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Ben F. Luisi - One of the best experts on this subject based on the ideXlab platform.
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In situ structure and assembly of the multidrug Efflux Pump AcrAB-TolC.
Nature communications, 2019Co-Authors: Xiaodong Shi, James M. Bell, Hans Wang, Isaac Forrester, Heather Villarreal, Joanita Jakana, Muyuan Chen, Ben F. LuisiAbstract:Multidrug Efflux Pumps actively expel a wide range of toxic substrates from the cell and play a major role in intrinsic and acquired drug resistance. In Gram-negative bacteria, these Pumps form tripartite assemblies that span the cell envelope. However, the in situ structure and assembly mechanism of multidrug Efflux Pumps remain unknown. Here we report the in situ structure of the Escherichia coli AcrAB-TolC multidrug Efflux Pump obtained by electron cryo-tomography and subtomogram averaging. The fully assembled Efflux Pump is observed in a closed state under conditions of antibiotic challenge and in an open state in the presence of AcrB inhibitor. We also observe intermediate AcrAB complexes without TolC and discover that AcrA contacts the peptidoglycan layer of the periplasm. Our data point to a sequential assembly process in living bacteria, beginning with formation of the AcrAB subcomplex and suggest domains to target with Efflux Pump inhibitors.
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In situ structure and assembly of the multidrug Efflux Pump AcrAB-TolC.
Nature Communications, 2019Co-Authors: Muyuan Chen, Dijun Du, Zhili Yu, James M. Bell, Hans Wang, Isaac Forrester, Heather Villarreal, Joanita Jakana, Ben F. LuisiAbstract:Multidrug Efflux Pumps actively expel a wide range of toxic substrates from the cell and play a major role in intrinsic and acquired drug resistance. In Gram-negative bacteria, these Pumps form tripartite assemblies that span the cell envelope. However, the in situ structure and assembly mechanism of multidrug Efflux Pumps remain unknown. Here we report the in situ structure of the Escherichia coli AcrAB-TolC multidrug Efflux Pump obtained by electron cryo-tomography and subtomogram averaging. The fully assembled Efflux Pump is observed in a closed state under conditions of antibiotic challenge and in an open state in the presence of AcrB inhibitor. We also observe intermediate AcrAB complexes without TolC and discover that AcrA contacts the peptidoglycan layer of the periplasm. Our data point to a sequential assembly process in living bacteria, beginning with formation of the AcrAB subcomplex and suggest domains to target with Efflux Pump inhibitors. Multidrug Efflux Pumps actively expel a wide range of toxic substrates from bacteria and play a major role in drug resistance. Here authors show the in situ structure of the Efflux Pump AcrAB-TolC obtained by electron cryo-tomography and subtomogram averaging.
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Structure of the AcrAB-TolC multidrug Efflux Pump
Nature, 2014Co-Authors: Dijun Du, Nathan R. James, Jarrod E. Voss, Ewa Klimont, Thelma Ohene-agyei, Henrietta Venter, Wah Chiu, Zhao Wang, Ben F. LuisiAbstract:The capacity of numerous bacterial species to tolerate antibiotics and other toxic compounds arises in part from the activity of energy-dependent transporters. In Gram-negative bacteria, many of these transporters form multicomponent 'Pumps' that span both inner and outer membranes and are driven energetically by a primary or secondary transporter component. A model system for such a Pump is the acridine resistance complex of Escherichia coli. This Pump assembly comprises the outer-membrane channel TolC, the secondary transporter AcrB located in the inner membrane, and the periplasmic AcrA, which bridges these two integral membrane proteins. The AcrAB-TolC Efflux Pump is able to transport vectorially a diverse array of compounds with little chemical similarity, thus conferring resistance to a broad spectrum of antibiotics. Homologous complexes are found in many Gram-negative species, including in animal and plant pathogens. Crystal structures are available for the individual components of the Pump and have provided insights into substrate recognition, energy coupling and the transduction of conformational changes associated with the transport process. However, how the subunits are organized in the Pump, their stoichiometry and the details of their interactions are not known. Here we present the pseudo-atomic structure of a complete multidrug Efflux Pump in complex with a modulatory protein partner from E. coli. The model defines the quaternary organization of the Pump, identifies key domain interactions, and suggests a cooperative process for channel assembly and opening. These findings illuminate the basis for drug resistance in numerous pathogenic bacterial species.
Dijun Du - One of the best experts on this subject based on the ideXlab platform.
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In situ structure and assembly of the multidrug Efflux Pump AcrAB-TolC.
