Mdrl Expression Research Articles

MdrL, a major facilitator superfamily efflux pump of Listeria monocytogenes involved in tolerance to benzalkonium chloride.

Efflux pumps are recognized as an important mechanism for decreased susceptibility of benzalkonium chloride (BC) in Listeria monocytogenes. Previous studies showed that the efflux pump MdrL was overexpressed in L. monocytogenes exposed to BC. In the present work, we aimed to clarify the role of MdrL in tolerance to BC and environmental stresses including acid, alkali, osmotic, ethanol, and oxidative stresses, as well as resistance to other antimicrobial agents in L. monocytogenes EGD-e. In addition, regulation of the expression of mdrL by LadR was investigated. Gene deletion mutants were constructed by homologous recombination strategy. For the wild-type and mutant strains, minimum inhibitory concentrations (MICs) of antimicrobial agents were determined by the agar dilution, and the growth and survival analysis were also performed. LadR was expressed and the interaction between LadR and the mdrL promoter was investigated by electrophoretic mobility shift assay (EMSA). Compared to the wild-type strain, the growth of ΔmdrL deletion mutant strain was impaired in the presence of sublethal concentration of BC. Moreover, the mutant showed a lower level of survival than that of the wild-type strain in the presence of lethal concentration of BC. However, the deletion of mdrL had no impact on cefotaxime resistance and ethidium bromide efflux. BC could induce the expression of mdrL in L. monocytogenes and the mdrL expression was regulated by LadR instead of SigmaB. LadR was able to specifically bind to the mdrL promoter. The results showed that efflux pump MdrL was associated with BC tolerance in L. monocytogenes EGD-e. Moreover, our results also provided strong evidence that LadR negatively regulated the expression of mdrL. Since BC is commonly used in the food industry, efflux pump MdrL is beneficial for L. monocytogenes to survive this stress in food processing environments.

Resistance to Biocides in Listeria monocytogenes Collected in Meat-Processing Environments.

The emergence of microorganisms exerting resistance to biocides is a challenge to meat-processing environments. Bacteria can be intrinsically resistant to biocides but resistance can also be acquired by adaptation to their sub-lethal concentrations. Moreover, the presence of biocide resistance determinants, which is closely linked to antibiotic resistance determinants, could lead to co-selection during disinfection practices along the food chain, and select cross-resistant foodborne pathogens. The purpose of this work was to test the resistance of wild strains of Listeria monocytogenes, isolated from pork meat processing plants, toward benzalkonium chloride (BC), used as proxy of quaternary ammonium compounds. Furthermore, the expression of two non-specific efflux pumps genes (lde and mdrL) under biocide exposure was evaluated. L. monocytogenes were isolated from five processing plants located in the Veneto region (northeast of Italy) before and after cleaning and disinfection (C&D) procedures. A total of 45 strains were collected: 36 strains before and nine after the C&D procedures. Collected strains were typed according to MLST and ERIC profiles. Strains sampled in the same site, isolated before, and after the C&D procedures and displaying the same MLST and ERIC profiles were tested for their sensitivity to different concentrations of BC, in a time course assay. The expression of non-specific efflux pumps was evaluated at each time point by qPCR using tufA gene as housekeeping. A differential expression of the two investigated genes was observed: lde was found to be more expressed by the strains isolated before C&D procedures while its expression was dose-dependent in the case of the post C&D procedures strain. On the contrary, the expression of mdrL was inhibited under low biocidal stress (10 ppm BC) and enhanced in the presence of high stress (100 ppm BC). These findings suggests a possible role for C&D procedures to select L. monocytogenes persisters, pointing out the importance of dealing with the identification of risk factors in food plants sanification procedures that might select more tolerant strains.

Celecoxib-induced apoptosis of gastric cancer cell line SGC7901/ADR by inhibiting nuclear factor-κB activity

