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We currently focus on studying bacterial antibiotic resistance mediated by active multi-drug efflux transporters. Our long-term goal is to elucidate the structures and fundamental mechanisms that give rise to multiple drug recognition and extrusion in these multi-drug transporters. In addition, our laboratory addresses fundamental questions regarding the nature of multi-drug recognition and gene regulation in transcriptional regulators. These regulators control the expression of multi-drug efflux transporters in bacteria by sensing the presence of a variety of structurally unrelated toxic compounds in the environment. Our experimental approach mainly utilizes x-ray crystallography to elucidate toxic chemical binding and export by these membrane efflux transporters.
Antibiotic efflux transporters are fundamental components in both intrinsic and acquired bacterial resistance to antimicrobials. Neisseria gonorrhoeae, a bacterium that is responsible for causing the infectious disease gonorrhea, harbors multiple antibiotic efflux transporters from a number of different families. In April 2007, the Centers for Diseases Control and Prevention (CDC) officially added Neisseria gonorrhoeae to the list of ‘Super Bugs’ that are now resistant to common antibiotics. The primary target of this project is the N. gonorrhoeae NorM transmembrane efflux pump, which is a member of the most recently classified multidrug and toxic compound extrusion (MATE) family. The structure and function of NorM is similar to YdhE from Escherichia coli. To determine the capacity of these two transporters to confer multiple antimicrobial resistances, we previously cloned, expressed, and purified these two efflux proteins, and characterized their functions both in vivo and vitro. In this study, we found that E. coli expressing norM and ydhE showed elevated (two-fold or greater) resistance to several antimicrobial agents, including fluoroquinolones, ethidium bromide, rhodamine 6G, acriflavin, proflavin, crystal violet, barberine, doxorubicin, novobiocin, enoxzcin, and tetraphenylphosphonium chloride. When expressed in E. coli, both transporters reduced ethidium bromide and norfloxacin accumulation through a mechanism requiring the proton-motive force and direct measurements of efflux confirmed that NorM behaves as a Na+-dependent transporter. The capacity of NorM and YdhE to recognize structurally divergent compounds was directly confirmed using steady-state fluorescence polarization assays. These results revealed, for the first time, that these transporters bind antimicrobials with dissociation constants in the micromolar region. We recently also crystallized the N. gonorrhoeae NorM multidrug efflux pump using hanging-drop vapor diffusion. Despite these recent advances, no detailed structure-function information is available for the MATE family of transporters. To fill these important gaps in our understanding of the active efflux systems in the MATE family, we thus plan to determine the X-ray structure of the N. gonorrhoeae NorM multidrug efflux pump, and identify important residues for multidrug recognition/extrusion in N. gonorrhoeae NorM and E. coli YdhE in this research project.
Bacteria such as Escherichia coli have developed various mechanisms to
overcome toxic environments that are unfavorable for their survival. One
important strategy that bacteria use to subvert toxic compounds, including
heavy-metal ions, is the expression of membrane transporters that recognize and
actively export these substances out of bacterial cells, thereby allowing the
bugs to survive in extremely toxic conditions. The project focuses on studying
the E. coli CusABC efflux system that recognizes silver and copper ions and
extrudes them out of the bacterial cell. CusA is an inner membrane
transporter, which belongs to the resistance-nodulation-division (RND) protein
superfamily. It consists of 1,047 amino acid residues. CusC, however, is a 457
amino acid polypeptide that forms an outer membrane channel in E. coli. In
conjunction with a 407 amino acid membrane fusion protein CusB, these proteins
assemble as a tripartite efflux complex to mediate the extrusion of heavy-metal
ions across both membranes of E. coli. It has been proposed that CusB may act
as an adaptor that interacts with CusA and CusC to form the CusABC complex.
The resulting CusABC complex (Figure 1) make direct contact with metal ions and
selectively expel them out of the cell. We recently cloned, expressed, and
purified the full-length CusA, CusC, and CusB efflux proteins. We will
crystallize these proteins in detergent solutions using vapor-diffusion. The
crystal structures will provide us direct information about how these proteins
handle heavy-metal ions.
