scholarly journals Target optimization for peptide nucleic acid (PNA)-mediated antisense inhibition of the CmeABC multidrug efflux pump in Campylobacter jejuni

2013 ◽  
Vol 69 (2) ◽  
pp. 375-380 ◽  
Author(s):  
E. Oh ◽  
Q. Zhang ◽  
B. Jeon
Keyword(s):  
Nucleic Acid ◽  
Campylobacter Jejuni ◽  
Peptide Nucleic Acid ◽  
Efflux Pump ◽  
Antisense Inhibition ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump

scholarly journals CmeR Functions as a Pleiotropic Regulator and Is Required for Optimal Colonization of Campylobacter jejuni In Vivo

2008 ◽  
Vol 190 (6) ◽  
pp. 1879-1890 ◽  
Author(s):  
Baoqing Guo ◽  
Ying Wang ◽  
Feng Shi ◽  
Yi-Wen Barton ◽  
Paul Plummer ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Capsular Polysaccharide ◽  
Efflux Pump ◽  
Loss Of Function ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Reverse Transcription Pcr ◽  
Dicarboxylate Transport

ABSTRACT CmeR functions as a transcriptional repressor modulating the expression of the multidrug efflux pump CmeABC in Campylobacter jejuni. To determine if CmeR also regulates other genes in C. jejuni, we compared the transcriptome of the cmeR mutant with that of the wild-type strain using a DNA microarray. This comparison identified 28 genes that showed a ≥2-fold change in expression in the cmeR mutant. Independent real-time quantitative reverse transcription-PCR experiments confirmed 27 of the 28 differentially expressed genes. The CmeR-regulated genes encode membrane transporters, proteins involved in C4-dicarboxylate transport and utilization, enzymes for biosynthesis of capsular polysaccharide, and hypothetical proteins with unknown functions. Among the genes whose expression was upregulated in the cmeR mutant, Cj0561c (encoding a putative periplasmic protein) showed the greatest increase in expression. Subsequent experiments demonstrated that this gene is strongly repressed by CmeR. The presence of the known CmeR-binding site, an inverted repeat of TGTAAT, in the promoter region of Cj0561c suggests that CmeR directly inhibits the transcription of Cj0561c. Similar to expression of cmeABC, transcription of Cj0561c is strongly induced by bile compounds, which are normally present in the intestinal tracts of animals. Inactivation of Cj0561c did not affect the susceptibility of C. jejuni to antimicrobial compounds in vitro but reduced the fitness of C. jejuni in chickens. Loss-of-function mutation of cmeR severely reduced the ability of C. jejuni to colonize chickens. Together, these findings indicate that CmeR governs the expression of multiple genes with diverse functions and is required for Campylobacter adaptation in the chicken host.

Global distribution, dissemination and overexpression of potent multidrug efflux pump RE-CmeABC in Campylobacter jejuni

2020 ◽  
Author(s):  
Hong Yao ◽  
Wenbo Zhao ◽  
Dian Jiao ◽  
Stefan Schwarz ◽  
Rongmin Zhang ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Campylobacter Jejuni ◽  
Promoter Region ◽  
Spanning Trees ◽  
Efflux Pump ◽  
Global Distribution ◽  
Multidrug Efflux ◽  
Rt Pcr ◽  
Multidrug Efflux Pump ◽  
Close Relationship

Abstract Objectives To investigate the global distribution, dissemination and overexpression of RE-CmeABC in Campylobacter jejuni. Methods WGS information for 433 RE-cmeABC-positive C. jejuni isolates (including 18 isolates sequenced in this study and 415 isolates from GenBank) was used for the generation of minimum-spanning trees with STs. WGS information for 95 representative RE-cmeABC-positive C. jejuni isolates was used for phylogenetic analysis. RT–PCR was conducted to evaluate the association between inverted repeat (IR) sequence diversity in the RE-CmeABC promoter region and RE-cmeABC gene expression. Results WGS analysis revealed the global distribution of RE-cmeABC among C. jejuni from more than 10 countries. MLST results indicated that various STs were involved in the dissemination of RE-cmeABC, with ST2109 being the most predominant ST. Phylogenetic analysis revealed the close relationship between RE-cmeABC-carrying C. jejuni isolates from poultry and humans. The IR polymorphism in the RE-CmeABC promoter region is associated with the overexpression of RE-cmeABC, which was demonstrated experimentally by RT–PCR. Conclusions To the best of our knowledge, our analysis represents the first view of the global distribution of RE-CmeABC, which is horizontally transferable and diffused regionally in a clonal manner. The close relationship of RE-cmeABC-positive C. jejuni from poultry and humans supports the potential of these isolates for zoonotic transmission. Overexpressed RE-CmeABC in C. jejuni will increase the fitness of the corresponding bacteria and be of advantage under antimicrobial selection.

