National Center for Biodefense and Infectious Diseases and School of Systems Biology, George Mason University Manassas, VA, USA.
U.S. Army Medical Research Institute of Infectious Diseases Frederick, MD, USA.
Front Microbiol. 2015 Feb 13;6:50. doi: 10.3389/fmicb.2015.00050. eCollection 2015.
Yersinia pestis (Yp) causes the re-emerging disease plague, and is classified by the CDC and NIAID as a highest priority (Category A) pathogen. Currently, there is no approved human vaccine available and advances in early diagnostics and effective therapeutics are urgently needed. A deep understanding of the mechanisms of host response to Yp infection can significantly advance these three areas. We employed the Reverse Phase Protein Microarray (RPMA) technology to reveal the dynamic states of either protein level changes or phosphorylation changes associated with kinase-driven signaling pathways during host cell response to Yp infection. RPMA allowed quantitative profiling of changes in the intracellular communication network of human lung epithelial cells at different times post infection and in response to different treatment conditions, which included infection with the virulent Yp strain CO92, infection with a derivative avirulent strain CO92 (Pgm-, Pst-), treatment with heat inactivated CO92, and treatment with LPS. Responses to a total of 111 validated antibodies were profiled, leading to discovery of 12 novel protein hits. The RPMA analysis also identified several protein hits previously reported in the context of Yp infection. Furthermore, the results validated several proteins previously reported in the context of infection with other Yersinia species or implicated for potential relevance through recombinant protein and cell transfection studies. The RPMA results point to strong modulation of survival/apoptosis and cell growth pathways during early host response and also suggest a model of negative regulation of the autophagy pathway. We find significant cytoplasmic localization of p53 and reduced LC3-I to LC3-II conversion in response to Yp infection, consistent with negative regulation of autophagy. These studies allow for a deeper understanding of the pathogenesis mechanisms and the discovery of innovative approaches for prevention, early diagnosis, and treatment of plague.
鼠疫耶尔森菌（Yp）引起再现疾病鼠疫，被疾病预防控制中心和国家过敏和传染病研究所归类为最高优先级（A 类）病原体。目前，尚无可用的人类疫苗，迫切需要在早期诊断和有效治疗方面取得进展。深入了解宿主对 Yp 感染的反应机制可以显著推进这三个领域。我们采用反相蛋白微阵列（RPMA）技术来揭示宿主细胞对 Yp 感染的反应过程中与激酶驱动的信号通路相关的蛋白水平变化或磷酸化变化的动态状态。RPMA 允许定量分析不同时间点感染后以及不同处理条件下（包括感染强毒力 Yp 菌株 CO92、感染无毒性衍生菌株 CO92（Pgm-，Pst-）、用灭活的 CO92 处理和用 LPS 处理）人肺上皮细胞内细胞通讯网络的变化。对总共 111 种经过验证的抗体进行了分析，发现了 12 种新的蛋白靶点。RPMA 分析还确定了以前在 Yp 感染背景下报道的几种蛋白靶点。此外，这些结果验证了以前在感染其他耶尔森氏菌种或通过重组蛋白和细胞转染研究表明与潜在相关性的背景下报道的几种蛋白。RPMA 结果表明，在宿主早期反应过程中强烈调节存活/凋亡和细胞生长途径，并且还暗示了自噬途径负调控的模型。我们发现 p53 的细胞质定位明显增加，并且响应于 Yp 感染 LC3-I 到 LC3-II 的转化减少，这与自噬的负调控一致。这些研究加深了对发病机制的理解，并为预防、早期诊断和治疗鼠疫发现了创新方法。
