MexEF-OprN multidrug efflux pump transporter negatively controls N-acyl-homoserine lactone accumulation in pseudomonas syringae pv. Tabaci 6605.

Our previous studies revealed that flagellar-motility-defective mutants such as ∆fliC of Pseudomonas syringae pv. tabaci 6605 (Pta6605) have remarkably reduced production of N-acyl-homoserine lactones (AHL), quorum-sensing molecules. To investigate the reason of loss of AHL production in ∆fliC mutant, we carried out transposon mutagenesis. Among approximately 14,000 transconjugants, we found 11 AHL production-recovered (APR) strains. In these APR strains, a transposon was inserted into either mexE or mexF, genes encoding for the multidrug efflux pump transporter MexEF-OprN, and mexT, a gene encoding a putative transcriptional activator for mexEF-oprN. These results suggest that MexEF-OprN is a negative regulator of AHL production. To confirm the negative effect of MexEF-OprN on AHL production, loss- and gain-of-function experiments for mexEF-oprN were carried out. The ∆fliC∆mexF and ∆fliC∆mexT double mutant strains recovered AHL production, whereas the mexT overexpressing strain abolished AHL production, although the psyI, a gene encoding AHL synthase, is transcribed as wild type. Introduction of a mexF or mexT mutation into another flagellar-motility- and AHL production-defective mutant strain, ∆motCD, also recovered the ability to produce AHL. Furthermore, introduction of the mexF mutation into other AHL production-defective mutant strains such as ∆gacA and ∆aefR also recovered AHL production but not to the ∆psyI mutant. These results indicate that MexEF-OprN is a decisive negative determinant of AHL production and accumulation.

[A case of inoperable advanced bile duct cancer treated effectively with combined chemotherapy of gemcitabine and S-1].

The patient was a 63-year-old male. He was admitted to our department due to obstructive jaundice and acute renal failure, and was diagnosed with a lower bile duct cancer. As a result of a stent placement into the bile duct and hemodialysis, jaundice and renal failure improved. As scattered metastases were recognized on the superior surface of both hepatic lobes in intraoperative findings, only a portoenterostomy was performed. After that, 1,000 mg/m(2) of gemcitabine(day 1)and 60 mg/m(2) day of S-1(days 1-7)were administered repeatedly every other week as a course. One year and four months after the start of chemotherapy, radiation therapy of 40 Gy was performed at the site considered to be the remaining primary tumor according to the PET-CT findings. While chemotherapy was continued without change thereafter, the time passed with no visualization of lesions by CT. Two years and five months after the start of chemotherapy, duodenal stenosis and a metastasis in the liver occurred, resulting thereafter in aggravated conditions and death. The entire course lasted two years and eight months. We considered that combined therapy of gemcitabine and S-1 would be a useful option in chemotherapy for biliary tract cancer.

Role of Two Sets of RND-Type Multidrug Efflux Pump Transporter Genes, mexAB-oprM and mexEF-oprN, in Virulence of Pseudomonas syringae pv. tabaci 6605.

Pseudomonas syringae pv. tabaci 6605 has two multidrug resistance (MDR) efflux pump transporters, MexAB-OprM and MexEF-OprN. To understand the role of these MDR efflux pumps in virulence, we generated deletion mutants, ∆mexB, ∆mexF, and ∆mexB∆mexF, and investigated their sensitivity to plant-derived antimicrobial compounds, antibiotics, and virulence. Growth inhibition assays with KB soft agar plate showed that growth of the wild-type (WT) was inhibited by 5 µl of 1 M catechol and 1 M coumarin but not by other plant-derived potential antimicrobial compounds tested including phytoalexins. The sensitivity to these compounds tended to increase in ∆mexB and ∆mexB∆mexF mutants. The ∆mexB∆mexF mutant was also sensitive to 2 M acetovanillone. The mexAB-oprM was constitutively expressed, and activated in the ∆mexF and ∆mexB∆mexF mutant strains. The swarming and swimming motilities were impaired in ∆mexF and ∆mexB∆mexF mutants. The flood inoculation test indicated that bacterial populations in all mutant strains were significantly lower than that of WT, although all mutants and WT caused similar disease symptoms. These results indicate that MexAB-OprM extrudes plant-derived catechol, acetovanillone, or coumarin, and contributes to bacterial virulence. Furthermore, MexAB-OprM and MexEF-OprN complemented each other's functions to some extent.