Morán-Barrio Jorgelina, Cameranesi María M, Relling Verónica, Limansky Adriana S, Brambilla Luciano, Viale Alejandro M
Instituto de Biología Molecular y Celular de Rosario (IBR), Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, CONICET, Universidad Nacional de Rosario (UNR), Rosario, Argentina.
Instituto de Biología Molecular y Celular de Rosario (IBR), Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, CONICET, Universidad Nacional de Rosario (UNR), Rosario, Argentina
Antimicrob Agents Chemother. 2017 Feb 23;61(3). doi: 10.1128/AAC.01737-16. Print 2017 Mar.
The number and type of outer membrane (OM) channels responsible for carbapenem uptake in are still not well defined. Here, we addressed these questions by using as a model species and a combination of methodologies aimed to characterize OM channels in their original membrane environment. Kinetic and competition analyses of imipenem (IPM) uptake by whole cells allowed us to identify different carbapenem-specific OM uptake sites. Comparative analyses of IPM uptake by wild-type (WT) cells and Δ mutants lacking CarO indicated that this OM protein provided a carbapenem uptake site displaying saturable kinetics and common binding sites for basic amino acids compatible with a specific channel. The kinetic analysis uncovered another carbapenem-specific channel displaying a somewhat lower affinity for IPM than that of CarO and, in addition, common binding sites for basic amino acids as determined by competition studies. The use of gene deletion mutants lacking OM proteins proposed to function in carbapenem uptake in indicated that CarO and OprD/OccAB1 mutants displayed low but consistent reductions in susceptibility to different carbapenems, including IPM, meropenem, and ertapenem. These two mutants also showed impaired growth on l-Arg but not on other carbon sources, further supporting a role of CarO and OprD/OccAB1 in basic amino acid and carbapenem uptake. A multiple-carbapenem-channel scenario may provide clues to our understanding of the contribution of OM channel loss or mutation to the carbapenem-resistant phenotype evolved by pathogenic members of the genus.
负责碳青霉烯类药物摄取的外膜（OM）通道的数量和类型在[具体物种未提及]中仍未明确界定。在此，我们以[具体物种未提及]作为模型物种，并结合多种方法来解决这些问题，旨在在其原始膜环境中表征OM通道。通过对[具体物种未提及]全细胞摄取亚胺培南（IPM）的动力学和竞争分析，我们能够识别出不同的碳青霉烯类药物特异性OM摄取位点。对野生型（WT）细胞和缺乏CarO的Δ突变体摄取IPM的比较分析表明，这种OM蛋白提供了一个碳青霉烯类药物摄取位点，该位点显示出饱和动力学以及与特定通道兼容的碱性氨基酸共同结合位点。动力学分析发现了另一个碳青霉烯类药物特异性通道，其对IPM的亲和力略低于CarO，此外，竞争研究确定其也有碱性氨基酸共同结合位点。使用缺乏被认为在[具体物种未提及]中参与碳青霉烯类药物摄取功能的OM蛋白的基因缺失突变体表明，CarO和OprD/OccAB1突变体对不同碳青霉烯类药物（包括IPM、美罗培南和厄他培南）的敏感性降低，但降低程度较低且一致。这两个突变体在l-精氨酸上的生长也受到损害，但在其他碳源上不受影响，进一步支持了CarO和OprD/OccAB1在碱性氨基酸和碳青霉烯类药物摄取中的作用。多碳青霉烯类药物通道的情况可能为我们理解OM通道丧失或突变对[具体属未提及]属致病成员所产生的碳青霉烯类耐药表型的贡献提供线索。
