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Genetic Basis and Functional Consequences of Differential Expression of the CmeABC Efflux Pump in Campylobacter jejuni Isolates.

Grinnage-Pulley T, Zhang Q - PLoS ONE (2015)

Bottom Line: Several examined amino acid substitutions in CmeR did not affect its binding to the cmeABC promoter, but a mutation that led to C-terminal truncation of CmeR abolished its DNA-binding activity.Overexpression of cmeABC did not affect the susceptibility of C. jejuni to most tested antimicrobials except for chloramphenicol, but promoted the emergence of ciprofloxacin-resistant mutants under antibiotic selection.These results link CmeABC overexpression in natural C. jejuni isolates to various mutations and indicate that this phenotypic change promotes the emergence of antibiotic-resistant mutants under selection pressure.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Microbiology and Preventive Medicine, College of Veterinary Medicine, Iowa State University, Ames, Iowa, United States of America.

ABSTRACT
The CmeABC multidrug efflux transporter of Campylobacter jejuni plays a key role in antimicrobial resistance and is suppressed by CmeR, a transcriptional regulator of the TetR family. Overexpression of CmeABC has been observed in laboratory-generated mutants, but it is unknown if this phenotype occurs naturally in C. jejuni isolates and if it has any functional consequences. To answer these questions, expression of cmeABC in natural isolates obtained from broiler chickens, turkeys and humans was examined, and the genetic mechanisms and role of cmeABC differential expression in antimicrobial resistance was determined. Among the 64 C. jejuni isolates examined in this study, 43 and 21 were phenotypically identified as overexpression (OEL) and wild-type expression (WEL) levels. Representative mutations of the cmeABC promoter and/or CmeR-coding sequence were analyzed using electrophoretic mobility shift assays and transcriptional fusion assays. Reduced CmeR binding to the mutated cmeABC promoter sequences or decreased CmeR levels increased cmeABC expression. Several examined amino acid substitutions in CmeR did not affect its binding to the cmeABC promoter, but a mutation that led to C-terminal truncation of CmeR abolished its DNA-binding activity. Interestingly, some OEL isolates harbored no mutations in known regulatory elements, suggesting that cmeABC is also regulated by unidentified mechanisms. Overexpression of cmeABC did not affect the susceptibility of C. jejuni to most tested antimicrobials except for chloramphenicol, but promoted the emergence of ciprofloxacin-resistant mutants under antibiotic selection. These results link CmeABC overexpression in natural C. jejuni isolates to various mutations and indicate that this phenotypic change promotes the emergence of antibiotic-resistant mutants under selection pressure. Thus, differential expression of CmeABC may facilitate Campylobacter adaptation to antibiotic treatments.

No MeSH data available.


Differential expression of CmeABC in clinical Campylobacter jejuni isolates.Expression was determined by immunoblotting of whole cell proteins from NCTC 11168 (lane 1), clinical isolates (lanes 2–11), and 11168ΔcmeR (lane 12) with anti-CmeB, anti-CmeC, anti-CmeA, and anti-major outer membrane protein (MOMP) antibodies. These broiler isolates in lanes 2 to 11 are CB1:6, CB1:14, CB 1:18, CB2:6, CB2:8, CB2:11, CB3:1, CB3:5, CB 3:14, and CB3:21. Isolates CB2:8, CB2:11, CB 3:1, CB3:5, CB3:14 and CB3:21 (lanes 6–11) were designated as having overexpression levels of CmeABC and isolates CB1:6, CB1:14, CB 1:18, and CB2:6 (lanes 2–5) as having wild-type levels of CmeABC. The major outer membrane protein (MOMP) was used as an internal control.
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pone.0131534.g001: Differential expression of CmeABC in clinical Campylobacter jejuni isolates.Expression was determined by immunoblotting of whole cell proteins from NCTC 11168 (lane 1), clinical isolates (lanes 2–11), and 11168ΔcmeR (lane 12) with anti-CmeB, anti-CmeC, anti-CmeA, and anti-major outer membrane protein (MOMP) antibodies. These broiler isolates in lanes 2 to 11 are CB1:6, CB1:14, CB 1:18, CB2:6, CB2:8, CB2:11, CB3:1, CB3:5, CB 3:14, and CB3:21. Isolates CB2:8, CB2:11, CB 3:1, CB3:5, CB3:14 and CB3:21 (lanes 6–11) were designated as having overexpression levels of CmeABC and isolates CB1:6, CB1:14, CB 1:18, and CB2:6 (lanes 2–5) as having wild-type levels of CmeABC. The major outer membrane protein (MOMP) was used as an internal control.

Mentions: Initial screening for phenotypic classification of CmeABC expression was done through immunoblotting (Fig 1) and real time RT-PCR for expression of cmeB. Analysis of the 64 C. jejuni isolates for cmeB expression identified 43 isolates with overexpression levels (OEL) of CmeABC and 21 isolates with wild-type expression levels (WEL) of CmeABC. The region spanning from cmeR to cmeA was sequenced for all OEL isolates, 4 of the WEL isolates, and C. jejuni strain 81–176 to identify genetic mutations that were potentially associated with the differential cmeABC expression (S1 Table). All isolates except 1 OEL isolate harbored mutations in the sequenced region compared to the same region in strain NCTC 11168.


