• Journal of Microbiology and Biotechnology
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• 제23권3호
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• pp.430-435
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• 2013

Multidrug resistance, especially multidrug efflux mechanisms that extrude structurally unrelated cytotoxic compounds from the cell by multidrug transporters, is a serious problem and one of the main reasons for the failure of therapeutic treatment of infections by pathogenic microorganisms as well as of cancer cells. Streptococcus mutans is considered one of the primary causative agents of dental caries and periodontal disease, which comprise the most common oral diseases. A fragment of chromosomal DNA from S. mutans KCTC3065 was cloned using Escherichia coli KAM32 as host cells lacking major multidrug efflux pumps. Although E. coli KAM32 cells were very sensitive to many antimicrobial agents, the transformed cells harboring a recombinant plasmid became resistant to several structurally unrelated antimicrobial agents such as tetracycline, kanamycin, rhodamin 6G, ampicillin, acriflavine, ethidium bromide, and tetraphenylphosphonium chloride. This suggested that the cloned DNA fragment carries a gene encoding a multidrug efflux pump. Among 49 of the multidrug-resistant transformants, we report the functional gene cloning and characterization of the function of one multidrug efflux pump, namely MdeA from S. mutans, which was expressed in E. coli KAM32. Judging from the structural and biochemical properties, we concluded that MdeA is the first cloned and characterized multidrug efflux pump using the proton motive force as the energy for efflux drugs.

• 한국미생물·생명공학회지
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• 제31권3호
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• pp.230-234
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• 2003

Small multidrug resistance(SMR) family(TC #2.A.7.1)에 속하는 막단백질 중 하나를 coding하는 Bacillus subtilis의 yvaE유전자의 발현을 유도한 결과, 발현된 YvaE단백질에 의해 대장균세포내에서 살균제를 포함한 다양한 양이온 약제에 대한 efflux활성을 촉매하는 것으로 확인되었다. 같은 operon내에 인접한 yaD유전자의 동시발현을 유도한 결과, 이러한 efflux활성은 억제가 되는 것으로 나타났다. Ethidium bromide를 기질로 하여 fluorimeter를 이용한 efflux transport실험결과, YvaE 단백질이 발현된 대장균세포의 경우 vector만을 함유한 대조세포에 비해 현저히 빠른 efflux활성을 보여주었다 따라서 YvaE 막단백질은 multidrug export를 촉매하는 SMR-type efflux pump임을 확인하였다.

• 대한의생명과학회지
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• 제27권2호
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• pp.59-68
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• 2021

Multidrug-resistant strain of Acinetobacter baumannii (MDRAB) is an emerging pathogen in health care facilities, preventing MDRAB is a public health concern. We conducted this experiment on a clinical isolate of A. baumannii with two main goals: the role of the efflux pump system in the stress provision of carbapenem and the response to the transcription level of the efflux pump gene. A total of 34 strains of A. baumannii was isolated from the Yangsan Hospital of Pusan National University. First, when we compared and observed the expression of the efflux pump gene and antibacterial resistance to carbapenem, a strong correlation was observed between carbapenem resistance and overexpression of adeB (P=0.0056). Second, a correlation between the efflux pump and concentration gradient and tolerance to carbapenem stress at the AdeABC efflux pump genes transcription level was confirmed. Our results revealed that the expression of the AdeABC efflux pump is an important resistance determinant in obtaining antibiotic resistance of the carbapenem group in A. baumannii.

• BMB Reports
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• 제56권6호
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• pp.326-334
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• 2023

Antibiotic resistance (AR) is a silent pandemic that kills millions worldwide. Although the development of new therapeutic agents against antibiotic resistance is in urgent demand, this has presented a great challenge, especially for Gram-negative bacteria that have inherent drug-resistance mediated by impermeable outer membranes and multidrug efflux pumps that actively extrude various drugs from the bacteria. For the last two decades, multidrug efflux pumps, including AcrAB-TolC, the most clinically important efflux pump in Gram-negative bacteria, have drawn great attention as strategic targets for re-sensitizing bacteria to the existing antibiotics. This article aims to provide a concise overview of the AcrAB-TolC operational mechanism, reviewing its architecture and substrate specificity, as well as the recent development of AcrAB-TolC inhibitors.

