GlycoNet scientist helps discover novel way to synthesize antibiotics

By Chardelle Prevatt – April 28, 2017

Antibiotics are drugs that either kill or inhibit the growth of bacteria. Therefore, antibiotics are used to treat or prevent bacterial infections. In the midst of growing concerns over antibiotic resistance, a new study has revealed the first steps in the biosynthesis of kanosamine—a known antibiotic and antifungal agent, which also serves as a building block for other widely used antibiotics.

In a paper published in the American Chemical Society’s journal—Biochemistry earlier this month, GlycoNet scientist Dr. David Palmer and PhD candidate Natasha Vetter, at the University of Saskatchewan’s Department of Chemistry, have revealed a relatively fast, simple, and effective way that certain bacteria use to produce kanosamine from glucose, using a short series of enzymatic reactions.

“This work attempts to understand how the enzyme glucose-6-phosphate 3-dehydrogenase (NtdC) helps to generate kanosamine in bacteria,” said Natasha Vetter, lead author of the paper. “NtdC is a relatively novel enzyme in that it makes a 3-keto sugar which is fairly uncommon in the biological world. There’s also an interesting coupling between this enzyme and another enzyme, 3-oxo-glucose-6-phosphate:glutamate aminotransferase, that allows for that transformation to happen.”

Despite kanosamine’s ability to suppress bacterial infections, the extent of its efficacy remains unclear.

Dr. Palmer added, “There is an emerging, interdisciplinary area of research called ‘synthetic biology’ where scientists construct pathways to make desired biological products. Data from our latest research can potentially be valuable to this new field.”

This work was funded by the Natural Sciences and Engineering Research Council of Canada (NSERC).

To view the full research paper, click here.

About NSERC
NSERC aims to make Canada a country of discoverers and innovators for the benefit of all Canadians. The agency supports university students in their advanced studies, promotes and supports discovery research, and fosters innovation by encouraging Canadian companies to participate and invest in postsecondary research projects. NSERC researchers are on the vanguard of science, building on Canada’s long tradition of scientific excellence.

GlycoNet scientist at McMaster finds potential new antibiotic use for old drug

By Chris Dall – March 7, 2017 

Early last week, the World Health Organization (WHO) released a list of 12 priority pathogens for which new antibiotics are urgently needed. The list was dominated by gram-negative bacteria, which have become resistant to multiple classes of antibiotics.

Although gram-negative bacteria have emerged as a major public health threat in part because of their ability to acquire and pass on mobile resistance elements, they also have what [GlycoNet scientist and] McMaster University biochemistry professor Eric Brown, PhD, calls an “intrinsic resistance” to antibiotics. An outer membrane on the gram-negative cell envelope provides an extra layer of protection against many antibiotics.

Looking for molecules that can breach this defense mechanism is what led Brown to discover a potential new use for an old drug. The results of his research appeared yesterday in Nature Microbiology.

‘Spectacular’ results in mouse models

A few years ago, Brown and his colleagues at the Brown Lab started looking at molecules that might be able to penetrate that outer membrane and allow antibiotics, even antibiotics that would normally be effective only against gram-positive bacteria, to do their job. These types of antibiotic adjuvants have long been sought, but none has ever made it to the clinic. Their research resulted in a screening platform that looks specifically for molecules that are active on the outer membrane of Escherichia coli.

Of the three compounds identified by the screen from a panel of 1,440 previously approved drugs, one in particular—the antifungal and antiprotozoal drug pentamidine—”really lit the assay up,” Brown told CIDRAP News.

Pentamidine, developed in 1937, is mainly used to treat Pneumocystis pneumonia, a fungal infection that often affects people who are immunosuppressed, such as HIV, AIDS, and cancer patients. It’s on the WHO’s list of essential medicines. The screening platform developed by Brown and his team revealed that it also can pierce the outer membrane of E coli.

In vitro testing indicated that when combined with pentamidine, antibiotics typically restricted to gram-positive bacteria—rifampicin, novobiocin, and erythromycin in particular—were effective against a range of gram-negative bacteria. One of the pathogens tested, Acinetobacter baumannii, tops the WHO’s list of priority pathogens. Mouse experiments yielded more impressive findings.

“The results were really spectacular,” Brown said, as most A baumannii–infected mice showed no signs of infection 7 days after treatment with a combination of pentamidine and novobiocin, even at low doses. “We basically sterilized an Acinetobacter baumanni infection in a mouse.” The combination also protected 10 of 11 mice from A baumannii resistant to the last-resort antibiotic colistin and led to total bacterial clearance from the spleen.

In addition, in vitro tests showed that pentamidine retained its ability to penetrate the outer membrane of gram-negative pathogens even when they were resistant to colistin. Brown said he and his colleagures were concerned that colistin resistance, which affects phosphates in the outer membrane of bacteria, would inhibit the activity of pentamidine. Emerging resistance to colistin in multidrug-resistant organisms has been a concern since the discovery of the colistin-resistance gene MCR-1 in China.

