Antibiotic’s killer strategy revealed (PNAS)

Marine algae
Satellite image of a E. huxleyi marine algae bloom. (Image: NASA)

By Tien Nguyen, Department of Chemistry

Using a special profiling technique, scientists at Princeton have determined the mechanism of action of a potent antibiotic, known as tropodithietic acid (TDA), leading them to uncover its hidden ability as a potential anticancer agent.

TDA is produced by marine bacteria belonging to the roseobacter family, which exist in a unique symbiosis with microscopic algae. The algae provide food for the bacteria, and the bacteria provide protection from the many pathogens of the open ocean.

“This molecule keeps everything out,” said Mohammad Seyedsayamdost, an assistant professor of chemistry at Princeton and corresponding author on the study published in the Proceedings of the National Academy of Science. “How could something so small be so broad spectrum? That’s what got us interested,” he said.

In collaboration with researchers in the laboratory of Zemer Gitai, an associate professor of molecular biology at Princeton, the team used a laboratory technique referred to as bacterial cytological profiling to investigate the mode of action of TDA. This method involves destroying bacterial cells with the antibiotic in the presence of a set of dyes, and then visually assessing the aftermath. “The key assumption is that dead cells that look the same probably died by the same mechanism,” he said.

marine algae
Scanning electron microscope image of E. huxleyi (Image credit. M. Seyedsayamdost)

The team used three dyes to evaluate 13 different features of the deceased cells, such as cell membrane thickness and nucleoid area, comprising TDA’s cytological profile. By comparing to profiles of known drugs, the researchers found a match with a class of compounds called polyethers, which possess anticancer activity.

Given their similar profiles, Seyedsayamdost and coworkers hypothesized that TDA might exhibit anticancer properties as well, and indeed observed its strong anticancer activity in a screen against 60 different cancer cell lines. “The strength of this profiling technique is that it tells you how to repurpose molecules,” Seyedsayamdost said.

The researchers were surprised by the compounds’ shared mode of action because unlike the small sized TDA, polyether compounds are quite large. But through different chemical reactions, they are both able to cause chemical disruptions in the cell membrane that render the bacterium unable to produce the energy needed to perform critical tasks, such as cell division and making proteins.

In addition to TDA’s killing mechanism, the researchers were interested in understanding the mechanism by which a bacterial strain could become resistant to the antibiotic. Particularly, they wondered how the marine roseobacter kept itself safe from the deadly antibiotic weapon that it produced.

The research team approached the task by probing the genes in roseobacter that synthesize TDA as well as the surrounding genes. They identified three nearby genes responsible for transport in and out of the cell, and upon transferring these specific genes to E. coli, were able to produce an elusive TDA resistant bacterial strain.

“We often look at natural products as black boxes,” said Seyedsayamdost, “but these molecules have evolved for millennia to fulfill a certain function. By linking the unusual structural features of TDA to its mode of action, we have begun to explain why TDA looks the way it does.”

Read the abstract:

Wilson, M. Z.; Wang, R.; Gitai, Z.; Seyedsayamdost, M. R. “Tropodithietic Acid: Mode of Action and Mechanism of Resistance.” Proc. Natl. Acad. Sci. 2016, Published online on January 22, 2016.

This work was supported by grants from the National Institutes of Health (GM 098299 and 1DPOD004389).

Study questions the prescription for drug resistance (Proceedings of the Royal Society B)

A drug-resistant strain of bacteria known as MRSA. Photo by James Gathany
A new study examines the question of aggressive versus moderate drug treatment on the emergence of drug-resistant pathogens. Shown is a strain of bacteria known as methicillin-resistant Staphylococcus aureus (MRSA). Photo by James Gathany

By Catherine Zandonella, Office of the Dean for Research

In response to the rise of drug-resistant pathogens, doctors are routinely cautioned against overprescribing antimicrobials. But when a patient has a confirmed bacterial infection, the advice is to treat aggressively to quash the infection before the bacteria can develop resistance.