Objective To investigate the effects of celecoxib on mdr1 expression, nuclear factorκB (NF-KB) activity and apoptosis of SGC7901/ADR. Methods Cultured SGC7901 cells were divided into four groups: SGC7901 group, SGC7901 + ceIecoxib group, SGC7901/ADR group and SGC79OI/ADR + celecoxib group. The expression of mdrl gene was detected by reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry. Electrophoretic mobility shift assay (EMSA) was used to measure the activity of NF-κB. Intracellular accumulation of Rh123 and apoptosis rate were measured by flow cytometry. Results The mdr1 gene expression and NF-κB activity were increased to 2. 4 fold and 3.2 fold respectively in SGC7901/ADR group as compared with SGC7901 group (P <0. 01 ). Celecoxib inhibited the mdrl expression and NF-KB activity in SGC7901 and SGC7901/ADR groups. Celecoxib also improved the intracellular accumulation of Rh123 in SGC7901 group (1.2-fold) and SGC7901/ADR group (1.9-fold).Celecoxib increased the apoptosis rate in SGC7901 group (1.65-fold) and SGC7901/ADR group (2. 98-fold).induced by ADR. Conclusion Celecoxib can inhibit the mdr1 expression and increase intracellular Rh123 accumulation by suppressing NF-κB activity and induce the apoptosis of SGC7901/ADR cells. Key words: Celecoxib; NF-κB; Gastric cancer

Effects of inhibition of nuclear factor-κB activity on mdr1 expression and apoptosis of SGC7901/ VCR cells

Objective To investigate the effects of inhibition of nuclear factor-κB (NF-κB) activity induced by mutated IκBα on mdrl expression and apoptosis of gastric carcinoma cell lines SGC7901/ VCR. Methods pCMV4-mκBα was transfected into SGC7901/VCR cells by lipofectamine 2000.The activity of NF-κB was detected by EMSA. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were used to detect the mdrl gene expression. The accumulation of Rhodamine123 (Rh123) in SGC7901/VCR cells was analyzed by fluorescence spectrophotometric method. The apoptosis of SGC7901/VCR cells was examined by FCS technique. Results The inhibition rate of NF-kB activity and mdrl expression in SGC7901/VCR cells transfected with pCMV4-mlκBα was 64% and 75% respectively as compared with the control cells. Twenty-four h after transfection of mlκBα plasmid, the intracellular Rhl23 concentration was increased by 2.3-fold. The apoptosis rate in mlκBα plasmid transfected goups (14.15 ±1.52) was increased significantly as compared with the control group (P<0.01). Conclusion Inhibition of NF-κB activity results in the decrease of mdrl gene expression and increase of apoptosis by inhibiting drugs efflux in SGC 7901/VCR cells. Key words: Carcinoma of stomach; NF-kB; mdrl; Apoptosis

Role of Efflux Pumps in Adaptation and Resistance of Listeria monocytogenes to Benzalkonium Chloride

In this study, potential mechanisms underlying resistance and adaptation to benzalkonium chloride (BC) in Listeria monocytogenes were investigated. Two groups of strains were studied. The first group consisted of strains naturally sensitive to BC which could be adapted to BC. The second group consisted of naturally resistant strains. For all adapted isolates, there was a correlation between the resistance to BC and ethidium bromide, but this was not the case for the naturally resistant isolates. To investigate the role of efflux pumps in adaptation or resistance, reserpine, an efflux pump inhibitor, was added to the strains. Addition of reserpine to the sensitive and adapted strains resulted in a decrease in the MIC for BC, whereas no such decrease was observed for the resistant strains, indicating that efflux pumps played no role in the innate resistance of certain strains of L. monocytogenes to this compound. Two efflux pumps (MdrL and Lde) have been described in L. monocytogenes. Studies showed low and intermediate levels of expression of the genes encoding the efflux pumps for two selected resistant strains, H7764 and H7962, respectively. Adaptation to BC of sensitive isolates of L. monocytogenes resulted in significant increases in expression of mdrl (P < 0.05), but no such increase was observed for lde for two adapted strains of L. monocytogenes, LJH 381 (P = 0.91) and C719 (P = 0.11). This indicates that the efflux pump Mdrl is at least partly responsible for the adaptation to BC.

LadR, a new PadR-related transcriptional regulator fromListeria monocytogenes, negatively regulates the expression of the multidrug efflux pump MdrL

The Listeria monocytogenes genome encodes putative multidrug efflux transporters but only the MdrL transporter has been partially characterized in the wild-type LO28 strain. Here, we show in the LO28 strain, that the expression of MdrL is repressed at the transcriptional level, under standard growth conditions, by the product of a new gene ladR (lmo1408), and the expression of MdrL is induced in the presence of rhodamine. Phylogenetic analysis in related firmicutes shows that LadR, conserved in all sequenced Listeria genomes, forms an independent group from the large and diverse PadR transcriptional regulator family (PF03551). This is the first report of a bacterial multidrug transporter controlled by a member of the PadR family.

