Tsai Yi-Lin, Wang Min-Cheng, Hsueh Po-Ren, Liu Ming-Che, Hu Rouh-Mei, Wu Yue-Jin, Liaw Shwu-Jen
Department and Graduate Institute of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China.
Departments of Laboratory Medicine and Internal Medicine, National Taiwan University Hospital, College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China.
PLoS One. 2015 Mar 10;10(3):e0120395. doi: 10.1371/journal.pone.0120395. eCollection 2015.
Proteus mirabilis isolates commonly have decreased susceptibility to imipenem. Previously, we found P. mirabilis hfq mutant was more resistant to imipenem and an outer membrane protein (OMP) could be involved. Therefore, we investigated the role of this OMP in carbapenem susceptibility. By SDS-PAGE we found this OMP (named ImpR) was increased in hfq mutant and LC-MS/MS revealed it to be the homologue of Salmonella YbfM, which is a porin for chitobiose and subject to MicM (a small RNA) regulation. We demonstrated that ImpR overexpression resulted in increased carbapenem MICs in the laboratory strain and clinical isolates. Chitobiose induced expression of chb (a chitobiose utilization operon). Real-time RT-PCR and SDS-PAGE were performed to elucidate the relationship of hfq, impR, chb and MicM in P. mirabilis. We found MicM RNA was decreased in hfq mutant and chbBC-intergenic region (chbBC-IGR) overexpression strain (chbIGRov), while impR mRNA was increased in hfq mutant, micM mutant and chbIGRov strain. In addition, mutation of hfq or micM and overexpression of chbBC-IGR increased ImpR protein level. Accordingly, chitobiose made wild-type have higher levels of ImpR protein and are more resistant to carbapenems. Hfq- and MicM-complemented strains restored wild-type MICs. Mutation of both impR and hfq eliminated the increase in carbapenem MICs observed in hfq mutant and ImpR-complementation of hfq/impR double mutant resulted in MICs as hfq mutant, indicating that the ImpR-dependent decreased carbapenem susceptibility of hfq mutant. These indicate MicM was antisense to impR mRNA and was negatively-regulated by chbBC-IGR. Together, overexpression of ImpR contributed to the decreased carbapenem susceptibility in P. mirabilis.
奇异变形杆菌分离株通常对亚胺培南的敏感性降低。此前，我们发现奇异变形杆菌hfq突变体对亚胺培南的耐药性更强，且可能涉及一种外膜蛋白（OMP）。因此，我们研究了这种OMP在碳青霉烯类敏感性中的作用。通过SDS-PAGE，我们发现这种OMP（命名为ImpR）在hfq突变体中增加，LC-MS/MS显示它是沙门氏菌YbfM的同源物，YbfM是一种几丁二糖孔蛋白，受MicM（一种小RNA）调控。我们证明ImpR的过表达导致实验室菌株和临床分离株中碳青霉烯类的最低抑菌浓度（MIC）增加。几丁二糖诱导了chb（一个几丁二糖利用操纵子）的表达。进行实时RT-PCR和SDS-PAGE以阐明hfq、impR、chb和MicM在奇异变形杆菌中的关系。我们发现MicM RNA在hfq突变体和chbBC基因间区域（chbBC-IGR）过表达菌株（chbIGRov）中减少，而impR mRNA在hfq突变体、micM突变体和chbIGRov菌株中增加。此外，hfq或micM的突变以及chbBC-IGR的过表达增加了ImpR蛋白水平。相应地，几丁二糖使野生型具有更高水平的ImpR蛋白，并且对碳青霉烯类更耐药。Hfq和MicM互补菌株恢复了野生型MIC。impR和hfq两者的突变消除了在hfq突变体中观察到的碳青霉烯类MIC的增加，并且hfq/impR双突变体的ImpR互补导致MIC与hfq突变体相同，表明hfq突变体中碳青霉烯类敏感性降低依赖于ImpR。这些表明MicM与impR mRNA反义，并且受chbBC-IGR负调控。总之，ImpR的过表达导致奇异变形杆菌中碳青霉烯类敏感性降低。
