Using crystal structures to battle antibiotic-resistant bacteria: a crystallographer's approach to fighting infection

This post is the first of a two-part feature on the work of the Sousa lab in Chemistry and Biochemistry.  Watch for another post about their exciting research in the coming weeks!

-------------
Jacob A. Greenberg, 3rd year PhD candidate in Organic Chemistry

Culture of Pseudomonas 
aeruginosa, a Gram negative 
bacteria. 
Source: faculty.ccbcmd.edu

At a young age when we learned to cover our mouths for coughs and sneezes, we learned not to spread germs. But the idea that germs, such as bacteria and other microorganisms, are responsible for human disease is still relatively recent.  

Louis Pasteur was the first scientist to hypothesize that microorganisms are responsible for disease in the 19^th century.   Since their discovery, physicians and scientists have worked to uncover exactly how bacteria work in order to devise strategies to keep us healthy.  As scientists learned more about bacteria, two major classes emerged: Gram positive and Gram negative. These two types of bacteria can be distinguished by the fact that the membranes surrounding them have different structures.  

While these two classes of bacteria have evolved very different strategies for survival, both Gram positive and Gram negative strains can cause human disease.  Because their different survival strategies have resulted in vastly different cellular architecture, scientists have put a lot of effort into developing unique treatments for each type. 

Gram negative bacteria are generally more difficult to treat in part because they have evolved an especially strong cell membrane that is made up of not one but two layers of molecular barriers to gain entry to the cell.   The “double layer” of defense makes it challenging for scientists to design drugs that are able to get inside the cell to treat the infections.   

This phenomenon has made it extremely difficult to treat infections that arise in patients with cystic fibrosis in particular, and as a result these Gram negative infections are the number one cause of mortality in those with cystic fibrosis.  One of the leading research groups in the field of Gram negative bacterial membranes is the Sousa Lab in the Department of Chemistry and Biochemistry, whose work we will be featuring over the next few weeks. 

Bacteria use many pathways to resist detection and to defend against antibiotics.  These lead to antibiotic resistant strains of bacteria, such as the infamous MRSA – or Methicillin-Resistant Staphloccocus aeureus.  Antibiotic resistant strains are extremely difficult to treat because traditional antibiotics no longer have the same effect they once had. Worse, bacteria that are resistant to multiple drugs are showing up more frequently, particularly in hospital settings where diseases are easily transmitted to individuals who are already sick.   

Antibiotic-resistant bacteria, and in particular multidrug resistant Gram negative strains such as Pseudomonas aeruginosa, Salmonella, and E.coli among others cause particular problems for patients with cystic fibrosis.   Although cystic fibrosis is a human genetic disease, the primary cause of mortality is not the disease itself but rather the persistent, chronic, drug-resistant infections in the lungs.  Because of this, researchers in the Sousa lab are particularly interested in understanding Gram negative bacterial membranes. A more thorough understanding of these membranes will lead to better treatments for these dangerous pathogens.

Gram negative cell membrane.  Both leaflets of the inner 
membrane are composed of phospholipids.  In the outer 
membrane, the inner leaflet is composed of phospholipids, 
but the outer leaflet is made upof lipopolysaccharide (LPS).  
This double membrane makes Gram negative bacteria much 
more difficult to treat.  Adapted from Nature Structural & 
Molecular Biology, 2012, 19, 1132-1138.

The Gram negative cell membrane is composed of an inner cell membrane and outer cell membrane.  Each membrane is composed of two layers, called the inner and the outer leaflets. The inner membrane is symmetric, with both leaflets composed of molecules known as phospholipids. The outer membrane is asymmetric; the inner leaflet is composed of phospholipids, while the outer leaflet is composed of a different molecule known as lipopolysaccharide (LPS).  A specific portion of the LPS molecule is known as lipid A.  Lipid A is an important piece of the molecule because it’s what the human body’s immune system recognizes as an invader, and signals to the body to attack.  

Many Gram negative bacterial strains are becoming increasingly difficult to treat because of their ability to modify lipid A by activating enzymatic pathways. An enzyme is a biological molecule that catalyzes molecular chemical reactions in the cell. An enzymatic pathway is carried out by a series of enzymes, which mediate chemical reactions to complete a task needed by the cell. In the case of Gram negative bacteria, they are able to use an enzymatic pathway to modify the outer membrane’s lipid A, which allows them to evade detection and avoid being attacked by our immune systems.  This also protects them from being targeted by polymyxins, a class of antibiotics that were developed to specifically target lipid A.

Dr. Myeongseon Lee, a research associate in the Sousa lab, investigated the pathway Gram negative bacteria use to modify lipid A, which is known as the arabinose synthesis (Arn) enzymatic pathway.  This pathway was previously known as the polymyxins resistance pathway, given that bacteria often used it to avoid being targeted by polymyxins antibiotics. The Arn pathway is a series of 8 steps carried out by 7 proteins. 

The Arn pathway has been studied for some time, but scientists still do not completely understand how it works.  In order to get a better view of it, Myeongseon generated X-ray crystal structures of one of the enzymes in the pathway. The use of X-rays to determine structure has been a monumental achievement in this field, and four Nobel prizes have been awarded to X-ray crystallographers – including for the discovery of DNA structure. X-ray crystal structures can be thought of as three dimensional snapshots of a protein. These protein snapshots can help scientists design drugs that will target these enzymes and inhibit their function in bacteria. 

ArnB Crystal structure pdb (1MDX). 
The blue ribbon is the structure of the protein and 
the multicolored molecule in the center is the 
chemical substrate that co-crystallized in the 
protein. Image rendered by Jacob Greenberg.

The second step in the Arn pathway is catalyzed by the protein ArnB, which is the protein Myeonseon was interested in studying.  He recently published an article that describes a new crystal structure of ArnB. To make the crystal structure, Myeongseon generated a version of ArnB with a mutation that stopped the enzymatic reaction in an intermediate state, allowing him to see this usually transient state.  

Because this crystal structure showed Myeongseon what the enzyme looks like as it is catalyzing its reaction, he or other scientists will be able to use the structure to design drug molecules that specifically block its activity.   Inhibition of ArnB would disrupt the entire pathway, which in turn would leave the bacteria more susceptible to attack from the immune system as well as from polymyxins. 

Myeongseon was also able to compare the active site of ArnB to active sites in human proteins. An active site is the location within the 3D structure where the chemical reaction takes place on an enzyme. Most enzyme proteins work like a lock: only one “key” molecule fits into the active site and allows the reaction to occur.  When designing a drug molecule, it is important that the structural design will not interfere with human enzymes that are structurally similar and perform similar functions.

By comparing the active sites, scientists design drug molecules to specifically disrupt bacterial proteins without interacting with similar human proteins.  Myeongseon’s paper compares the active site of ArnB to two of the most similar human enzymes. He found human enzymes’ active sites bind the molecule in a completely different fashion compared to ArnB. This is evidence that ArnB would be an excellent target for drug design.

Crystal structures of enzymes involved in bacterial antibiotic resistance allow scientists to develop new drugs in the ever-present war against antibiotic resistant bacterial infections.  The development of new drugs for ArnB, in particular, could be powerful for treating drug resistant Gram negative bacterial infections — such as those that frequently occur in patients with cystic fibrosis. These new data will allow scientists in the Sousa lab and around the globe to make progress in finding treatments for these increasingly challenging drug resistant infections. 

-------------

Dr. Myeongseon Lee has recently moved to the Stowell lab in Molecular, Cellular and Developmental Biology, where he is studying eukaryotic membrane proteins.

To access Myeongseon's full article, click here.