Ehrlichia chaffeensis proliferation begins with NtrY/NtrX and PutA/GlnA upregulation and CtrA degradation induced by proline and glutamine uptake.
Ehrlichia chaffeensis proliferation begins with NtrY/NtrX and PutA/GlnA upregulation and CtrA degradation induced by proline and glutamine uptake.
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DOI：
10.1128/mbio.02141-14
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发表时间：
2014-11-25
期刊：
影响因子：
6.4
通讯作者：
Rikihisa Y
中科院分区：
文献类型：
--
作者：
Cheng Z;Lin M;Rikihisa Y
专性细胞内细菌查菲埃里希体如何在进入人单核细胞后开始复制尚不清楚。在这里，我们研究了氨基酸在启动细胞内复制中的潜在作用。PutA将脯氨酸转化为谷氨酸，GlnA将谷氨酸转化为谷氨酰胺。查菲大肠杆菌PutA和GlnA互补大肠杆菌putA和glnA突变体。谷氨酰胺合成酶抑制剂甲磺酰亚胺抑制查菲埃立克体GlnA活性和查菲埃立克体感染人细胞。将查菲埃立克体与人细胞一起孵育，迅速诱导putA和glnA表达，在孵育后24 h达到峰值。查菲肠球菌摄取脯氨酸和谷氨酰胺，但不摄取谷氨酸。用脯氨酸转运体抑制剂（鱼精蛋白）、谷氨酰胺转运体抑制剂（组氨酸）或脯氨酸类似物预处理查菲埃立克体抑制查菲埃立克体感染，而用脯氨酸或谷氨酰胺预处理增强感染，并且上调putA和glnA的速度比不处理或谷氨酸预处理快。putA和glnA表达的时间响应类似于NtrY和NtrX的时间响应，NtrX是一个双组分系统，电泳迁移率变动分析显示重组查菲埃立克体NtrX（rNtrX）与查菲埃立克体putA和glnA的启动子区域特异性结合。此外，rNtrX反式激活大肠杆菌中的查菲埃立克氏菌putA和glnA启动子-lacZ融合体。脯氨酸和谷氨酰胺的生长促进活性也伴随着DNA结合蛋白CtrA的快速降解。我们的研究结果表明，脯氨酸和谷氨酰胺的摄取调节putA和glnA的表达，通过NtrY/NtrX，并促进CtrA的降解，启动一个新的周期的查菲埃里希生长。人单核细胞埃立克体病（HME）是美国最流行的、威胁生命的新发人畜共患传染病之一。HME是由查菲埃立克次体感染引起的，查菲埃立克次体是立克次体目中的一种专性细胞内细菌，包括几种B/C类病原体，例如引起落基山斑疹热和流行性斑疹伤寒的病原体。对真核细胞内控制细菌生长的机制的有限理解继续阻碍了对立克次体疾病新治疗靶点的鉴定。细胞外立克次体不能复制，但立克次体进入真核宿主细胞后复制增强。我们的研究结果将提供一个新的机制的双组分系统，调节E. chaffeensis在人类单核细胞的生长启动的见解。这一结果也很重要，因为人们对任何细菌中的NtrY/NtrX双组分系统知之甚少，更不用说专性细胞内细菌了。我们的研究结果将推进该领域目前的概念范式的强制性细胞内营养，代谢和生长的调节。
How the obligatory intracellular bacterium Ehrlichia chaffeensis begins to replicate upon entry into human monocytes is poorly understood. Here, we examined the potential role of amino acids in initiating intracellular replication. PutA converts proline to glutamate, and GlnA converts glutamate to glutamine. E. chaffeensis PutA and GlnA complemented Escherichia coli putA and glnA mutants. Methionine sulfoximine, a glutamine synthetase inhibitor, inhibited E. chaffeensis GlnA activity and E. chaffeensis infection of human cells. Incubation of E. chaffeensis with human cells rapidly induced putA and glnA expression that peaked at 24 h postincubation. E. chaffeensis took up proline and glutamine but not glutamate. Pretreatment of E. chaffeensis with a proline transporter inhibitor (protamine), a glutamine transporter inhibitor (histidine), or proline analogs inhibited E. chaffeensis infection, whereas pretreatment with proline or glutamine enhanced infection and upregulated putA and glnA faster than no treatment or glutamate pretreatment. The temporal response of putA and glnA expression was similar to that of NtrY and NtrX, a two-component system, and electrophoretic mobility shift assays showed specific binding of recombinant E. chaffeensis NtrX (rNtrX) to the promoter regions of E. chaffeensis putA and glnA. Furthermore, rNtrX transactivated E. chaffeensis putA and glnA promoter-lacZ fusions in E. coli. Growth-promoting activities of proline and glutamine were also accompanied by rapid degradation of the DNA-binding protein CtrA. Our results suggest that proline and glutamine uptake regulates putA and glnA expression through NtrY/NtrX and facilitates degradation of CtrA to initiate a new cycle of E. chaffeensis growth. Human monocytic ehrlichiosis (HME) is one of the most prevalent, life-threatening emerging infectious zoonoses in the United States. HME is caused by infection with E. chaffeensis, an obligatory intracellular bacterium in the order Rickettsiales, which includes several category B/C pathogens, such as those causing Rocky Mountain spotted fever and epidemic typhus. The limited understanding of the mechanisms that control bacterial growth within eukaryotic cells continues to impede the identification of new therapeutic targets against rickettsial diseases. Extracellular rickettsia cannot replicate, but rickettsial replication ensues upon entry into eukaryotic host cells. Our findings will provide insights into a novel mechanism of the two-component system that regulates E. chaffeensis growth initiation in human monocytes. The result is also important because little is known about the NtrY/NtrX two-component system in any bacteria, let alone obligatory intracellular bacteria. Our findings will advance the field’s current conceptual paradigm on regulation of obligatory intracellular nutrition, metabolism, and growth.