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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.


Expression of CmeR in various isolates and its correlation with CmeABC expression.(A) Immunoblotting of whole cell proteins from NCTC 11168 (lane 1), clinical isolates (lanes 2–9), and 11168ΔcmeR (lane 10) with the anti-CmeR antibody. The clinical isolates in lanes 2 to 9 are M63885, CT9:7, CB2:6, CB2:8, CB2:11, S13530, T37957A, and X7199, respectively. (B) Immunoblotting of whole cell proteins from 11168ΔcmeR (lane 1), CT2:2 (lane 2), and NCTC 11168 (lanes 3) with anti-CmeR, anti-CmeB, and anti-CmeA antibodies.
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pone.0131534.g004: Expression of CmeR in various isolates and its correlation with CmeABC expression.(A) Immunoblotting of whole cell proteins from NCTC 11168 (lane 1), clinical isolates (lanes 2–9), and 11168ΔcmeR (lane 10) with the anti-CmeR antibody. The clinical isolates in lanes 2 to 9 are M63885, CT9:7, CB2:6, CB2:8, CB2:11, S13530, T37957A, and X7199, respectively. (B) Immunoblotting of whole cell proteins from 11168ΔcmeR (lane 1), CT2:2 (lane 2), and NCTC 11168 (lanes 3) with anti-CmeR, anti-CmeB, and anti-CmeA antibodies.

Mentions: Several DNA polymorphisms were detected in the cmeR gene of the clinical isolates, resulting in amino acid changes in this regulatory protein (Table 3). Immunoblotting of whole cell proteins was performed to determine the CmeR expression level from 8 OEL isolates and 1 WEL isolate harboring representative mutations. The anti-CmeR antibody detected the CmeR protein from 8 of the 9 cmeR mutants (Fig 4A). The remaining cmeR mutant isolate (CT2:2) did not produce a band reactive with the antibody (Fig 4B, lane 2), suggesting that the CmeR protein was not translated in this isolate. CT2:2 contained a T insertion after base 583 in cmeR, resulting in a frame shift and premature truncation (Table 3). Additional immunoblotting failed to detect any portion of the truncated CmeR from CT 2:2.


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)

Expression of CmeR in various isolates and its correlation with CmeABC expression.(A) Immunoblotting of whole cell proteins from NCTC 11168 (lane 1), clinical isolates (lanes 2–9), and 11168ΔcmeR (lane 10) with the anti-CmeR antibody. The clinical isolates in lanes 2 to 9 are M63885, CT9:7, CB2:6, CB2:8, CB2:11, S13530, T37957A, and X7199, respectively. (B) Immunoblotting of whole cell proteins from 11168ΔcmeR (lane 1), CT2:2 (lane 2), and NCTC 11168 (lanes 3) with anti-CmeR, anti-CmeB, and anti-CmeA antibodies.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4488513&req=5

pone.0131534.g004: Expression of CmeR in various isolates and its correlation with CmeABC expression.(A) Immunoblotting of whole cell proteins from NCTC 11168 (lane 1), clinical isolates (lanes 2–9), and 11168ΔcmeR (lane 10) with the anti-CmeR antibody. The clinical isolates in lanes 2 to 9 are M63885, CT9:7, CB2:6, CB2:8, CB2:11, S13530, T37957A, and X7199, respectively. (B) Immunoblotting of whole cell proteins from 11168ΔcmeR (lane 1), CT2:2 (lane 2), and NCTC 11168 (lanes 3) with anti-CmeR, anti-CmeB, and anti-CmeA antibodies.
Mentions: Several DNA polymorphisms were detected in the cmeR gene of the clinical isolates, resulting in amino acid changes in this regulatory protein (Table 3). Immunoblotting of whole cell proteins was performed to determine the CmeR expression level from 8 OEL isolates and 1 WEL isolate harboring representative mutations. The anti-CmeR antibody detected the CmeR protein from 8 of the 9 cmeR mutants (Fig 4A). The remaining cmeR mutant isolate (CT2:2) did not produce a band reactive with the antibody (Fig 4B, lane 2), suggesting that the CmeR protein was not translated in this isolate. CT2:2 contained a T insertion after base 583 in cmeR, resulting in a frame shift and premature truncation (Table 3). Additional immunoblotting failed to detect any portion of the truncated CmeR from CT 2:2.

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.