• Journal of Microbiology and Biotechnology
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• 제30권7호
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• pp.982-995
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• 2020

A putative multidrug efflux gene, yddA, was cloned from the Escherichia coli K-12 strain. A drug-sensitive strain of E. coli missing the main multidrug efflux pump AcrB was constructed as a host and the yddA gene was knocked out in wild-type (WT) and drug-sensitive E. coliΔacrB to study the yddA function. Sensitivity to different substrates of WT E.coli, E. coliΔyddA, E. coliΔacrB and E. coliΔacrBΔyddA strains was compared with minimal inhibitory concentration (MIC) assays and fluorescence tests. MIC assay and fluorescence test results showed that YddA protein was a multidrug efflux pump that exported multiple substrates. Three inhibitors, ortho-vanadate, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and reserpine, were used in fluorescence tests. Ortho-vanadate and reserpine significantly inhibited the efflux and increased accumulation of ethidium bromide and norfloxacin, while CCCP had no significant effect on YddA-regulated efflux. The results indicated that YddA relies on energy released from ATP hydrolysis to transfer the substrates and YddA is an ABC-type multidrug exporter. Functional study of unknown ATP-binding cassette (ABC) superfamily transporters in the model organism E. coli is conducive to discovering new multidrug resistance-reversal targets and providing references for studying other ABC proteins of unknown function.

• BMB Reports
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• 제48권6호
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• pp.348-353
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• 2015

In the present study, we investigated the characteristics of drug efflux transporter expressions following status epilepticus (SE). In the hippocampus and piriform cortex (PC), vasogenic edema peaked 3-4 days after SE. The expression of breast cancer resistance protein (BCRP), multidrug resistance protein-4 (MRP4), and p-glycoprotein (p-GP) were decreased 4 days after SE when vasogenic edema was peaked, but subsequently increased 4 weeks after SE. Multidrug resistance protein-1 (MRP1) expression gradually decreased in endothelial cells until 4 weeks after SE. These findings indicate that SE-induced vasogenic edema formation transiently reduced drug efflux pump expressions in endothelial cells. Subsequently, during recovery of vasogenic edema drug efflux pump expressions were differentially upregulated in astrocytes, neuropils, and endothelial cells. Therefore, we suggest that vasogenic edema formation may be a risk factor in pharmacoresistent epilepsy. [BMB Reports 2015; 48(6): 348-353]

• Journal of Microbiology and Biotechnology
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• 제26권12호
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• pp.2043-2050
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• 2016

Exploring novel antibiotics is necessary for multidrug-resistant pathogenic bacteria. Because the probiotics in soybean food have antimicrobial activities, we investigated their effects on multidrug-resistant Acinetobacter baumannii. Nineteen multidrug-resistant A. baumannii strains were clinically isolated as an experimental group and 11 multidrug-sensitive strains as controls. The growth rates of all bacteria were determined by using the analysis for xCELLigence Real-Time Cell. The combination of antibiotics showed synergistic effects on the strains in the control group but no effect on the strains in the experimental group. Efflux pump gene adeS was absent in all the strains from the control group, whereas it exists in all the strains from the experimental group. Furthermore, all the strains lost multidrug resistance when an adeS inhibitor was used. One strain of probiotics isolated from soybean food showed high antimicrobial activity for multidrug-resistant A. baumannii. The isolated strain belongs to Bacillus subtilis according to 16S RNA analysis. Furthermore, E. coli showed multidrug resistance when it was transformed with the adeS gene from A. baumannii whereas the resistant bacteria could be inhibited completely by isolated Bacillus subtilis. Thus, probiotics from soybean food provide potential antibiotics against multidrug-resistant pathogenic bacteria.

• 미생물학회지
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• 제49권2호
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• pp.126-130
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• 2013