Nature Communications, 2019Co-Authors: Muyuan Chen, Dijun Du, Zhili Yu, James M. Bell, Hans Wang, Isaac Forrester, Heather Villarreal, Joanita Jakana, Ben F. LuisiAbstract:Multidrug Efflux Pumps actively expel a wide range of toxic substrates from the cell and play a major role in intrinsic and acquired drug resistance. In Gram-negative bacteria, these Pumps form tripartite assemblies that span the cell envelope. However, the in situ structure and assembly mechanism of multidrug Efflux Pumps remain unknown. Here we report the in situ structure of the Escherichia coli AcrAB-TolC multidrug Efflux Pump obtained by electron cryo-tomography and subtomogram averaging. The fully assembled Efflux Pump is observed in a closed state under conditions of antibiotic challenge and in an open state in the presence of AcrB inhibitor. We also observe intermediate AcrAB complexes without TolC and discover that AcrA contacts the peptidoglycan layer of the periplasm. Our data point to a sequential assembly process in living bacteria, beginning with formation of the AcrAB subcomplex and suggest domains to target with Efflux Pump inhibitors. Multidrug Efflux Pumps actively expel a wide range of toxic substrates from bacteria and play a major role in drug resistance. Here authors show the in situ structure of the Efflux Pump AcrAB-TolC obtained by electron cryo-tomography and subtomogram averaging.
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Structure of the AcrAB-TolC multidrug Efflux Pump
Nature, 2014Co-Authors: Dijun Du, Nathan R. James, Jarrod E. Voss, Ewa Klimont, Thelma Ohene-agyei, Henrietta Venter, Wah Chiu, Zhao Wang, Ben F. LuisiAbstract:The capacity of numerous bacterial species to tolerate antibiotics and other toxic compounds arises in part from the activity of energy-dependent transporters. In Gram-negative bacteria, many of these transporters form multicomponent 'Pumps' that span both inner and outer membranes and are driven energetically by a primary or secondary transporter component. A model system for such a Pump is the acridine resistance complex of Escherichia coli. This Pump assembly comprises the outer-membrane channel TolC, the secondary transporter AcrB located in the inner membrane, and the periplasmic AcrA, which bridges these two integral membrane proteins. The AcrAB-TolC Efflux Pump is able to transport vectorially a diverse array of compounds with little chemical similarity, thus conferring resistance to a broad spectrum of antibiotics. Homologous complexes are found in many Gram-negative species, including in animal and plant pathogens. Crystal structures are available for the individual components of the Pump and have provided insights into substrate recognition, energy coupling and the transduction of conformational changes associated with the transport process. However, how the subunits are organized in the Pump, their stoichiometry and the details of their interactions are not known. Here we present the pseudo-atomic structure of a complete multidrug Efflux Pump in complex with a modulatory protein partner from E. coli. The model defines the quaternary organization of the Pump, identifies key domain interactions, and suggests a cooperative process for channel assembly and opening. These findings illuminate the basis for drug resistance in numerous pathogenic bacterial species.
David C Hooper - One of the best experts on this subject based on the ideXlab platform.
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tet38 Efflux Pump contributes to fosfomycin resistance in staphylococcus aureus
Antimicrobial Agents and Chemotherapy, 2018Co-Authors: Que Chi Truongbolduc, Yan Wang, David C HooperAbstract:: Fosfomycin inhibits MurA following uptake by the GlpT transporter of glycerol-3-phosphate in Escherichia coli In Staphylococcus aureus, plasmid overexpression of the Tet38 Efflux Pump and a glpT mutant resulted in increased MICs and decreased accumulation of fosfomycin, with MICs affected by glycerol-3-phosphate. In contrast, a tet38 mutant had a lower MIC and increased accumulation of fosfomycin, suggesting that Tet38 acts as an Efflux transporter of fosfomycin.
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identification of a staphylococcus aureus Efflux Pump regulator using a dna protein affinity technique
Methods of Molecular Biology, 2018Co-Authors: Que Chi Truongbolduc, David C HooperAbstract:: In this chapter, we describe the step-by-step identification of a putative regulator protein and demonstrate the function of this protein as a repressor of the expression of a specific Efflux Pump, causing resistance to quinolones in Staphylococcus aureus. We show that the knockout gene mutant has an increase in transcript levels of the target Efflux Pump when compared to that of the S. aureus parental strain RN6390. We provide a detailed protocol that includes the identification of the DNA-binding transcriptional regulatory protein from S. aureus cell extracts using DNA sequences linked to magnetic beads. In addition, we describe the real-time qRT-PCR assays and MIC testing to evaluate the effects of the regulator on S. aureus drug resistance phenotype.