scholarly journals CmeABC Functions as a Multidrug Efflux System in Campylobacter jejuni

2002 ◽  
Vol 46 (7) ◽  
pp. 2124-2131 ◽  
Author(s):  
Jun Lin ◽  
Linda Overbye Michel ◽  
Qijing Zhang
Keyword(s):  
Campylobacter Jejuni ◽  
Antimicrobial Agents ◽  
Efflux Pump ◽  
Wild Type ◽  
Multidrug Efflux ◽  
Intrinsic Resistance ◽  
Multidrug Efflux Pump ◽  
Efflux Pump Inhibitor ◽  
Gram Negative ◽  
Multidrug Efflux Pumps

ABSTRACT Campylobacter jejuni, a gram-negative organism causing gastroenteritis in humans, is increasingly resistant to antibiotics. However, little is known about the drug efflux mechanisms in this pathogen. Here we characterized an efflux pump encoded by a three-gene operon (designated cmeABC) that contributes to multidrug resistance in C. jejuni 81-176. CmeABC shares significant sequence and structural homology with known tripartite multidrug efflux pumps in other gram-negative bacteria, and it consists of a periplasmic fusion protein (CmeA), an inner membrane efflux transporter belonging to the resistance-nodulation-cell division superfamily (CmeB), and an outer membrane protein (CmeC). Immunoblotting using CmeABC-specific antibodies demonstrated that cmeABC was expressed in wild-type 81-176; however, an isogenic mutant (9B6) with a transposon insertion in the cmeB gene showed impaired production of CmeB and CmeC. Compared to wild-type 81-176, 9B6 showed a 2- to 4,000-fold decrease in resistance to a range of antibiotics, heavy metals, bile salts, and other antimicrobial agents. Accumulation assays demonstrated that significantly more ethidium bromide and ciprofloxacin accumulated in mutant 9B6 than in wild-type 81-176. Addition of carbonyl cyanide m-chlorophenylhydrazone, an efflux pump inhibitor, increased the accumulation of ciprofloxacin in wild-type 81-176 to the level of mutant 9B6. PCR and immunoblotting analysis also showed that cmeABC was broadly distributed in various C. jejuni isolates and constitutively expressed in wild-type strains. Together, these findings formally establish that CmeABC functions as a tripartite multidrug efflux pump that contributes to the intrinsic resistance of C. jejuni to a broad range of structurally unrelated antimicrobial agents.

scholarly journals N-linked glycans confer protein stability and modulate multidrug efflux pump assemblyin Campylobacter jejuni

2019 ◽  
Author(s):  
Sherif Abouelhadid ◽  
John Raynes ◽  
Tam T.T. Bui ◽  
Jon Cuccui ◽  
Brendan W. Wren
Keyword(s):  
Campylobacter Jejuni ◽  
Protein Modification ◽  
Efflux Pump ◽  
Protein Complexes ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Post Translational Modifications ◽  
Protein Protein Interaction ◽  
Domains Of Life ◽  
Glycosylation Pathway

AbstractIt is now apparent that nearly all bacteria species have at least a single glycosylation system, but the direct effect(s) of these protein post translational modifications are unresolved. In this study, we used the generalN-linked glycosylation pathway fromCampylobacter jejunito investigate the biophysical roles of protein modification on the CmeABC multidrug efflux pump complex. The study reveals the multifunctional role ofN-linked glycans in enhancing protein thermostability, stabilising protein complexes and the promotion of protein-protein interaction. Our findings demonstrate, for the first time, that regardless of glycan diversification among domains of life,N-linked glycans confer a common evolutionary intrinsic role.

N-glycosylation of the CmeABC multidrug efflux pump is needed for optimal function in Campylobacter jejuni

Glycobiology ◽  
2019 ◽  
Vol 30 (2) ◽  
pp. 105-119 ◽  
Author(s):  
Rajinder K Dubb ◽  
Harald Nothaft ◽  
Bernadette Beadle ◽  
Michele R Richards ◽  
Christine M Szymanski
Keyword(s):  
Campylobacter Jejuni ◽  
Protein Modification ◽  
Efflux Pump ◽  
Protein Structures ◽  
Protein Glycosylation ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Modification System ◽  
Glycosylation Pathway