원유시료에서 분리한 장알균속 세균 245균주의 항생제 내성에 관여하는 다중약물 유출 펌프 유전자 분포도와 항생제 내성 패턴을 연구하였다. 그 결과 245 장알균속 균주의 ampicillin에 대한 내성률은 44.1%, erythromycin에 대한 내성률은 79.2%, tetracycline에 대한 내성률은 76.3%, chloramphenicol에 대한 내성률은 36.3%였으며 vancomycin과 ciprofloxacin에 대해서는 모두 감수성임을 알 수 있었다. 내성 관련 유전자 중 MFS타입의 eme(A)는 82.1%의 장알균에 분포하였으며, ABC 타입의 유전자인 efr(A)는 72.7%, efr(B)는 77.1%, lsa는 71.8%의 장알균에 분포하였다. 특히 이러한 유전자의 기원 세균인 Enterococcus faecalis의 경우 eme(A)는 92.5%, efr(A)는 87.4%, efr(B)는 88.4%, lsa는 88.4%의 분포도를 나타내었다. 한편 동일한 장알균속이지만 종이 다른 장알균에서 eme(A)는 E. faecium 4균주, E. avium 7균주, E. durans 4균주 및 E. raffinosus 2균주에 분포하고 있었다. efr(A)는 E. faecium 2균주와 E. durans 2균주에 분포하였으며, efr(B)는 E. faecium 4균주, E. avium 5균주 및 E. durans 4균주에 분포하였다. 본 연구는 우리나라 원유시료에서 분리한 장알균속의 여러 종의 세균에서 동일한 다중약물 유출 펌프(multidrug efflux pump)의 분포에 대한 첫 번째 보고라 사료되며 E. faecalis 이외의 장알균속에서 이러한 유전자의 분포는 서로 다른 종간의 유전자의 수평적인 이동의 가능성을 시사한다.

• Molecules and Cells
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• 제38권2호
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• pp.180-186
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• 2015

Escherichia coli AcrAB-TolC is a multidrug efflux pump that expels a wide range of toxic substrates. The dynamic nature of the binding or low affinity between the components has impeded elucidation of how the three components assemble in the functional state. Here, we created fusion proteins composed of AcrB, a transmembrane linker, and two copies of AcrA. The fusion protein exhibited acridine pumping activity, suggesting that the protein reflects the functional structure in vivo. To discern the assembling mode with TolC, the AcrBA fusion protein was incubated with TolC or a chimeric protein containing the TolC aperture tip region. Three-dimensional structures of the complex proteins were determined through transmission electron microscopy. The overall structure exemplifies the adaptor bridging model, wherein the funnel-like AcrA hexamer forms an intermeshing cogwheel interaction with the ${\alpha}$-barrel tip region of TolC, and a direct interaction between AcrB and TolC is not allowed. These observations provide a structural blueprint for understanding multidrug resistance in pathogenic Gram-negative bacteria.

• Asian Pacific Journal of Cancer Prevention
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• 제13권9호
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• pp.4807-4814
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• 2012

Purpose: The chemoresistance of human hepatocellular carcinoma (HCC) to cytotoxic drugs, especially intrinsic or acquired multidrug resistance (MDR), still remains a major challenge in the management of HCC. In the present study, possible mechanisms involved in MDR of HCC were identified using a 5-fluorouracil (5-FU)-resistant human HCC cell line. Methods: BEL-7402/5-FU cells were established through continuous culturing parental BEL-7402 cells, imitating the pattern of chemotherapy clinically. Growth curves and chemosensitivity to cytotoxic drugs were determined by MTT assay. Doubling times, colony formation and adherence rates were calculated after cell counting. Morphological alteration, karyotype morphology, and untrastructure were assessed under optical and electron microscopes. The distribution in the cell cycle and drug efflux pump activity were measured by flow cytometry. Furthermore, expression of potential genes involved in MDR of BEL-7402/5-FU cells were detected by immunocytochemistry. Results: Compared to its parental cells, BEL-7402/5-FU cells had a prolonged doubling time, a lower mitotic index, colony efficiency and adhesive ability, and a decreased drug efflux pump activity. The resistant cells tended to grow in clusters and apparent changes of ultrastructures occurred. BEL-7402/5-FU cells presented with an increased proportion in S and G2/M phases with a concomitant decrease in G0/G1 phase. The MDR phenotype of BEL-7402/5-FU might be partly attributed to increased drug efflux pump activity via multidrug resistance protein 1 (MRP1), overexpression of thymidylate synthase (TS), resistance to apoptosis by augmentation of the Bcl-xl/Bax ratio, and intracellular adhesion medicated by E-cadherin (E-cad). P-glycoprotein (P-gp) might play a limited role in the MDR of BEL-7402/5-FU. Conclusion: Increased activity or expression of MRP1, Bcl-xl, TS, and E-cad appear to be involved in the MDR mechanism of BEL-7402/5-FU.