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Identification of a Staphylococcus aureus Efflux Pump Regulator Using a DNA–Protein Affinity Technique
Methods of Molecular Biology, 2017Co-Authors: Que Chi Truong-bolduc, David C HooperAbstract:In this chapter, we describe the step-by-step identification of a putative regulator protein and demonstrate the function of this protein as a repressor of the expression of a specific Efflux Pump, causing resistance to quinolones in Staphylococcus aureus. We show that the knockout gene mutant has an increase in transcript levels of the target Efflux Pump when compared to that of the S. aureus parental strain RN6390. We provide a detailed protocol that includes the identification of the DNA-binding transcriptional regulatory protein from S. aureus cell extracts using DNA sequences linked to magnetic beads. In addition, we describe the real-time qRT-PCR assays and MIC testing to evaluate the effects of the regulator on S. aureus drug resistance phenotype.
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oqxab encoding a multidrug Efflux Pump in human clinical isolates of enterobacteriaceae
Antimicrobial Agents and Chemotherapy, 2009Co-Authors: Chi Hye Park, George A Jacoby, Minghua Wang, David C HooperAbstract:The genes for multidrug Efflux Pump OqxAB, which is active on fluoroquinolones, were found in human clinical isolates on a plasmid in Escherichia coli and on the chromosome of Klebsiella pneumoniae. IS26-like sequences flanked the plasmid-mediated oqxAB genes, suggesting that they had been mobilized as part of a composite transposon.
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norc a new Efflux Pump regulated by mgra of staphylococcus aureus
Antimicrobial Agents and Chemotherapy, 2006Co-Authors: Que Chi Truongbolduc, Jacob Strahilevitz, David C HooperAbstract:NorC, a new Efflux Pump, like NorB, contributes to quinolone resistance that includes resistance to moxifloxacin and sparfloxacin in Staphylococcus aureus. norC expression, like that of norB and tet38, is negatively regulated by MgrA, and overexpression of both norC and norB contributes to the quinolone resistance phenotype of an mgrA mutant.
Muyuan Chen - One of the best experts on this subject based on the ideXlab platform.
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In situ structure and assembly of the multidrug Efflux Pump AcrAB-TolC.
Nature Communications, 2019Co-Authors: Muyuan Chen, Dijun Du, Zhili Yu, James M. Bell, Hans Wang, Isaac Forrester, Heather Villarreal, Joanita Jakana, Ben F. LuisiAbstract:Multidrug Efflux Pumps actively expel a wide range of toxic substrates from the cell and play a major role in intrinsic and acquired drug resistance. In Gram-negative bacteria, these Pumps form tripartite assemblies that span the cell envelope. However, the in situ structure and assembly mechanism of multidrug Efflux Pumps remain unknown. Here we report the in situ structure of the Escherichia coli AcrAB-TolC multidrug Efflux Pump obtained by electron cryo-tomography and subtomogram averaging. The fully assembled Efflux Pump is observed in a closed state under conditions of antibiotic challenge and in an open state in the presence of AcrB inhibitor. We also observe intermediate AcrAB complexes without TolC and discover that AcrA contacts the peptidoglycan layer of the periplasm. Our data point to a sequential assembly process in living bacteria, beginning with formation of the AcrAB subcomplex and suggest domains to target with Efflux Pump inhibitors. Multidrug Efflux Pumps actively expel a wide range of toxic substrates from bacteria and play a major role in drug resistance. Here authors show the in situ structure of the Efflux Pump AcrAB-TolC obtained by electron cryo-tomography and subtomogram averaging.
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In situ structure and assembly of the multidrug Efflux Pump AcrAB-TolC.
Nature communications, 2019Co-Authors: Xiaodong Shi, James M. Bell, Hans Wang, Isaac Forrester, Heather Villarreal, Joanita Jakana, Muyuan Chen, Ben F. LuisiAbstract:Multidrug Efflux Pumps actively expel a wide range of toxic substrates from the cell and play a major role in intrinsic and acquired drug resistance. In Gram-negative bacteria, these Pumps form tripartite assemblies that span the cell envelope. However, the in situ structure and assembly mechanism of multidrug Efflux Pumps remain unknown. Here we report the in situ structure of the Escherichia coli AcrAB-TolC multidrug Efflux Pump obtained by electron cryo-tomography and subtomogram averaging. The fully assembled Efflux Pump is observed in a closed state under conditions of antibiotic challenge and in an open state in the presence of AcrB inhibitor. We also observe intermediate AcrAB complexes without TolC and discover that AcrA contacts the peptidoglycan layer of the periplasm. Our data point to a sequential assembly process in living bacteria, beginning with formation of the AcrAB subcomplex and suggest domains to target with Efflux Pump inhibitors.
