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Principal Investigator: EDWARD W YU
Organization: IOWA STATE UNIVERSITY
Fiscal Year: 2024
Award: $626,007
Funding agency: National Institute of Allergy and Infectious Diseases
PROJECT SUMMARY
Bacterial multidrug efflux transporters confer resistance to structurally diverse antimicrobials, which is one of the
major causes for clinical treatment failure. Campylobacter jejuni is a major enteric pathogen and has developed
various mechanisms for antibiotic resistance. Recently, both the World Health Organization and the Centers for
Disease Prevention and Control have designated Campylobacter as a “serious antibiotic resistance threat”. In
Campylobacter, the multidrug efflux pump CmeABC, a RND-type efflux system, plays a key role in the resistance
to various antimicrobials and in intestinal colonization by mediating bile resistance. In CmeABC, the three
proteins assemble to form a powerful tripartite machinery, allowing direct efflux of substrates across both
membranes of the Gram-negative cellular envelope. CmeABC is essential for C. jejuni as it has an important
natural function for bile resistance, which is required for Campylobacter colonization in animal intestine. Typically,
CmeABC requires to function cooperatively with other resistance mechanisms (such as target mutations) to
confer clinically relevant antibiotic resistance. However, a “super” resistance-enhancing variant of CmeABC (RE-
CmeABC) has recently emerged in clinical isolates of C. jejuni. This variant pump has a distinct CmeB sequence
and is much more potent in conferring multidrug resistance. Additionally, we found that RE-CmeABC is
increasingly prevalent in clinical isolates and mediates exceedingly high-level resistance to fluoroquinolone, a
clinically important antibiotic for treating campylobacteriosis. Our preliminary data further suggest the enhanced
efflux function of RE-CmeABC is due to sequence variations in the RE-CmeB transporter. To begin to understand
how CmeABC extrudes antimicrobials, we have initiated work to decipher the structural basis of CmeABC-
mediated efflux. Our preliminary crystallization data indicate that CmeB forms a homotrimer, where individual
protomers bind to and export substrates independently. Based on the solid preliminary data, we propose in this
application to pursue three specific aims to 1) identify the specific mutations responsible for the enhanced efflux
function in RE-CmeABC, 2) define the structural basis of CmeB-mediated antibiotic efflux and how sequence
polymorphisms affect the structure-function relationship, and 3) determine the horizontal spread of RE-cmeABC
and its impact on C. jejuni fitness in the absence and presence of antibiotic selection pressure. We will use a
high throughput mapping strategy, a CRISPR-Cas9 based technique for efficient gene editing and replacement,
in vitro and animal model systems, x-ray crystallography, and single-molecule FRET to achieve the goals of the
three specific aims. The team of investigators have strong and complementary expertise, and are uniquely
positioned to conduct the proposed work, which is expected to reveal novel mechanisms used by an RND-type
transporter for antibiotic extrusion and enhanced multidrug resistance. This gained knowledge should be
transferrable to other bacterial efflux pumps, and the findings may facilitate the development of new strategies
to control the emergence and spread of multidrug resistant Campylobacter.
Terms: <Affect><Amino Acids><Animal Model><Animal Models and Related Studies><Animals><Antibiotic Agents><Antibiotic Drugs><Antibiotic Resistance><Antibiotics><Bile><Bile Juice><Bile fluid><Binding><Biologic Models><Biological Models><C jejuni><C. jejuni><CRISPR approach><CRISPR based approach><CRISPR method><CRISPR methodology><CRISPR technique><CRISPR technology><CRISPR tools><CRISPR-CAS-9><CRISPR-based method><CRISPR-based technique><CRISPR-based technology><CRISPR-based tool><CRISPR/CAS approach><CRISPR/Cas method><CRISPR/Cas technology><CRISPR/Cas9><CRISPR/Cas9 technology><Campylobacter><Campylobacter infection><Campylobacter jejuni><Campylobacteriosis><Carbapenems><Cas nuclease technology><Centers for Disease Control><Centers for Disease Control and Prevention><Centers for Disease Control and Prevention (U.S.)><Chimera Protein><Chimeric Proteins><Clinical><Clinical Treatment><Clustered Regularly Interspaced Short Palindromic Repeats approach><Clustered Regularly Interspaced Short Palindromic Repeats method><Clustered Regularly Interspaced Short Palindromic Repeats methodology><Clustered Regularly Interspaced Short Palindromic Repeats technique><Clustered Regularly Interspaced Short Palindromic Repeats technology><Co-culture><Cocultivation><Coculture><Coculture Techniques><Colimycin><Colisticin><Colistin><Crystallization><Culture Media><Data><Data Bases><Databases><Development><Diarrhea><Drug Efflux><Drug-resistant Campylobacter><Drugs><Fusion Protein><GI colonization><GeneHomolog><Genes><Genetic><Genetic Alteration><Genetic Change><Genetic Polymorphism><Genetic defect><Goals><Homolog><Homologous Gene><Homologue><Human><Image><In Vitro><Individual><Intervention><Intervention Strategies><Intestinal><Intestines><Investigators><Ion Channel><Ionic Channels><Knowledge><Ligands><Maps><Mediating><Medication><Membrane><Membrane Channels><Membrane Fusion><Membrane Transport Proteins><Membrane Transporters><Miscellaneous Antibiotic><Model System><Modern Man><Molecular Interaction><Multi-Drug Resistance><Multidrug Resistance><Multiple Drug Resistance><Multiple Drug Resistant><Mutation><Pathogen detection><Periplasmic Space><Pharmaceutical Preparations><Physiology><Play><Polymyxin E><Position><Positioning Attribute><Prevalence><Proteins><Protomer><Public Health><Pump><Research Personnel><Researchers><Resistance><Resistance to Multi-drug><Resistance to Multidrug><Resistance to Multiple Drug><Resistance to antibiotics><Resistant to Multiple Drug><Resistant to antibiotics><Resistant to multi-drug><Resistant to multidrug><Single Crystal Diffraction><Solid><Structure><Structure-Activity Relationship><System><Techniques><Transmission><Treatment Failure><United States Centers for Disease Control><United States Centers for Disease Control and Prevention><Variant><Variation><Work><World Health Organization><X Ray Crystallographies><X-Ray Crystallography><X-Ray Diffraction Crystallography><X-Ray/Neutron Crystallography><Xray Crystallography><aminoacid><anti-microbial><antibiotic drug resistance><antibiotic efflux><antibiotic export><antibiotic resistant><antimicrobial><bacteria pathogen><bacterial fitness><bacterial pathogen><bowel><chemical structure function><clinical relevance><clinically relevant><co-infection><coinfection><conformational conversion><conformational transition><coping><data base><develop therapy><developmental><drug/agent><efflux pump><enteral pathogen><enteric pathogen><enteropathogen><entire genome><experiment><experimental research><experimental study><experiments><fitness><fluoroquinolone resistance><fluoroquinolone resistant><full genome><gastrointestinal tract colonization><genome mutation><genome sequencing><growth media><gut colonization><human pathogen><imaging><in vivo><insight><intervention development><interventional strategy><intestinal colonization><intestinal pathogen><intestine pathogen><membrane structure><model of animal><multi-drug resistant><multidrug resistant><novel><pathogenic bacteria><periplasm><polymorphism><pressure><resistance mechanism><resistance to fluoroquinolone><resistant><resistant mechanism><resistant to fluoroquinolone><single-molecule FRET><single-molecule fluorescence resonance energy transfer><smFRET><structure function relationship><therapy development><therapy failure><transmission process><treatment development><trial regimen><trial treatment><whole genome>