Hope in combination therapies

Brown’s study also revealed, however, that pentamidine is not the “perfect adjuvant” for antibiotic combination therapies. For one, it wasn’t effective against all the gram-negative pathogens tested. In particular, the pentamidine/rifampicin combination did not work against Pseudomonas aeruginosa, another one of the WHO’s high-priority pathogens. Brown said this could be caused by P aeruginosa having a different outer membrane structure. In addition, Brown explained, pentamidine has some toxicity issues.

But given the challenge of finding and developing new antibiotics, Brown and his colleagues think that combination approaches could be significant weapons in the fight against multidrug-resistant gram-negative pathogens, and they are continuing to use their screening platform to look for more molecules with outer-membrane activity.

While drug companies are reluctant to pursue combination therapies because of the challenge of working with more than one agent, Brown said more people in the research community are coming around to the strategy. “Combination therapies are going to be the way we get out of this mess,” he said.

Hybrid Antibiotic Created With Molecular ‘Rope’ Kills Resistant Bacteria

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Due to increasing antibiotic resistance, microbiologists are on the lookout for unconventional ways to kill bacteria. Atypical methods range from phage therapy, in which bacteria-killing viruses are unleashed upon the microbes, to the use of “bed-of-nails” surfaces that physically rip bacteria apart.

Such out-of-the-box thinking was displayed yet again by a team of scientists from the University of Manitoba [including two GlycoNet scientists Drs. George Zhanel and Frank Schweizer] who created a hybrid antibiotic by tying together two different antibiotics with a molecular “rope.”

One strategy to fight resistance is to hit a bacterium with multiple antibiotics at the same time. The reasoning is that, while it is relatively simple for a bacterium to spontaneously mutate to avoid the lethal effect of one antibiotic, it is very unlikely that multiple mutations will occur simultaneously that protect it against more than one. In other words, a bacterium may become resistant to antibiotic A or antibiotic B if they are used separately; but if used together, antibiotics A + B present a formidable challenge to resistance.

To create their hybrid, the researchers subjected the antibiotic tobramycin to a series of chemical reactions that (1) attached a molecular “rope” (a chain of 12 carbon atoms) to the center of the molecule and (2) tied the rope to a second antibiotic, moxifloxacin. (See diagram at the top of this article.)

Then, they tested how the hybrid antibiotic performed against clinically isolated samples of Pseudomonas aeruginosa, a bacterium that is both highly resistant to multiple antibiotics and is commonly transmitted in hospitals. (See below.)

Susceptibility to antibiotics is measured using the minimum inhibitory concentration (MIC, measured in micrograms per milliliter), which indicates the lowest amount of antibiotic necessary to prevent the bacteria from growing. (Thus, the higher the number, the more resistant the bacteria are to that antibiotic.)

Note that most clinical isolates of P. aeruginosa were highly resistant to both moxifloxacin (red box) and tobramycin (blue box) but that the hybrid antibiotic (purple box) was quite effective at inhibiting bacterial growth. The authors inferred that whatever molecular mechanisms allow these bacteria to be resistant to each antibiotic individually do not apply to the hybrid antibiotic.

So the team was curious to discover how exactly their hybrid antibiotic worked. Their investigation suggested that it operated in a completely different way than the original antibiotics. Instead of blocking protein synthesis (like tobramycin) or inhibiting DNA replication (like moxifloxacin), the hybrid exhibited an entirely new mode of action: It pokes holes in a bacterium’s outer membrane and disrupts the electrical field potential (called the proton motive force) associated with the inner membrane. The former causes the bacterium to literally disintegrate (see below), and the latter disrupts various aspects of bacterial physiology.

Additionally, the hybrid antibiotic rescued moths infected with P. aeruginosa. Even better, when the authors tried to coax P. aeruginosa into becoming resistant to their hybrid antibiotic, they were unable to. This bizarre molecule, at least for the time being, is too much for this microbe to handle.

Source: Bala Kishan Gorityala et al. “Hybrid Antibiotic Overcomes Resistance in P. aeruginosa by Enhancing Outer Membrane Penetration and Reducing Efflux.” J Med Chem 59 (18): 8441–8455. Published: 13-Aug-2016. DOI: 10.1021/acs.jmedchem.6b00867

About the Author:

alex-berezowDr. Alex Berezow joined the American Council on Science and Health as Senior Fellow of Biomedical Science in May 2016. He is a prolific science journalist whose work regularly appears in BBC News, The Economist, and USA Today, where he serves as a member of the Board of Contributors. With Hank Campbell in 2012, he co-authored the book Science Left Behind, which was an environmental policy bestseller.

Formerly, he was the founding editor of RealClearScience. He holds a Ph.D. in microbiology.