A new study questions the accepted wisdom that aggressive treatment with high drug dosages and long durations is always the best way to stem the emergence and spread of resistant pathogens. The review of nearly 70 studies of antimicrobial resistance, which was authored by researchers at Princeton and other leading institutions and published last week in the journal Proceedings of the Royal Society B, reveals the lack of evidence behind the practice of aggressive treatment in many cases.

“We found that while there are many studies that test for resistance emergence between different drug regimes, surprisingly few have looked at the topic of how varying drug dosage might affect the emergence and spread of resistance,” said Ruthie Birger, a Princeton graduate student who works with C. Jessica Metcalf, an assistant professor of ecology and evolutionary biology and public affairs at Princeton’s Woodrow Wilson School, and Bryan Grenfell, the Kathryn Briger and Sarah Fenton Professor of Ecology and Evolutionary Biology and Public Affairs in Princeton’s Woodrow Wilson School. Birger, Metcalf and Grenfell coauthored the paper with colleagues from 16 universities. “We are a long way from having the evidence for the best treatment decisions with respect to resistance for a range of diseases,” Birger said.

Microbes such as bacteria and parasites can evade today’s powerful drugs by undergoing genetic mutations that enable them to avoid being killed by the drug. For example, bacteria can develop enzymes that degrade certain antibiotics. The logic behind aggressive treatment goes something like this: kill off as many microbes as you can so that few will be around to evolve into resistant forms.

But some scientists have observed a different outcome in mice infected with both an already-resistant strain of malaria and a non-resistant strain. The high-dose drug treatment killed off the non-resistant malarial parasites, leaving the resistant strains to multiply and make the mice even sicker.

The idea that aggressive treatment may backfire against malarial parasites led the authors of the current study to comb the scientific literature to examine whether the same may be true for other types of microbes such as bacteria. The few studies that they found — mostly in laboratory cell cultures rather than animal models or patients — suggest that the picture is complicated, and depends on whether the resistance is new or existing, how many mutations are necessary for the pathogen to become resistant, and how long the drugs have been in use. “It’s remarkable how little we know about this topic,” said Metcalf. “The malaria study conducted by Silvie Huijben and colleagues at Pennsylvania State University is an inspiring step towards developing an evidence base for these important issues.”

In the current analysis, the study authors found that drug resistance is governed by two factors: the abundance of the pathogen and the strength of the selection pressure that drives the pathogen to evolve. Aggressive treatment deals with the first factor by killing off as much pathogen as possible, while moderate treatment may, for some pathogens, reduce the ability for the resistant pathogen to thrive (for example, by maintaining the competitive advantage of a co-infecting drug-sensitive strain of the pathogen) but still reduce total pathogen levels sufficiently that the patient can recover.

Finding the ideal dose and duration of treatment, one that cures the patient without aiding the spread of resistance, will likely be done on a disease by disease basis, the authors found.

One possibility is that moderate treatment might be best used against already-resistant microbes to prevent their spread. Moderate treatment may also be best for drugs that have been on the market for several years with plenty of time for resistant strains to develop.

Aggressive treatment might be best for pathogens that develop resistance slowly, over the course of several mutations. High doses early in the process could be effective at heading off the development of resistance.

Read the abstract.

Roger D. Kouyos, C. Jessica E. Metcalf, Ruthie Birger, Eili Y. Klein, Pia Abel zur Wiesch, Peter Ankomah, Nimalan Arinaminpathy, Tiffany L. Bogich, Sebastian Bonhoeffer, Charles Brower, Geoffrey Chi-Johnston, Ted Cohen, Troy Day, Bryan Greenhouse, Silvie Huijben, Joshua Metlay, Nicole Mideo, Laura C. Pollitt, Andrew F. Read, David L. Smith, Claire Standley, Nina Wale and Bryan Grenfell. Proc. R. Soc. B: Biological Sciences, 281, 20140566. Published Sept. 24, 2014

The work emerged from two workshops held at Princeton University and funded by the RAPIDD program of the Science and Technology Directorate, Department of Homeland Security and the Fogarty International Center, National Institutes of Health; Science and Technology Directorate, Department of Homeland Security; contract HSHQDC-12-C-00058