Characterization of the ebp(fm) pilus-encoding operon of Enterococcus faecium and its role in biofilm formation and virulence in a murine model of urinary tract infection.
Characterization of the ebp(fm) pilus-encoding operon of Enterococcus faecium and its role in biofilm formation and virulence in a murine model of urinary tract infection.
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DOI：
10.4161/viru.1.4.11966
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发表时间：
2010-07
期刊：
影响因子：
5.2
通讯作者：
Murray BE
中科院分区：
文献类型：
--
作者：
Sillanpää J;Nallapareddy SR;Singh KV;Prakash VP;Fothergill T;Ton-That H;Murray BE
我们最近在粪肠球菌TX16 （DO）基因组中发现了15个基因编码具有msrmrm和/或菌毛特征的表面蛋白，包括4个预测的菌毛编码基因簇；我们还证明了其中一个ebpccfm被转录为一个操纵子，其假定的主要菌毛亚基EbpCfm（也称为PilB）被聚合成高分子量复合物，并且在临床大肠杆菌分离株中富集。在这里，我们在心内膜衍生的粪肠杆菌菌株（TX82）中创建了ebpABCfm操纵子的缺失，并通过全细胞ELISA、流式细胞术、免疫印迹和免疫金电镜的组合显示，这种缺失消除了EbpCfm的表达，并从细胞表面消除了含有EbpCfm的菌毛。然而，下游分选酶bpsfm的转录不受影响。重要的是，与野生型相比，ebpABCfm缺失导致生物膜形成（p < 0.0001）和初始粘附（p < 0.0001）显著减少；在反式中补充EbpCfm后，两者都恢复了，这也恢复了EbpCfm的细胞表面表达和菌毛的产生。此外，在两个独立的混合感染小鼠尿路实验中，缺失突变体显著减弱，即在肾脏（p = 0.0003和< 0.0001）和膀胱（p = 0.0003和= 0.002）中，缺失突变体的数量超过了野生型。总之，我们已经证明ebpABCfm位点编码粪肠TX82细胞表面的毛，并提供了第一个证据，证明这种新兴病原体的毛对其形成生物膜和引起上升UTI模型感染的能力很重要。
We recently identified 15 genes encoding putative surface proteins with features of MSCRAMMs and/or pili in the Enterococcus faecium TX16 (DO) genome, including four predicted pilus-encoding gene clusters; we also demonstrated that one of these, ebpABCfm, is transcribed as an operon, that its putative major pilus subunit, EbpCfm (also called PilB), is polymerized into high molecular weight complexes, and that it is enriched among clinical E. faecium isolates. Here, we created a deletion of the ebpABCfm operon in an endocarditis-derived E. faecium strain (TX82) and showed, by a combination of whole-cell ELISA, flow cytometry, immunoblot and immunogold electron microscopy, that this deletion abolished EbpCfm expression and eliminated EbpCfm-containing pili from the cell surface. However, transcription of the downstream sortase, bpsfm, was not affected. Importantly, the ebpABCfm deletion resulted in significantly reduced biofilm formation (p < 0.0001) and initial adherence (p < 0.0001) versus the wild-type; both were restored by complementing ebpABCfm in trans, which also restored cell surface expression of EbpCfm and pilus production. Furthermore, the deletion mutant was significantly attenuated in two independent mixed infection mouse urinary tract experiments, i.e., outnumbered by the wild-type in kidneys (p = 0.0003 and < 0.0001, respectively) and urinary bladders (p = 0.0003 and = 0.002). In conclusion, we have shown that the ebpABCfm locus encodes pili on the E. faecium TX82 cell surface and provide the first evidence that pili of this emerging pathogen are important for its ability to form biofilm and to cause infection in an ascending UTI model.