Identification of a new locus in Listeria monocytogenes involved in cellobiose-dependent repression of hly expression

Expression of the PrfA-controlled virulence gene hly (encoding the pore-forming cytolysin listeriolysin) is down-regulated by readily metabolized carbon sources in Listeria monocytogenes. We isolated a Tn 917-insertional mutant of L. monocytogenes (strain LO28), which expressed a hemolytic phenotype in the presence of cellobiose. Using hly fusions to luxAluxB genes, we show that hly expression was derepressed in the presence of cellobiose at the transcriptional level. Surprisingly, hly expression was still repressed by glucose, as observed for the parental strain. Genetic analysis of the Tn 917-flanking regions indicated that the transposon had inserted in a non-coding region located between two genes in opposite orientations. These two newly identified genes were designated orfA and mdrL. The insertion occurred immediately upstream of orfA, likely into its promoter region. Transcriptional analysis of orfA and mdrL revealed that Tn 917 had abolished orfA expression whereas it had activated expression of mdrL. orfA encodes a putative protein of 176 amino acids homologous to YfiO of Bacillus subtilis (28% identity), a protein of unknown function. mdrL codes for a putative protein of 398 amino acids homologous to Bmr and Blt of B. subtilis (21–24% identity), two members of the multidrug resistance efflux pump family. Our results indicate that we have identified a new locus which plays a crucial role in the cellobiose-dependent repression of hly expression.

Identification of a new locus in Listeria monocytogenes involved in cellobiose-dependent repression of hly expression.

Expression of the PrfA-controlled virulence gene hly (encoding the pore-forming cytolysin listeriolysin) is down-regulated by readily metabolized carbon sources in Listeria monocytogenes. We isolated a Tn917-insertional mutant of L. monocytogenes (strain LO28), which expressed a hemolytic phenotype in the presence of cellobiose. Using hly fusions to luxAluxB genes, we show that hly expression was derepressed in the presence of cellobiose at the transcriptional level. Surprisingly, hly expression was still repressed by glucose, as observed for the parental strain. Genetic analysis of the Tn917-flanking regions indicated that the transposon had inserted in a non-coding region located between two genes in opposite orientations. These two newly identified genes were designated orfA and mdrL. The insertion occurred immediately upstream of orfA, likely into its promoter region. Transcriptional analysis of orfA and mdrL revealed that Tn917 had abolished orfA expression whereas it had activated expression of mdrL. orfA encodes a putative protein of 176 amino acids homologous to YfiO of Bacillus subtilis (28% identity), a protein of unknown function. mdrL codes for a putative protein of 398 amino acids homologous to Bmr and Blt of B. subtilis (21-24% identity), two members of the multidrug resistance efflux pump family. Our results indicate that we have identified a new locus which plays a crucial role in the cellobiose-dependent repression of hly expression.

Structural and functional analysis of the cytidine deaminase gene in patients with acute myeloid leukaemia.

Gene transfer of the cytidine deaminase (CDD) cDNA has recently been shown to induce cellular resistance to cytarabine (AraC) in vitro. To investigate the role for CDD in acute myeloid leukaemia (AML) we analysed the CDD activity and CDD gene structure in blast material from well-defined patients with untreated and AraC refractory (RF) AML. Median CDD activity in previously untreated AML was significantly lower than in RF-AML blasts (P=0.015) and was significantly lower in patients with complete remission than with blast persistence following induction chemotherapy (P=0.043). Structural investigation of the CDD gene by Southern analyses and RT-PCR showed no detectable aberrations. Sequence analysis of the CDD cDNA from nine RF-AML patients showed inconsistent aberrations in three patients. Semiquantitative assessment of CDD mRNA expression revealed a significant correlation with CDD activity. In conclusion, concordant with another recent study our data suggest a correlation of pretherapeutic CDD activity with induction treatment response. Besides the previously described prognostic impact of mdrl expression, this result could be useful for the development of risk-adapted AML treatment strategies and warrants further studies of CDD activity in well-defined cohorts of AML patients and of the mechanisms involved in the regulation of CDD activity.

Expression of multidrug resistance-related genes (mdrl, MRP, GST-pi and DNA topoisomerase II) in urothelial cancers.