Qijing Zhang - One of the best experts on this subject based on the ideXlab platform.
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Bile salts modulate expression of the CmeABC multidrug Efflux Pump in Campylobacter jejuni.
Journal of bacteriology, 2005Co-Authors: Jun Lin, Cédric Cagliero, Sophie Payot, Baoqing Guo, Yi-wen Barton, Marie-christine Maurel, Qijing ZhangAbstract:CmeABC, a multidrug Efflux Pump, is involved in the resistance of Campylobacter jejuni to a broad spectrum of antimicrobial agents and is essential for Campylobacter colonization in animal intestine by mediating bile resistance. Previously, we have shown that expression of this Efflux Pump is under the control of a transcriptional repressor named CmeR. Inactivation of CmeR or mutation in the cmeABC promoter (PcmeABC) region derepresses cmeABC, leading to overexpression of this Efflux Pump. However, it is unknown if the expression of cmeABC can be conditionally induced by the substrates it extrudes. In this study, we examined the expression of cmeABC in the presence of various antimicrobial compounds. Although the majority of the antimicrobials tested did not affect the expression of cmeABC, bile salts drastically elevated the expression of this Efflux operon. The induction was observed with both conjugated and unconjugated bile salts and was in a dose- and time-dependent manner. Experiments using surface plasmon resonance demonstrated that bile salts inhibited the binding of CmeR to PcmeABC, suggesting that bile compounds are inducing ligands of CmeR. The interaction between bile salts and CmeR likely triggers conformational changes in CmeR, resulting in reduced binding affinity of CmeR to PcmeABC. Bile did not affect the transcription of cmeR, indicating that altered expression of cmeR is not a factor in bile-induced overexpression of cmeABC. In addition to the CmeR-dependent induction, some bile salts (e.g., taurocholate) also activated the expression of cmeABC by a CmeR-independent pathway. Consistent with the elevated production of CmeABC, the presence of bile salts in culture media resulted in increased resistance of Campylobacter to multiple antimicrobials. These findings reveal a new mechanism that modulates the expression of cmeABC and further support the notion that bile resistance is a natural function of CmeABC.
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cmer functions as a transcriptional repressor for the multidrug Efflux Pump cmeabc in campylobacter jejuni
Antimicrobial Agents and Chemotherapy, 2005Co-Authors: Masato Akiba, Orhan Sahin, Qijing ZhangAbstract:CmeABC, a resistance-nodulation-division (RND) type of Efflux Pump, contributes to Campylobacter resistance to a broad spectrum of antimicrobial agents and is also essential for Campylobacter colonization of the animal intestinal tract by mediation of bile resistance. As one of the main systems for Campylobacter adaptation to different environments, CmeABC is likely subject to control by regulatory elements. We describe the identification of a transcriptional repressor for CmeABC. Insertional mutagenesis of cmeR, an open reading frame immediately upstream of the cmeABC operon, resulted in overexpression of cmeABC, as determined by transcriptional fusion (PcmeABC-lacZ) and immunoblotting with CmeABC-specific antibodies. Overexpression of the Efflux Pump was correlated with a moderate increase in the level of resistance of the cmeR mutant to several antimicrobials. In vitro, recombinant CmeR bound specifically to the promoter region of cmeABC, precisely, to the inverted repeat sequences in the cmeABC promoter. A single nucleotide deletion between the two half sites of the inverted repeat reduced the level of CmeR binding to the promoter sequence and resulted in overexpression of cmeABC. Together, these findings indicate that cmeR encodes a transcriptional repressor that directly interacts with the cmeABC promoter and modulates the expression of cmeABC. Mutation either in CmeR or in the inverted repeat impedes the repression and leads to enhanced production of the MDR Efflux Pump.
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Bile salts modulate expression of the CmeABC multidrug Efflux Pump in Campylobacter jejuni
Journal of Bacteriology, 2005Co-Authors: Jun Lin, Cédric Cagliero, Sophie Payot, Baoqing Guo, Yi-wen Barton, Marie-christine Maurel, Qijing ZhangAbstract:Bile salts modulate expression of the CmeABC multidrug Efflux Pump in Campylobacter jejuni
