Badger J L, Wass C A, Kim K S
Division of Infectious Diseases, Childrens Hospital Los Angeles, CA 90027, USA. University of Southern California School of Medicine, Los Angeles, CA 90027, USA.
Mol Microbiol. 2000 Apr;36(1):174-82. doi: 10.1046/j.1365-2958.2000.01840.x.
Most cases of Escherichia coli K1 meningitis arise as a result of haematogenous spread, however there is a limited understanding of the mechanisms by which circulating E. coli K1 cross the blood-brain barrier. We have previously shown that environmental growth conditions both positively and negatively influence the capabilities of E. coli K1 to invade brain microvascular endothelial cells (BMEC), for example growth in media supplemented with 50% newborn bovine serum (NBS) increased BMEC invasion, whereas growth in media supplemented with 0.2 M NaCl repressed invasion in vitro and in vivo. In this study, differential fluorescence induction (DFI) was used to identify E. coli K1 genes involved in this differentially expressed invasion phenotype. E. coli K1 promoter libraries were constructed and screened for gfp expression in a manner analogous to the above growth conditions. Twenty-four clones were isolated that showed fluorescence induction when grown under the invasion-enhancing condition (i.e. NBS). Four of these clones also demonstrated repression or no induction of fluorescence when grown under the invasion-repressing condition (i.e. 0.2 M NaCl). One such clone, containing a ygdP promoter and an open reading frame (ORF), showed significant homology to Bartonella bacilliformis IalA (invasion associated locus). Among the other NBS-inducing loci, finPtraJ was identified as well as several clones with no homology to other known genes. When ygdP, finPtraJ and several of the unique loci were disrupted in E. coli K1, there was a significant decrease in human BMEC (HBMEC) invasion. RNA transcript analysis determined that these newly identified invasion loci were differentially regulated at the transcriptional level. This is the first demonstration of using DFI to identify E. coli K1 genes contributing to HBMEC invasion.
大多数大肠杆菌K1脑膜炎病例是血行播散所致，然而对于循环中的大肠杆菌K1穿越血脑屏障的机制了解有限。我们之前已表明，环境生长条件对大肠杆菌K1侵袭脑微血管内皮细胞（BMEC）的能力有正负两方面影响，例如在添加50%新生牛血清（NBS）的培养基中生长可增加对BMEC的侵袭，而在添加0.2 M NaCl的培养基中生长则在体外和体内均抑制侵袭。在本研究中，采用差异荧光诱导（DFI）来鉴定参与这种差异表达侵袭表型的大肠杆菌K1基因。构建了大肠杆菌K1启动子文库，并以类似于上述生长条件的方式筛选绿色荧光蛋白（gfp）表达。分离出24个克隆，这些克隆在侵袭增强条件（即NBS）下生长时显示出荧光诱导。其中4个克隆在侵袭抑制条件（即0.2 M NaCl）下生长时也表现出荧光抑制或无诱导。一个这样的克隆，包含一个ygdP启动子和一个开放阅读框（ORF），与杆状巴尔通体IalA（侵袭相关基因座）具有显著同源性。在其他NBS诱导基因座中，鉴定出了finPtraJ以及几个与其他已知基因无同源性的克隆。当在大肠杆菌K1中破坏ygdP、finPtraJ和几个独特基因座时，对人BMEC（HBMEC）的侵袭显著降低。RNA转录分析确定这些新鉴定的侵袭基因座在转录水平上受到差异调节。这是首次证明使用DFI来鉴定有助于HBMEC侵袭的大肠杆菌K1基因。
