Discovery of the extracellular loops being essential for the proper function of the multidrug efflux pump suggests that the loops may be an excellent target for the pump inhibitor

铜绿假单胞菌是一种机会致病菌，它对多种抗生素表现出高度的耐药性，这种耐药性主要是由该细菌中的多药外排泵介导的。虽然在铜绿假单胞菌基因组中编码了几个RND外排泵基因，但MexAB-OprM基因仅在成分上表达。MexAB-OprM是一种多组分的外排泵：MexB作为药物/质子逆向转运体，OprM作为外膜药物的通道，MexA作为MexB和OprM之间的连接物。我们重点研究了OprM通道，并对其功能和结构进行了研究。(1)通过交联法研究了OprM及其同系物OprJ和OprN的低聚结构。OprM和OprN形成三聚体，但出人意料地发现OprJ形成四聚体，这表明具有相同功能的同源外膜通道可以呈现不同的寡聚体结构。(2)OprM(468个氨基酸)由三个结构域(α-螺旋周质结构域，β-桶跨膜DO…)组成P100~p109(EL1)和R311~G320(EL2)。为了阐明EL1和EL2的功能，我们将EL1和EL2的氨基酸残基交换为半胱氨酸，并检测了MexAB突变体OprM的活性。结果表明，EL1或EL2突变导致泵功能障碍，说明EL1和EL2在泵排出药物过程中起着关键作用。此外，令人惊讶的是，EL1和EL2被证明是泵识别底物的位点。(3)基于以上结果，我们认为EL可能成为外排泵抑制剂的良好靶点，因为EL功能的抑制可能导致泵的功能障碍。由于EL是从细胞外进入的，我们认为可以用单抗来干扰EL的功能。然后，我们合成了与EL2氨基酸序列相同的多肽，并将其作为抗原。我们制备了杂交瘤细胞，并对其上清液进行了ELISA检测，以检测其与多肽的结合活性。到目前为止，已有17株杂交瘤细胞分泌与该多肽具有结合活性的单抗。较少

Pseudomonas aeruginosa, an opportunistic pathogen, exhibits a high-level resistance to a wide variety of antibiotics that is mainly mediated by the multidrug efflux pumps in this organism. Although several RND efflux pump genes are coded in the P. aeruginosa genome, MexAB-OprM gene is only constitutively expressed. MexAB-OprM is a multicomponent efflux pump : MexB functions as a drug/proton antiporter, OprM as a channel for drugs in the outer membrane, and MexA as a linker between MexB and OprM. We focused on the OprM channel and examined its function and structure.(1)We examined the oligomeric structure of OprM and its homologs such as OprJ and OprN by crosslinking. OprM and OprN were shown to form a trimer, but unexpectedly OprJ was found to form a tetramer, indicating that homologous outer membrane channels with the same function can assume different oligomeric structures.(2)OprM (468 amino acids) consists of the three domains (α-helical periplasmic domain, β-barrel transmembrane do … More main and two extracellular loop (EL)), and two ELs consist of P100〜P109 (EL1) and R311〜G320 (EL2), respectively. In order to elucidate the function of the EL1 and EL2, we exchanged the amino acid residues of these ELs to Cys and examined the activity of MexAB-mutant OprM. It was clearly demonstrated that the mutation in the EL1 or EL2 caused the dysfunction of the pump, indicating that both EL1 and EL2 play the pivotal role in the drug extrusion by the pump. Furthermore, to be surprised, EL1 and EL2 are shown to be the sites contributing to the substrate recognition by the pump.(3)Based on the above results, we considered that EL might become an excellent target site for the inhibitors of the efflux pump since the inhibition of the EL function could lead to the dysfunction of the pump. As EL is accessible from the outside of the cell, we though that the monoclonal antibody (mAb) can be used to interfere the EL function. Then we synthesized the peptide having the same amino acid sequence as that of EL2 and used it as an antigen. We generated the hybridomas and then their supernatants were subjected to ELISA to test the binding activity to the peptide. Up to now, we could produce 17 hybridomas secreting mAb with the binding activity to the peptide. Less