How to Find a Needle in a Haystack, Fast

Antibiotic resistance has become a medical crisis. As bacteria continue to outsmart the current arsenal of antibiotics and become resistant, infections that were once easily treated can now become life-threatening.

This crisis is driving a major push by the scientific community to identify new drug targets that are less likely to result in resistant bacteria. It is a massive undertaking that can sometimes leave researchers feeling like they are searching for a needle in a haystack. Luckily, Prof. Anthony Clarke and graduate student Ashley Brott in the Department of Molecular and Cellular Biology are speeding up the hunt with a new high throughput screening technique to help identify a new kind of antibiotic that will turn a bacterium’s own machinery against it.

Bacteria run a tight ship. Every process is tightly regulated, including those that require making cuts in the cell wall when needed (such as during cell division, or adding flagella to allow the bacterium to swim). Every bacterium has a special group of enzymes whose job it is to make holes in cell wall when needed. But, says Brott, “if you don’t control where they make a hole then they will just cut everywhere.”

To avoid this, a bacterium will add chemical groups (called acetyl groups) to the sections of the cell wall where it does not want holes. The process of adding these chemical groups is called O-acetylation and is carried out by another group of enzymes called O-acetyltransferases.

Clarke and Brott knew that if they could find a way to block the O-acetylation process, it would lead to uncontrolled holes in the cell wall – something that would eventually kill the bacterium. This meant testing a rather large array of potential compounds – 145,000 of them – to determine if any could inhibit the enzymes in question. Faced with such a daunting task, they set out to develop the first high throughput screening technique for O-acetylation inhibitors.

The resulting automated process allows dozens of compounds to be screened simultaneously. First, the O-acetylation enzyme is pumped into a plate of small wells by a machine. The plate is then flipped upside down and moved on top of another plate that contains the different test compounds. The machine then uses sound waves to move the compounds up into the plate containing the enzyme. After the compounds are mixed with the enzyme, the researchers can observe the reactions to see if the mixture lights up or “fluoresces”. If it lights up, that means the enzyme is still active and functioning properly. However, if the mixture doesn’t light up, then the compound in question has prevented the enzyme from functioning. It is these compounds that are of interest to researchers as potential antimicrobials.

Using the high throughput screen, Brott and her colleagues identified a number of compounds with potential antibiotic activity. Follow up tests found that one compound in particular (known as compound 89224) could reduce the growth of the bacteria that causes gonorrhea by 90 percent, but had no effect on the growth of Escherichia coli.

This specificity is exactly what they were looking for, says Brott. “Some antibiotics target a wide range of bacteria, which unfortunately means they will also kill the microflora (good bacteria) in your gut. The benefit of targeting the O-acetylation process is that it isn’t a common process in gut bacteria; it occurs mostly in pathogenic bacteria.”

While the new high throughput technique needs further development before it can become clinically useful, Brott and her colleagues are thrilled that it has provided important proof of concept.

“The screen shows that O-acetyltransferases are great targets for future drug development,” says Brott.

This study was funded by the Canadian Institutes of Health Research and the Canadian Glycomics Network.

Read the full study in the journal Antibiotics.

Read about other CBS Research Highlights.