Multi-drug efflux pumps interfere significantly with cancer
chemotherapy and the treatment of bacterial infections, by recognizing a number
of structurally unrelated toxic compounds and actively extruding them from
cells. One of our ongoing projects is to study the Escherichia coli AcrB
transmembrane efflux pump, which shows the widest substrate specificity among
all known multi-drug transporters. We have determined the X-ray structures of
AcrB in the presence of four structurally different toxic compounds (Figure 2).
The crystal structures illustrate that three ligand molecules bind
simultaneously to the extremely large central cavity of 5000 cubic Angstroms,
primarily by hydrophobic, aromatic stacking and van der Waals interactions.
Each ligand uses a slightly different subset of AcrB residues for binding. The
subsequent study of the efflux pump carried out by crystallizing a mutant AcrB
with five structurally diverse ligands indicates that AcrB consists of two
distinct binding sites. These five ligands not only bind to various positions
of the central cavity, but also to residues lining the deep external depression
formed by the C-terminal periplasmic domain. The structures also suggest that
AcrB assembles as a trimer of three identical channels for the extrusion of
drugs. In the current phase, we are determining the structure and function of
the AcrB transporter using X-ray crystallography and site-directed mutagenesis.
The planned work also focuses on co-crystallizing AcrB with the periplasmic
membrane fusion protein AcrA, and determines the X-ray structure of the AcrAB
co-crystal complex.
Campylobacter jejuni is the leading bacterial cause of food-borne
diarrhea in the USA as well as other developed countries. It is also in the
list of the NIAID Category B Priority Pathogens. C. jejuni is able to infect
the host and colonize the intestinal tract. To withstand the various
deleterious environments, this microorganism contains 13 antibiotic efflux
transporters that may be recruited to extrude antimicrobial compounds. The
primary contributor to antibiotic resistance in C. jejuni is the CmeABC
antibiotic efflux transporter, which is tightly regulated by the
transcriptional regulator CmeR. We recently crystallized the CmeR regulator
and have its x-ray structure determined (Figure 3). The crystal structure have
provided us valuable clues about how this regulator controls the production of
the CmeABC antibiotic efflux transporter in C. jejuni. We are currently
co-crystallizing CmeR in the presence of bile acids and its target DNA. We
expect that these structures will give us novel insight into the mechanisms of
multiple ligand recognition and gene regulation in CmeR. The finding may also
help to improve our knowledge of multi-drug resistance in Campylobacter.
This project addresses fundamental questions regarding the nature of
multi-ligand recognition and gene regulation in transcriptional regulators.
The primary target is the Escherichia coli AcrR repressor that regulates the
multidrug transporter AcrB. The 215-residue AcrR consists of two domains, the
C-terminal multi-ligand binding and the N-terminal DNA binding domains. Upon
binding a wide variety of structurally diverse ligands in the C-terminal
region, it triggers conformational change at the N-terminal domain, prohibiting
the binding of AcrR to its target DNA. The net result is the over-expression
of the AcrB transporter, which, in turn, promotes efflux from the cell, thus
protecting it from toxic substances. How can AcrR and other transcriptional
repressors recognize a variety of toxic chemicals? How do they respond to
changing environmental conditions? An understanding of these repressors may
allow us to uncover the mechanisms that these proteins use to bind multiple
ligands, regulate gene expression, and transmit signal from one binding site to
the other. We have determined the x-ray crystallographic structure of AcrR
(Figure 4), we will use this crystal structure to guide us to alter the
function of AcrR and convert it into toxic chemical sensors. In this project,
we will convert AcrR to respond to chemicals that cannot be recognized by the
regulator. Based on a simple Acr system network, we will develop a whole cell
green fluorescence biosensor for multiple chemicals. In addition, we will
study the structural and functional relationships of the AcrR transcriptional
regulator using X-ray crystallography and site-directed mutagenesis.
1605 Gilman Hall, Ames, Iowa 50011-3111, 1-800-521-CHEM, rmharris [at] iastate [dot] edu
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