Exposing a possible Achilles’ heel in Gram-negative bacteria

Pseudomonas_aeruginosa_Gram neg
Pseudomonas aeruginosa, a Gram-negative bacteria that can cause 
pneumonia as well as other diseases.

Multidrug resistant bacterial infections are a major biomedical problem. In the US, drug resistant bacteria infect more than 2 million people, resulting in over 20,000 deaths. For this reason, the development of new antibiotics that target these organisms is a priority for the pharmaceutical industry and government agencies.

In the clinic, a class of bacteria known as Gram-negative cause some of the most difficult to treat multidrug resistant infections. This is partially because Gram-negative bacteria have two membranes, or walls of lipid and protein molecules, protecting them from the environment. Like our skin, these membranes are a barrier against damaging agents in the environment and are the bacteria’s main line of defense. When a threatening environment does damage to these protective layers, the cell must repair the wall by replenishing its components. Gram-negative bacteria do this by inserting new proteins into the outermost membrane, a process that requires specialized equipment. This job is the responsibility of a group of proteins that function together as a molecular machine known as the β-Barrel Assembly Machinery, or BAM complex. Understanding how this complex works could lead to new ways of killing Gram-negative bacteria, thereby effectively treating many multidrug resistant bacterial infections.

BamABamBmodel
A schematic of the BAM complex embedded within the 
Gram-negative outer membrane. 

Though it’s known that the BAM complex helps restore Gram-negative bacteria’s outer cell wall by inserting proteins into the membrane, it’s not clear exactly how the machinery works. Katarina Jansen, a newly minted Ph.D. from Marcelo Sousa’s lab in the Department of Chemistry and Biochemistry, focused her thesis work on trying to figure this out.

“We try to understand what the role of each BAM protein is and how this molecular machine is able to interact with its [specific partners],” says Katarina. “Once we understand the molecular process, we will be able to design drugs to block this machine, which is our ultimate goal. Gram- negative bacteria cannot survive without a functional BAM.”

What does a functional BAM complex look like? Katarina used a technique known as x-ray crystallography to take three-dimensional molecular “snapshots” of the proteins that make up the BAM complex. The pictures that x-ray crystallography produces are known as crystal structures and are used to answer many biological questions. For her investigation, Katarina took two sets of pictures. The first set was of individual BAM proteins, sort of like having your portrait taken, and the second set was of BAM proteins interacting with each other, like a candid family photo. With her 3-D pictures, Katarina was able to highlight the interaction between two important members of the complex, proteins known simply as BamA and BamB.

Taking these pictures isn’t as easy as snapping a selfie with your smartphone. Crystallography requires very strong molecular interactions to capture the proteins. Because the interaction between BamA and BamB in the cell is temporary, it is difficult to generate a crystal structure of the two “in action.” To overcome this problem, Katarina cleverly joined the proteins together by a molecular attachment known as a linker.  This resulted in a permanent tether, and allowed her to generate a crystal structure of the two proteins as they interacted.

One caveat to x-ray crystallography is that the structures are determined in an artificial environment. It’s possible that a protein’s structure is different in the environment required for x-ray crystallography compared to inside the cell. Such a change could alter the way proteins like BamA and BamB interact with each other, so knowing whether the interaction is the same inside the cells is vital. Katarina therefore followed up her crystallography studies with another experiment. She used the crystal structure to identify which amino acids, the smaller individual molecules that make up proteins, are responsible for the interaction between BamA and BamB. Then, she changed these amino acids, and tested whether the modified proteins still interacted inside the cell.  The crystal structure showed that BamB interacts with a specific part of BamA through a large network of hydrogen bonds, a very close-range but easily disrupted interaction. In addition to the spatial organization of the complex, the crystal structure revealed the three-dimensional rearrangements that BamB undergoes in order to interact with BamA. In other words, BamB must change its shape just to make connections with BamA. Importantly, this interaction is required for the BAM complex to do its job. Such a finding would likely not have been possible if Katarina had only looked at each protein individually. Her ability to observe the two proteins together revealed new information about how the BAM complex operates.

She found that the altered amino acids disrupted key interactions in living cells and as a result BamA and BamB did not behave the same way as in the unmodified cells. This experiment confirmed that the interactions she observed using x-ray crystallography are indeed the same as those occurring inside the bacteria. Furthermore, this experiment exposed an Achilles’ heel in the BAM complex – the crucial but easily disrupted interaction between BamA and BamB – that could be used as a target for new antibiotics.

Katarina’s findings help the Sousa lab come one step closer to their overarching goal of designing drugs against this molecular machine.

Katarina published her results in the Journal of Biological Chemistry and now works as a research scientist at Enzymatics in Boulder, CO.

By Roni Dengler, Jacob A. Greenberg, and Joan G. Marcano-Velazquez

Leave a Reply

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out / Change )

Twitter picture

You are commenting using your Twitter account. Log Out / Change )

Facebook photo

You are commenting using your Facebook account. Log Out / Change )

Google+ photo

You are commenting using your Google+ account. Log Out / Change )

Connecting to %s