标还将探索5‘非编码区如何促进蛋白质表达的增加。目的2将确定增加的InlB表达如何促进胎盘和胎儿的细菌定植。实验将以怀孕的小鼠为模型，比较具有不同InlB活性水平的菌株。荧光显微镜结合免疫组织化学将确定In1B高表达菌株是否增强了对特定胎盘细胞类型的侵袭，或者通过新的进入途径获得进入。RNAScope原位杂交和流式细胞术将确定细菌定位如何影响胎盘免疫反应的性质。上述目标的最终目标将是建立LM菌株导致严重胎儿和新生儿疾病的机制。这些研究可能最终允许改进对严重LM感染的治疗，并采取更有效的策略来保护孕妇免受LM引起的妊娠并发症的影响。

Abstract Pregnant women are more likely to be infected with the intracellular pathogen Listeria monocytogenes (Lm) than the general population, and the bacterium can be vertically transmitted leading to fetal infection and pregnancy loss. Despite its public/health/relevance, the determinants of maternofetal transmission remain undefined. Examination of clinical Lm isolates indicates that subpopulations of strains possess an enhanced ability for vertical transmission. One of these strains, 07PF0776, was first noted for its ability to target the heart, and this cardiotropism requires InlB, a bacterial surface protein known to be important for the invasion of mammalian cells. Experimental evidence indicates that InlB is similarly required for vertical transmission and that high InlB expression alone is sufficient to significantly enhance vertical transmission in an unrelated strain. The goal for this project is to elucidate the mechanisms by which InlB enhances the vertical transmission of hypervirulent Lm strains and of Lm generally. The project aims are designed to connect strain-specific genetic variations to altered bacterial behavior, host pathology, and immune responses. The current working hypothesis is that enhanced translation of inlB occurs in Lm hypervirulent subpopulations as the result of a trinucleotide substitution in the 5’ untranslated region (UTR) of the inlB transcript that promotes ribosome binding. It is also hypothesized that amino acid variations within InlB enhance InlB binding to the bacterial cell wall and thus increase InlB surface abundance. Aim 1 will determine how InlB levels are altered in 07PF0776 by looking at InlB synthesis, degradation, and cell wall affinity. This aim will also explore how the 5’ UTR contributes to increased protein expression. Aim 2 will determine how increased InlB expression promotes bacterial colonization of the placenta and fetus. Experiments will use pregnant mice as a model and compare strains with varying levels of InlB activity. Fluorescence microscopy combined with immuno-histochemistry will determine if InlB-high-expressing strains have enhanced invasion of specific placental cell types, or alternatively gain access through new portals of entry. RNAScope in situ hybridization and flow cytometry will establish how bacterial localization impacts the nature of the placental immune response. The ultimate goal of the above aims will be to establish the mechanisms by which strains of Lm cause severe fetal and neonatal disease. These studies may eventually allow for improved treatment of severe Lm infection and more effective strategies to protect pregnant women from Lm-induced pregnancy complications.