Genetic Basis and Functional Consequences of Differential Expression of the CmeABC Efflux Pump in Campylobacter jejuni Isolates.

Grinnage-Pulley T, Zhang Q - PLoS ONE (2015)

Differential expression of CmeABC in clinical Campylobacter jejuni isolates.Expression was determined by immunoblotting of whole cell proteins from NCTC 11168 (lane 1), clinical isolates (lanes 2–11), and 11168ΔcmeR (lane 12) with anti-CmeB, anti-CmeC, anti-CmeA, and anti-major outer membrane protein (MOMP) antibodies. These broiler isolates in lanes 2 to 11 are CB1:6, CB1:14, CB 1:18, CB2:6, CB2:8, CB2:11, CB3:1, CB3:5, CB 3:14, and CB3:21. Isolates CB2:8, CB2:11, CB 3:1, CB3:5, CB3:14 and CB3:21 (lanes 6–11) were designated as having overexpression levels of CmeABC and isolates CB1:6, CB1:14, CB 1:18, and CB2:6 (lanes 2–5) as having wild-type levels of CmeABC. The major outer membrane protein (MOMP) was used as an internal control.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4488513&req=5

pone.0131534.g001: Differential expression of CmeABC in clinical Campylobacter jejuni isolates.Expression was determined by immunoblotting of whole cell proteins from NCTC 11168 (lane 1), clinical isolates (lanes 2–11), and 11168ΔcmeR (lane 12) with anti-CmeB, anti-CmeC, anti-CmeA, and anti-major outer membrane protein (MOMP) antibodies. These broiler isolates in lanes 2 to 11 are CB1:6, CB1:14, CB 1:18, CB2:6, CB2:8, CB2:11, CB3:1, CB3:5, CB 3:14, and CB3:21. Isolates CB2:8, CB2:11, CB 3:1, CB3:5, CB3:14 and CB3:21 (lanes 6–11) were designated as having overexpression levels of CmeABC and isolates CB1:6, CB1:14, CB 1:18, and CB2:6 (lanes 2–5) as having wild-type levels of CmeABC. The major outer membrane protein (MOMP) was used as an internal control.
Mentions: Initial screening for phenotypic classification of CmeABC expression was done through immunoblotting (Fig 1) and real time RT-PCR for expression of cmeB. Analysis of the 64 C. jejuni isolates for cmeB expression identified 43 isolates with overexpression levels (OEL) of CmeABC and 21 isolates with wild-type expression levels (WEL) of CmeABC. The region spanning from cmeR to cmeA was sequenced for all OEL isolates, 4 of the WEL isolates, and C. jejuni strain 81–176 to identify genetic mutations that were potentially associated with the differential cmeABC expression (S1 Table). All isolates except 1 OEL isolate harbored mutations in the sequenced region compared to the same region in strain NCTC 11168.

Bottom Line: Several examined amino acid substitutions in CmeR did not affect its binding to the cmeABC promoter, but a mutation that led to C-terminal truncation of CmeR abolished its DNA-binding activity.Overexpression of cmeABC did not affect the susceptibility of C. jejuni to most tested antimicrobials except for chloramphenicol, but promoted the emergence of ciprofloxacin-resistant mutants under antibiotic selection.These results link CmeABC overexpression in natural C. jejuni isolates to various mutations and indicate that this phenotypic change promotes the emergence of antibiotic-resistant mutants under selection pressure.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Microbiology and Preventive Medicine, College of Veterinary Medicine, Iowa State University, Ames, Iowa, United States of America.

ABSTRACT
The CmeABC multidrug efflux transporter of Campylobacter jejuni plays a key role in antimicrobial resistance and is suppressed by CmeR, a transcriptional regulator of the TetR family. Overexpression of CmeABC has been observed in laboratory-generated mutants, but it is unknown if this phenotype occurs naturally in C. jejuni isolates and if it has any functional consequences. To answer these questions, expression of cmeABC in natural isolates obtained from broiler chickens, turkeys and humans was examined, and the genetic mechanisms and role of cmeABC differential expression in antimicrobial resistance was determined. Among the 64 C. jejuni isolates examined in this study, 43 and 21 were phenotypically identified as overexpression (OEL) and wild-type expression (WEL) levels. Representative mutations of the cmeABC promoter and/or CmeR-coding sequence were analyzed using electrophoretic mobility shift assays and transcriptional fusion assays. Reduced CmeR binding to the mutated cmeABC promoter sequences or decreased CmeR levels increased cmeABC expression. Several examined amino acid substitutions in CmeR did not affect its binding to the cmeABC promoter, but a mutation that led to C-terminal truncation of CmeR abolished its DNA-binding activity. Interestingly, some OEL isolates harbored no mutations in known regulatory elements, suggesting that cmeABC is also regulated by unidentified mechanisms. Overexpression of cmeABC did not affect the susceptibility of C. jejuni to most tested antimicrobials except for chloramphenicol, but promoted the emergence of ciprofloxacin-resistant mutants under antibiotic selection. These results link CmeABC overexpression in natural C. jejuni isolates to various mutations and indicate that this phenotypic change promotes the emergence of antibiotic-resistant mutants under selection pressure. Thus, differential expression of CmeABC may facilitate Campylobacter adaptation to antibiotic treatments.

No MeSH data available.