Mechanisms of placental infection by food-borne pathogens

描述（由申请人提供）：孕妇是侵袭性沙门氏菌感染的高危人群，可导致母体和胎儿并发症。我们发现，由于胎盘滋养细胞（tbc）中大量细菌增殖，妊娠小鼠感染沙门氏菌可导致胎儿和母体快速死亡。在体外，沙门氏菌在人滋养层细胞衍生的绒毛膜癌细胞中繁殖。然而，关于怀孕期间沙门氏菌感染易感性增加的机制，我们的知识还存在差距。我们的长期目标是制定能够控制孕妇细胞内感染结果的策略。本应用程序的总体目标是确定使tbc对沙门氏菌高度敏感的分子机制。我们的中心假设是，胎盘tbc被沙门氏菌有效感染，并提供了一个独特的细胞内生态位，由于无效的免疫反应，允许不受控制的毒性沙门氏菌复制，导致胎盘死亡。我们的中心假设将通过三个特定目标进行验证，这些目标将确定：1)沙门氏菌在tbc中不受控制的细胞内生长机制，2)IFN-γ在tbc对沙门氏菌的敏感性中的作用，以及3)沙门氏菌诱导的胎盘炎症机制。研究将在体外使用原代分离的人类滋养细胞和胎盘外植体，在体内使用怀孕小鼠模型。特异性目标1将测试沙门氏菌在滋养细胞的独特细胞内环境中茁壮成长的假设。我们将阐明不同胎龄的滋养细胞亚群是否由于特定的细胞-细胞相互作用而特异性地促进沙门氏菌的复制。我们还将确定细菌注射毒力因子在促进进入tbc中的作用以及滋养细胞吞噬作用的相对作用。最后，我们将描述沙门氏菌增殖的细胞内环境（亚细胞液泡）的分子特征。特异性目标2将验证滋养细胞对IFN-γ的低反应性有助于沙门氏菌易感性的假设。我们将测试IFN-γ预处理tbc是否可以抑制沙门氏菌感染，沙门氏菌如何调节tbc中的JAK-STAT-1信号通路，以及敲除IFN-γ信号的负反馈抑制剂（如SOCS表达）是否有助于tbc -沙门氏菌感染的解决。特异性目的3将验证st诱导的TBC死亡机制与其他感染细胞不同的工作假设，并有助于明显的炎症。我们将确定不同的炎症（IL-1ß， IL-18, caspase）和/或细胞死亡信号（凋亡，热亡或坏死）作为TBC对沙门氏菌易感性的原因。总的来说，我们希望确定在胎盘破坏中独特的滋养细胞特征与沙门氏菌毒力因素的相对重要性。这一贡献将大大重塑我们对怀孕期间感染风险的理解。这项研究的成功完成将代表着制定合理的妊娠期胎盘感染治疗方案所需的连续研究的关键的第一步。

DESCRIPTION (provided by applicant): Pregnant women are at high risk for invasive Salmonella infection, which can cause both maternal and fetal complications. We found that Salmonella infection in pregnant mice caused rapid fetal and maternal death due to massive bacterial proliferation in placental trophoblast cells (TBCs). In vitro, Salmonella flourished in human trophoblast-derived choriocarcinoma cells. However, there is a gap in our knowledge regarding the mechanism of increased susceptibility to Salmonella infection during pregnancy. Our long-term objective is to develop strategies that can control the outcome of intracellular infections in pregnant women. The overall objective of this application is to identify the molecular mechanisms that render TBCs highly susceptible to Salmonella. Our central hypothesis is that placental TBCs are productively infected by Salmonella and provide a unique intra- cellular niche that permits uncontrolled virulent Salmonella replication due to an ineffectie immune response, resulting in placental death. Our central hypothesis will be tested by three Specific Aims which will identify: 1) the mechanism of uncontrolled intracellular growth of Salmonella in TBCs, 2) the role(s) of IFN-γ in susceptibility of TBCs to Salmonella, and 3) the mechanism of Salmonella-induced placental inflammation. Studies will be performed in vitro using primary isolated human trophoblast cells and placental explants, and in vivo using pregnant mouse models. Specific Aim 1 will test the hypothesis that Salmonella thrive within a unique intracellular environment of trophoblast cells. We will elucidate whether different trophoblast subpopulations of differing gestational age specifically promote Salmonella replication due to specific cell-cell interactions. We will also identify the role of bacterial injectisome virulence factors in facilitating entry into TBCs and the relative role of trophoblast phagocytosis. Lastly, we will characterize the molecular features of the intracellular environment (sub-cellular vacuole) in which Salmonella proliferates. Specific Aim 2 will test the hypothesis that trophoblastic hypo-responsiveness to IFN-γ contributes to susceptibility to Salmonella. We will test whether IFN-γ pre- treatment of TBCs can contain Salmonella infection, how Salmonella modulates the JAK-STAT-1 signaling pathway in TBCs, and if knocking down negative-feedback inhibitors of IFN-γ signaling such as SOCS expression can aid resolution of TBC-Salmonella infection. Specific Aim 3 will test the working hypothesis that the mechanism of ST-induced TBC death is distinct from that encountered in other infected cells and contributes to overt inflammation. We will identify differential inflammation (IL-1ß, IL-18, caspase) and/or cell death signaling (apoptosis, pyroptosis or necroptosis) as a cause of TBC susceptibility to Salmonella. Overall we expect to identify the relative importance of unique trophoblast features versus Salmonella virulence factors in destruction of the placenta. This contribution will significantly reshape our understanding of infection risk during pregnancy. Successful completion of this study will represent the critical first step in the continuum of research requird to develop rational treatment regimens for management of placental infections during pregnancy.