Structural Biochemistry/Membrane Proteins/Multidrug-Resistance Pumps
Even though there are numerous drugs to kill bacteria, there always remains a small percentage that cannot be killed with the available medicines. Scientists have found intercellular proteins in the cell membranes, called multidrug-resistance (MDR) pumps, which are believed to be self-defense mechanisms. MDRs arise from three different gene families and are widespread in bacteria.
In bacteria,multidrug-resistance pumps confer resistance to chemically unrelated amphipathic toxins. A major challenge in developing efficacious antibiotics is identifying antimicrobial compounds that are not rapidly pumped out of bacterial cells. The plant antimicrobial berberine, the active component of the medicinal plants echinacea and golden seal, is a cation that is readily extruded by bacterial MDRs, thereby rendering it relatively ineffective as a therapeutic agent.
Multidrug efflux pumps have other physiological functions: AcrB of E.Coli, main physiological function is to protect bacteria against bile salts since E.Coli can be found in the intestines. In addition, bile salts have affinity to AcrB transporters
These pumps are found in almost all living organisms, and have a multitude of roles, including moving the body’s natural molecules in and out of cells. In humans, they can be found within the membranes of the brain, liver, kidneys, and digestive tract.
These MDR pumps not only work in bacteria, but unfortunately, also in cancerous cells. The cancerous cells can fend off the chemotherapy drugs by pumping them out of the cell, allowing evasion of cell death.
Kim Lewis of Northeastern University in Boston conducted an experiment to test the hypothesis that the MDR pump is indeed responsible for bacterial evasion of antibiotics. In his experiment, he genetically changed the bacterium Staphylococcus aureus to not have the MDR pump. He then treated this altered bacteria with berberine antibiotic, which is normally very futile against the unaltered bacteria. He saw that the antibiotic actually worked against these bacteria. Lewis also saw that if this usually weak antibiotic was given concurrently with MDR pump inhibitors, unaltered Staphylococcus aureus bacteria would be killed. These results implicate that the missing or deactivated MDR pump prove very critical in the bacteria’s protection and survival.
AcrB and its homologues are the major multidrug efflux transporter systems, and it captures some of its substrates from the periplasm in E. coli and other Gram-negative organisms. AcrB forms a complex with AcrA and an outer membrane protein channel, TolC, which harnesses proton-motive force, to export a wide variety of compounds across the periplasmic space to the exterior of the cell. Although AcrB is a homotrimer, it can undergo structural changes in which each subunit exhibits different conformations that interconvert to move toxic compounds from the initial binding site out of the transporter.
AcrB works with an outer membrane channel TolC and a membrane fusion protein AcrA. This complex removes many types of chemicals that may be toxic to the cell in a reaction that is powered by proton-motive force.
AcrB transports drugs by a three-step functionally rotating mechanism in which drugs undergo an ordered change in binding. The first conformation is the access state. This is where the vestibule is open to the periplasm to allow the substrate to enter into the complex. The second conformation is the binding state. In this state, the binding pocket is expanded and the substrate binds to different locations in the pocket. The third conformation is the extrusion state. The vestibule is close and the exit is opened. The bound drug is pushed out into the top of the funnel allowing the AcrB protomer to shrink and return to the first stage of the mechanism, the access state.
These conformational changes in the trimer are powered by the proton motive force across the membrane through the involvement of three chraged residues, Aspartate-407, 408, and Lysine-940, making charge pair in the membrane-embedded region. In the access and binding state, the side chain of Lysine-940 forms a salt bridge with the carboxy-groups of Aspartate-407 and Aspartate-408. This salt bridge is then dissociated in the extrustion state and the Lysine side chain is turned and tilted to form a new polar bond with Threonine-978 where a proton may be attached. To return to the access state, the bound proton is dissociated. These residues,Aspartate-407, 408, and Lysine-940, are essential for exporting drugs and without them the cell would be at a complete loss of drug resistance.
Because this complex has a trimer of AcrB, it expels three drugs consecutively. As the first protomer of AcrB is in the access state, the second protomer is in the binding state, and the third protomer is in the extrustion state. The change in conformation of one protomer affects the conformation of another. This conformation cycle where one protomer affects another can be explained by the principle of cooperativity.
Multidrug resistance came about after the development of drugs because bacteria soon became anti resistant. There are two forms of multidruge resistance occurring. The first way is by genes coding for resistance on R plasmids or transposons. The second way is by efflux pumps which are able to pump out one or more drugs at a time. Bacteria can also become resistant due to mutations that distort the protein so that it is less susceptible to the drug. Sometimes resistance can be transferred by cells passing it to other cells on their plasmids. Other times, the resistance is due to target modification such as a substrate binding onto the protein and altering its shape. For example, Tet(M) or Tet(S) protein bind to ribosomes and change the conformation to prevent the drug from binding to them. Resistance genes also came from microorganisms in the soil because of the evolutionary origin of degradation genes because they use antibiotics as nutrients. An example of when genes are expressed from plasmids are integrons, which contains a gene coding, and is catalytic for the insertion of resistance genes at certain sites on the R plasmids. ISCR delivers resistant genes to integrons to make more resistance genes. Sometimes R plasmids are stable and have a killer element which if the plasmid is loss, then the cell dies. Lastly, multidrug resistance occurs in antibiotic treated patients because when they are sick, they may get into a resistant state without genetic change. Genetic change and mutations is what makes bacteria become resistant to drugs.
"Multidrug Resistance in Bacteria." National Institutions of Health.http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2839888/
- "Medicine by Design." National Institutions of Health. http://www.nigms.nih.gov. July 2006.
- Von Heijne and Rees. Current Opinion in Structural Biology ,18:405. August 2008.
- "Multidrug efflux transporter, AcrB-the pumping mecahnism." Murakami, Satoshi. Current Opinion in Structural Biology, 18:459-465. August 2008.