To characterize the multidrug resistance (MDR) phenotype in human urothelial cancers, the expression levels of four MDR-related genes (multidrug resistance, mdrl; multidrug resistance-associated protein, MRP; glutathione S-transferase-pi, GST-pi; and DNA topoisomerase II, topo II) were analysed in urothelial cancers. Fifty-two tumour tissue and three normal urothelial mucosa samples were obtained from 44 patients with urothelial cancers. The expression of each gene was analysed with a reverse-transcription polymerase chain reaction (RT-PCR) method using beta 2-microglobulin (b2m) mRNA as an endogenous control. Levels of expression were expressed as the ratio of the specific products of the target gene to those specific to b2m. In primary urothelial cancer tissues, the mean (SD) expression of mdrl, MRP, GST-pi and topo II relative to b2m expression were 0.067 (0.061), 0.27 (0.23), 0.35 (0.31) and 0.12 (0.05), respectively. The mean expressions of MRP and GST-pi were higher than those of mdrl and topo II. The mean ratios of mdrl/b2m, MRP/b2m, GST-pi/b2m and topo II/b2m in normal urothelial mucosa were 0.06 (0.03), 0.12 (0.09), 0.30 (0.32) and 0.14 (0.01), respectively. There was no significant association of the expression of each gene with either the grade or extent of the primary tumour. The level of MRP expression in each sample was correlated significantly with the expression of mdrl and GST-pi in the urothelial cancers (r = 0.637 and 0.537, respectively). Chemotherapy did not markedly influence the induction of expression of the MDR-related genes, except for one case in which mdrl expression was 15 times greater than before chemotherapy. The expression of GST-pi in the patients not receiving chemotherapy was significantly higher than in those that did. These results suggest that the activation of MRP and GST-pi expression occurs during the tumorigenesis of urothelial cancers and that it may confer de novo and acquired drug resistance on urothelial cancers. These results should provide further insight into the complex role postulated for MDR-related genes in chemotherapy, carcinogenesis and tumour progression.

150 P - Expression of the drug resistance genes MDR1, MRP, GSTπ and MT2 A in human primary gastric carcinomas

Multidrug-resistance (MDR) can be due to the overexpression of the MDRl gene or the multidrug-resistance associated protein (MRP) gene. In addition, alterations In the expression and activity of glutathione-S-transferase π (GSTπ) can cause MDR. Metallothionein (MT) as antitoxic factor may also be involved in MDR. To evaluate the clinically active mechanisms of drug resistance in gastric carcinomas, we have evaluated the expression of MDRl, MRP, GSTπ and MT2 A in primary gastric carcinoma specimens by RT-PCR. RNA was isolated from tumor specimens and cDNA was synthesized by standard techniques. MRP RNA was detected in 50/72 (69%.) and MRP RNA in 59/72 (82%) of the tumors. GSTπ and MT2 A were expressed in all tumor specimens studied (n = 26). Thus several drug resistance genes are expressed in gastric carcinomas and might contribute to the clinical drug resistance but their relative quantitative contribution to clinical drug resistance will have to be determined in future studies. (Supported by the “Medizinisch-wissenschaftlichen Fonds des Burgermeisters der Bundeshauptstadt Wien”).

Expression of mdrl, mrp, topoisomerase IIα/β, and cyclin A in primary or relapsed states of acute lymphoblastic leukaemias

In a series of 60 ALL samples drawn during different stages of the disease we used a cDNA-PCR approach to analyse the relative mRNA levels of the MDR-associated genes encoding mdr1/P-glycoprotein, mrp, and the topoisomerase II isozymes alpha and beta. Expression analysis of the cyclin A gene was included to examine cellular proliferation activity. The expression of gapdh served as an internal standard. Calculating the mean values we found: (i) a distinctly lower mdr1 gene expression in primary ALL and first relapses compared to bone marrow from healthy donors, (ii) no change in mdr1 and mrp, but a decreased topoisomerase II alpha gene expression in first relapses of ALL compared to the primary leukaemia, and (iii) increased mdr1 and mrp levels combined to decreased topoisomerase II alpha levels in recurrent relapses of ALL showing significant correlations (mdr1/mrp: rs = +0.6833, P < 0.05; mdr1/topoII alpha: rs = -0.6727, P < 0.05). The expression of the topoisomerase II alpha gene was correlated to that of cyclin A, indicating a link of its expression to cellular proliferation. Our findings suggest that a multifactorial MDR including mrp appears particularly in recurrent relapses of ALL, which often do not respond to chemotherapy. Nonetheless, some individual samples showed gene expression levels very different from the mean values calculated for a particular state of the leukaemia, indicating the need of an individual expression analysis of MDR-associated genes.