Abstract Campylobacter jejuni is a prevalent gastrointestinal pathogen associated with increasing rates of antimicrobial resistance development. It was also the first bacterium demonstrated to possess a general N-linked protein glycosylation pathway capable of modifying > 80 different proteins, including the primary Campylobacter multidrug efflux pump, CmeABC. Here we demonstrate that N-glycosylation is necessary for the function of the efflux pump and may, in part, explain the evolutionary pressure to maintain this protein modification system. Mutants of cmeA in two common wildtype (WT) strains are highly susceptible to erythromycin (EM), ciprofloxacin and bile salts when compared to the isogenic parental strains. Complementation of the cmeA mutants with the native cmeA allele restores the WT phenotype, whereas expression of a cmeA allele with point mutations in both N-glycosylation sites is comparable to the cmeA mutants. Moreover, loss of CmeA glycosylation leads to reduced chicken colonization levels similar to the cmeA knock-out strain, while complementation fully restores colonization. Reconstitution of C. jejuni CmeABC into Escherichia coli together with the C. jejuni N-glycosylation pathway increases the EM minimum inhibitory concentration and decreases ethidium bromide accumulation when compared to cells lacking the pathway. Molecular dynamics simulations reveal that the protein structures of the glycosylated and non-glycosylated CmeA models do not vary from one another, and in vitro studies show no change in CmeA multimerization or peptidoglycan association. Therefore, we conclude that N-glycosylation has a broader influence on CmeABC function most likely playing a role in complex stability.

scholarly journals Bile Salts Modulate Expression of the CmeABC Multidrug Efflux Pump in Campylobacter jejuni

2005 ◽  
Vol 187 (21) ◽  
pp. 7417-7424 ◽  
Author(s):  
Jun Lin ◽  
Cédric Cagliero ◽  
Baoqing Guo ◽  
Yi-Wen Barton ◽  
Marie-Christine Maurel ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Conformational Changes ◽  
Bile Salts ◽  
Antimicrobial Agents ◽  
Efflux Pump ◽  
Dependent Manner ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Altered Expression ◽  
Bile Resistance

ABSTRACT 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 (P cmeABC ) 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 P cmeABC , 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 P cmeABC . 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.

scholarly journals Quantitative Analyses Reveal Novel Roles forN-Glycosylation in a Major Enteric Bacterial Pathogen

mBio ◽  
2019 ◽  
Vol 10 (2) ◽  
Author(s):  
Sherif Abouelhadid ◽  
Simon J. North ◽  
Paul Hitchen ◽  
Prerna Vohra ◽  
Cosmin Chintoan-Uta ◽  
...  
Keyword(s):  
Quality Control ◽  
Campylobacter Jejuni ◽  
Posttranslational Modification ◽  
Protein Quality ◽  
Efflux Pump ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Content Type ◽  
General Protein

ABSTRACTIn eukaryotes, glycosylation plays a role in proteome stability, protein quality control, and modulating protein function; however, similar studies in bacteria are lacking. Here, we investigate the roles of general protein glycosylation systems in bacteria using the enteropathogenCampylobacter jejunias a well-defined example. By using a quantitative proteomic strategy, we were able to monitor changes in theC. jejuniproteome when glycosylation is disrupted. We demonstrate that inC. jejuni, N-glycosylation is essential to maintain proteome stability and protein quality control. These findings guided us to investigate the role ofN-glycosylation in modulating bacterial cellular activities. In glycosylation-deficientC. jejuni, the multidrug efflux pump and electron transport pathways were significantly impaired. We demonstrate thatin vivo, fully glycosylation-deficientC. jejunibacteria were unable to colonize its natural avian host. These results provide the first evidence of a link between proteome stability and complex functions via a bacterial general glycosylation system.IMPORTANCEAdvances in genomics and mass spectrometry have revealed several types of glycosylation systems in bacteria. However, why bacterial proteins are modified remains poorly defined. Here, we investigated the role of generalN-linked glycosylation in a major food poisoning bacterium,Campylobacter jejuni. The aim of this study is to delineate the direct and indirect effects caused by disrupting this posttranslational modification. To achieve this, we employed a quantitative proteomic strategy to monitor alterations in theC. jejuniproteome. Our quantitative proteomic results linked general proteinN-glycosylation to maintaining proteome stability. Functional analyses revealed novel roles for bacterialN-glycosylation in modulating multidrug efflux pump, enhancing nitrate reduction activity, and promoting host-microbe interaction. This work provides insights on the importance of general glycosylation in proteins in maintaining bacterial physiology, thus expanding our knowledge of the emergence of posttranslational modification in bacteria.

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