The Cyclic AMP Receptor Protein, CRP, Is Required for Both Virulence and Expression of the Minimal CRP Regulon in Yersinia pestis Biovar microtus
The Cyclic AMP Receptor Protein, CRP, Is Required for Both Virulence and Expression of the Minimal CRP Regulon in Yersinia pestis Biovar microtus
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环 AMP 受体蛋白 CRP 是鼠疫耶尔森氏菌微小 CRP 调节子的毒力和表达所必需的
DOI：
10.1128/iai.00370-08
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发表时间：
2008-11-01
影响因子：
3.1
通讯作者：
Yang, Ruifu
中科院分区：
文献类型：
--
作者：
Zhan, Lingjun;Han, Yanping;Yang, Ruifu
环AMP受体蛋白（CRP）是一种细菌调节剂，控制100多种启动子，包括参与分解代谢产物抑制的启动子。在本研究中，构建了鼠疫耶尔森氏菌bv.田鼠品系201。基因芯片表达分析显示，至少有6%的Y.鼠疫菌基因受此突变的影响。进一步的逆转录-PCR和电泳迁移率变动分析揭示了一组37个基因或推定的操纵子是CRP的直接靶点，因此它们构成了Y中最小的CRP调节子。鼠疫随后的引物延伸和DNA酶I足迹分析映射的转录起始位点，核心启动子元件，CRP结合位点内的DNA区域上游的PLA和PST，揭示了这两个横向收购的质粒基因的CRP阳性和直接控制。crp的破坏影响了突变体的体外和体内生长，并导致皮下感染后毒力损失> 15，000倍，但静脉接种的50%致死剂量增加< 40倍。因此，CRP是Y的毒力所必需的。鼠疫杆菌，特别是对皮下接种的感染更重要。它可以进一步得出结论，减少在体内生长表型的crp突变体应有助于，至少部分地，它的毒力衰减的两种途径的感染。这与之前对Y.佩斯特斯公司lacZ报告基因融合分析表明crp缺失导致pla启动子活性几乎完全丧失。由pla编码的纤溶酶原激活剂先前被证明特异性地促进Y。通过外周感染途径传播鼠疫（皮下感染[跳蚤叮咬]或吸入）。上述证据支持这样的观点，即除了降低体内生长表型，crp突变体中pla表达的缺陷将极大地导致该突变株在皮下感染中毒力的巨大损失。
The cyclic AMP receptor protein (CRP) is a bacterial regulator that controls more than 100 promoters, including those involved in catabolite repression. In the present study, a null deletion of the crp gene was constructed for Yersinia pestis bv. microtus strain 201. Microarray expression analysis disclosed that at least 6% of Y. pestis genes were affected by this mutation. Further reverse transcription-PCR and electrophoretic mobility shift assay analyses disclosed a set of 37 genes or putative operons to be the direct targets of CRP, and thus they constitute the minimal CRP regulon in Y. pestis. Subsequent primer extension and DNase I footprinting assays mapped transcriptional start sites, core promoter elements, and CRP binding sites within the DNA regions upstream of pla and pst, revealing positive and direct control of these two laterally acquired plasmid genes by CRP. The crp disruption affected both in vitro and in vivo growth of the mutant and led to a > 15,000-fold loss of virulence after subcutaneous infection but a < 40-fold increase in the 50% lethal dose by intravenous inoculation. Therefore, CRP is required for the virulence of Y. pestis and, particularly, is more important for infection by subcutaneous inoculation. It can further be concluded that the reduced in vivo growth phenotype of the crp mutant should contribute, at least partially, to its attenuation of virulence by both routes of infection. Consistent with a previous study of Y. pestis bv. medievalis, lacZ reporter fusion analysis indicated that the crp deletion resulted in the almost absolute loss of pla promoter activity. The plasminogen activator encoded by pla was previously shown to specifically promote Y. pestis dissemination from peripheral infection routes (subcutaneous infection [flea bite] or inhalation). The above evidence supports the notion that in addition to the reduced in vivo growth phenotype, the defect of pla expression in the crp mutant will greatly contribute to the huge loss of virulence of this mutant strain in subcutaneous infection.