The role of RND-type efflux pumps in multidrug-resistant mutants of Klebsiella pneumoniae.

The emergence of multidrug-resistant Klebsiella pneumoniae is a worldwide problem. K. pneumoniae possesses numerous resistant genes in its genome. We isolated mutants resistant to various antimicrobials in vitro and investigated the importance of intrinsic genes in acquired resistance. The isolation frequency of the mutants was 10-7-10-9. Of the multidrug-resistant mutants, hyper-multidrug-resistant mutants (EB256-1, EB256-2, Nov1-8, Nov2-2, and OX128) were identified, and accelerated efflux activity of ethidium from the inside to the outside of the cells was observed in these mutants. Therefore, we hypothesized that the multidrug efflux pump, especially RND-type efflux pump, would be related to changes of the phenotype. We cloned all RND-type multidrug efflux pumps from the K. pneumoniae genome and characterized them. KexEF and KexC were powerful multidrug efflux pumps, in addition to AcrAB, KexD, OqxAB, and EefABC, which were reported previously. It was revealed that the expression of eefA was increased in EB256-1 and EB256-2: the expression of oqxA was increased in OX128; the expression of kexF was increased in Nov2-2. It was found that a region of 1,485 bp upstream of kexF, was deleted in the genome of Nov2-2. K. pneumoniae possesses more potent RND-multidrug efflux systems than E. coli. However, we revealed that most of them did not contribute to the drug resistance of our strain at basic levels of expression. On the other hand, it was also noted that the overexpression of these pumps could lead to multidrug resistance based on exposure to antimicrobial chemicals. We conclude that these pumps may have a role to maintain the intrinsic resistance of K. pneumoniae when they are overexpressed. The antimicrobial chemicals selected many resistant mutants at the same minimum inhibitory concentration (MIC) or a concentration slightly higher than the MIC. These results support the importance of using antibiotics at appropriate concentrations at clinical sites.

Hexamethylene bisacetamide can convert nonpermissive human cells to a permissive state for expressing the major immediate-early genes of human cytomegalovirus by up-regulating NF-kappaB activity.

Expression of the major immediate-early (MIE) genes of human cytomegalovirus (HCMV) in the human thyroid papillary carcinoma cell line TPC-1 is repressed at the transcriptional level. However, treatment of these cells with hexamethylene bisacetamide (HMBA), a chemical inducer of differentiation, for 12 to 24 h before infection enabled the cells to support IE1 and IE2 gene expression and consequently HCMV replication. In HMBA-treated cells the transcription factor NF-kappaB was induced and the MIE promoter (MIEP) was activated. The presence of a NF-kappaB inhibitory peptide SN-50 or expression of a dominant negative IkappaBalpha protein during the HMBA pretreatment period efficiently prevented the HMBA-induced MIEP activation and MIE protein synthesis. Moreover, introduction of mutations into the NF-kappaB binding sites in the MIEP in a plasmid expressing the IE1 protein diminished its ability to express the protein in HMBA-treated cells. Therefore, the NF-kappaB activity previously induced in HMBA-treated cells and the NF-kappaB sites in the MIEP were shown to be essential for HCMV to respond to HMBA action and to express the MIE genes. Investigation of the mechanisms by which HMBA activates NF-kappaB revealed that degradation of IkappaBalpha and translocation of the phosphorylated NF-kappaB p65 subunit to the nucleus, both of which are known to be critical steps in NF-kappaB activation, are stimulated in the HMBA-treated cells. These results indicate that treatment of nonpermissive TPC-1 cells with HMBA induces MIE gene permissiveness by up-regulating NF-kappaB activity.

Geldanamycin, a potent and specific inhibitor of Hsp90, inhibits gene expression and replication of human cytomegalovirus.

The effect of geldanamycin (GA), a specific inhibitor of heat shock protein 90 (Hsp90), on gene expression and replication of human cytomegalovirus (HCMV) was studied in human embryonic lung (HEL) fibroblasts. Kinetic analysis indicated that GA delayed synthesis of major immediate early (MIE), early and late viral proteins, and blocked a second tier of the synthesis of these proteins that occurred in untreated cells after 48 h post-infection (pi). Moreover, when HCMV-infected HEL cells were maintained with medium containing 40 nM GA for 6 days, with medium changes at 2-day intervals, the virus yield was reduced to an undetectable level. On a molecular level, the cellular kinase Akt and the transcription factor NFkappaB were activated in HCMV-infected cells within 30 min pi. NFkappaB was shown to be essential for MIE gene expression. However, in GA-treated cells, activation of both Akt and NFkappaB was greatly inhibited. Because LY294002, an inhibitor of cellular phosphatidylinositol 3-kinase (PI3-K), also prohibited HCMV-mediated activation of Akt and NFkappaB and synthesis of the MIE proteins, PI3-K signalling was necessary for expressing the MIE genes. These results suggest that the inhibitory effect of GA on HCMV replication is primarily caused by the disruption of the PI3-K signalling pathway, leading to the activation of NFkappaB, which plays a crucial role in expression of the critical MIE genes.

Decidualization induces the expression and activation of an extracellular protease neuropsin in mouse uterus.

Uterine decidualization is accompanied by the remodeling of the cell-matrix and cell-cell interactions around the endometrial stromal cells to allow an appropriate invasion of trophoblasts. This remodeling is thought to require the proteolysis of extracellular matrix proteins or cell adhesion molecules; however, the molecular mechanism remains poorly understood. In this study, decidualization induced the expression and activation of an extracellular serine protease neuropsin in the mouse uterus. Although nonpregnant uteri contained little neuropsin, the protein content and enzymatic activity increased markedly and peaked at the midgestational period in pregnant uteri. Neuropsin expression and activity was also upregulated in artificially induced deciduomata but not in nondecidualized pseudopregnant uteri. Neuropsin is the first extracellular protease to show the evident induction of expression and activity by decidualization and might contribute to the remodeling of extracellular components after decidualization.