What doesn’t kill you makes you stronger: Bacteria that withstand fungal ‘bully’ gain resistance to last-resort antibiotic

This can happen in cases like chronic wounds or cystic fibrosis. Hsieh chose to study two opportunistic pathogens that often co-infect lungs of patients with cystic fibrosis: the fungus Candida albicans and the bacterium Pseudomonas aeruginosa, a gram-negative bacterium with lipopolysaccharide (LPS)-containing outer cell membrane. (Gram-positive bacteria lack this LPS-containing membrane.)

While most previous work focused on how bacteria attack fungi, “Fungi must have some way to suppress bacteria,” Hsieh said.

She wanted to know how.

This desire inspired an entirely new research direction in the lab of her mentor, Fred Hutch microbiologist and Howard Hughes Medical Institute Investigator Harmit Malik, PhD. She initiated a collaboration with (and co-mentorship from) UW physician-scientist Ajai Dandekar, MD, PhD, who studies Pseudomonas infections. Hsieh also collaborated with the Hogan Lab at Dartmouth Geisel School of Medicine.

Microbes face off over magnesium

To better understand what biological processes are critical in the battle between fungi and bacteria, Hsieh focused on identifying bacterial genes that bacteria use to defend against fungi.

“If we know the genes that are really required [to survive co-existence with fungi], it will tell us what kind of stress the fungi are imposing on the bacteria,” she said.

To identify these genes, Hsieh grew 100,000 different P. aeruginosa mutants either alone or with C. albicans. Then she compared how each mutant fared with and without fungi.

Working with Malik Lab research technician Wendy Sun, Hsieh pinpointed a three-gene region that was critical for the survival of bacteria threatened by fungi. The only gene in the triad with a known function encoded a magnesium transporter that enhances that mineral’s uptake. She showed that the same gene also helps other gram-negative bacterial species withstand fungi.

Hsieh suspected that the importance of the magnesium transporter meant that C. albicans must sop up magnesium that bacteria need. Sure enough, she showed that the fungus reduced magnesium levels in culture medium; mutant bacteria that lacked the magnesium transporter gene suffered the consequences. However, she could boost survival of mutant bacteria by adding more magnesium to offset the fungus’ effects. 

Bacteria turn a loss into a win

Gram-negative bacteria need magnesium for many reasons, including to stabilize their cell membrane. LPS carries a negative charge, which can be neutralized by the binding of positively charged magnesium ions. Polymyxin antibiotics, often used as last-resort therapies for multi-drug-resistant bacterial infections, destroy bacterial cell membranes by displacing magnesium.

This destabilizes the membrane and eventually kills the bacterium as its internal contents leak away. But when magnesium is low, bacteria can modify their LPS to maintain cell-wall structure — and resist polymyxins like colistin.

“We have known for several decades that polymyxin antibiotics like colistin require magnesium,” said Malik. “Phoebe combined this prior knowledge with her findings about this novel competition for magnesium to ask whether fungal co-culture would alter how bacteria acquire colistin resistance.”

Indeed, Hsieh found that bacteria instantaneously became more colistin-resistant in the presence of fungus.

Long-term exposure to antibiotics also breeds resistance — through different means. Hsieh showed that she could promote further colistin resistance in P. aeruginosa by challenging the bacteria with the drug only, or with both drug and C. albicans for several generations. The bacteria exposed only to the drug acquired genetic changes, but the bacteria co-cultured with the fungus acquired resistance through different pathways.

“We have thus uncovered a novel resistance pathway that depends on the presence of the fungus,” Hsieh said.

When she removed C. albicans, the bacteria became just as sensitive to colistin as they had been before.

Tracing how bacteria become resistant

“I am really excited about Phoebe’s findings and the multiple avenues of research they open,” Malik said. “Phoebe’s project is a fantastic example of curiosity-driven basic sciences opening up new avenues that may lead to clinical intervention.”

Hsieh is now working to trace the evolutionary steps between the beginning of co-culture and when the bacteria acquire polymyxin resistance. She hopes to learn more about the biological processes that are important to bacteria — which may reveal potential microbicidal targets.

Although she cautioned that her findings are entirely preclinical, she is excited about several interesting avenues for approaching infection treatment. One obvious follow-up would be that strategies to overcome antibiotic resistance should consider how bacteria acquired their drug resistance, whether through exposure to fungi or the drug.

It may mean that different drugs are warranted in each situation. Or, it may mean that even in cases where antibiotic treatment has apparently “failed,” there may be potential to rescue antibiotic treatment success in poly-microbial infections by simultaneously targeting the fungal infection. There are also hints from clinical observations of patients with cystic fibrosis that magnesium could be an important player in patient health.

“I think it’s a very exciting discovery in terms of basic mechanism, and we hope it could inspire people working in the clinic to think about whether it’s in line with what they see — and whether that could improve treatments,” Hsieh said.

This work was funded by the National Institutes of Health, the Cystic Fibrosis Foundation, the Howard Hughes Medical Institute and the Burroughs-Wellcome Fund.