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June 14, 2017

Researchers Uncover How Microbes Pump Up the Resistance

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    In the periplasm, the space between the inner and outer membranes of a bacteria's cell wall, defensive proteins that detect a poison assemble like barrel staves to form a tunnel between pumps in the cell's inner and outer membranes to eject the intruders. [Ace George Santiago/Cornell University]

    A group of investigators led by scientists at Cornell University have just published new data elucidating some of the underlying cellular mechinsams associated with antibiotic resistance. By tagging a cell's proteins with fluorescent beacons, the research team found how E. coli bacteria defend themselves against antibiotics and other poisons. When the microbes come in contact with the unwanted molecules, the bacterial cell opens a tunnel though its cell wall and effluxes, or pumps out, the harmful molecules.

    The findings from the new study were published recently in PNAS through an article entitled “Adaptor protein mediates dynamic pump assembly for bacterial metal efflux.”

    "Dynamic assembly of these tunnels has long been hypothesized," explained senior study investigator Peng Chen, Ph.D., professor of chemistry and chemical biology at Cornell. "Now we see them."

    The researchers are optimistic that their findings could lead to new ways to combat antibiotic-resistant bacteria with a "cocktail" of drugs. "One is to inhibit the assembly of the tunnel, the next is to kill the bacteria," Dr. Chen noted.

    To study bacteria's defensive process, the researchers selected a strain of E. coli known to pump out copper atoms that would otherwise poison the bacteria. The researchers genetically engineered the bacteria with a GFP version of the defensive protein called CusB, so they could visually track how the microbes were getting rid of toxic molecules, such as copper ions.

    Using single-molecule superresolution imaging the scientists were able to visit the proteins movements after they exposed a bacterial cell to an environment containing copper atoms. CusB resides in the periplasm, the space between the inner and outer membranes that make up the bacteria's cell wall. When CusB binds to an intruder—in this experiment, a copper atom—that has passed through the porous outer membrane, it changes its shape so that it will attach itself between two related proteins in the inner and outer membranes to form a complex known as CusCBA that acts as a tunnel through the cell wall. The inner protein has a mechanism to grab the intruder and push it through.

    “We showed that in Escherichia coli, the tripartite complex CusCBA for Cu+ and Ag+ efflux exists as a dynamic structure and shifts toward the assembled form in response to metal stress,” the authors wrote. “Unexpectedly, the periplasmic adaptor protein CusB is a key metal-sensing protein that mediates the complex assembly. This adaptor protein-mediated dynamic pump assembly allows the bacterial cell for efficient efflux on cellular demand while still maintaining periplasmic plasticity; it can be broadly relevant to other multicomponent efflux systems.”

    Interestingly, the tunnel locks the inner and outer membranes together, making the periplasm less flexible and interfering with its normal functions. Moreover, the researchers note that the ability to assemble the tunnel only when needed, rather than having it permanently in place, gives the cell an advantage.

    The investigators concluded that their newly described mechanism for defending against toxic metals may also explain how bacteria develop resistance to antibiotics, by mutating their defensive proteins to recognize them. The team is currently investigating if similar mechanisms can be found in other species of bacteria.

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