An international team of researchers has modified a probiotic yeast to make it safer for use by immunocompromised people, older adults and infants. Testing in an animal model found the modified yeast is less likely to cause infection than unmodified strains of the same organism.
"The yeast we're working with is called Saccharomyces boulardii, which is marketed as a probiotic to promote gut health," says Alexandra Imre, first author of a paper on the work and a postdoctoral researcher at North Carolina State University. "However, there have been cases in which immunocompromised patients, infants and elderly people - who are especially vulnerable to infections - have gotten bloodstream infections due to probiotic usage. While these cases are rarely reported, they can be serious - even fatal.
"We wanted to learn more about what is contributing to these infections," Imre says. "We were also wondering whether it is possible to genetically modify this yeast to make it less virulent, thus safer for immunosuppressed patient groups."
The researchers worked with S. boulardii yeast cell lines that were isolated from multiple sources. These "isolates" came from commercially available probiotics and samples obtained from human patients, including two patients who had S. boulardii bloodstream infections.
The researchers used these yeast cell lines to infect mice that were immunosuppressed in order to determine which isolates were most virulent. The researchers then isolated multiple yeast cell lines - called sublineages - from these mice.
The researchers tested these sublineages to see how they responded to various stress factors in order to identify adaptations that could be making the yeast more virulent.
"We found that the isolates that were most virulent in our mouse model were also the ones that were most tolerant to osmotic stress," Imre says. "In other words, the yeast lines that were most likely to increase virulence were also best able to survive in environments that have high concentrations of salts.
"We focused on two genes, ENA1 and NHA1, that play a substantial role in making the yeast more tolerant of osmotic stress," says Imre.
The researchers genetically edited both the commercial and clinical S. boulardii isolates to delete those two genes and then repeated both the virulence testing in mice and the stress testing.
"We found that deleting NHA1 made very little difference, but deleting ENA1 made a big difference," says Imre. "Before deleting ENA1, mice infected by the most virulent yeast isolate had a survival rate of 30-40%. After deleting ENA1, the survival rate increased to 100%, during our six-day-long mouse infection experiment. We also found that deleting ENA1 resulted in impaired growth of the yeast strains when exposed to osmotic stress.
"Overall, our results showed that there is a clear correlation between osmotic stress tolerance and virulence," Imre says. "However, more research is going to be necessary to reveal the exact metabolic mechanisms behind this phenomenon; virulence attributes of emerging pathogens are under-researched."
The researchers also conducted tests in a mouse model and in vitro antimicrobial assays to see if modifying S. boulardii affected its utility as a probiotic. The researchers found the genetically modified S. boulardii were just as effective as the commercially available probiotic lines at inhibiting bacterial growth of bacteria species that often act as pathogens in immunosuppressed patients - and were just as able to survive in the gut, proving its potential application as a probiotic supplement.
"These are not exhaustive studies of probiotic efficacy, but our findings suggest the probiotic qualities of S. boulardii are not significantly impacted by deleting the genes associated with osmotic stress," says Nathan Crook, co-author of the paper and an associate professor of chemical and biomolecular engineering at NC State.
"Many people with gut disease also have compromised immune systems, which means probiotic treatment is often not an option for them," Crook says. "This study demonstrates the potential for creating engineered probiotic therapies for use by immunocompromised patients. Further work is certainly necessary, but we're excited about this."
The paper, "ENA1 deficiency attenuates Saccharomyces 'boulardii' probiotic yeast virulence in immunosuppressed mouse fungaemia model," is published in Nature's open-access journal Communications Biology. Corresponding author of the paper is Walter Pfliegler of the University of Debrecen, whose lab initiated this line of research. The paper was co-authored by Renátó Kovács, Ágnes Jakab, Andrea Harmath, Bálint Németh, Fruzsina Nagy, Lajos Forgács, Dávid Balázsi, László Majoros, Zsigmond Benkő and István Pócsi of the University of Debrecen.
The University of Debrecen and North Carolina State University have submitted an international PCT patent application for commercial application of the findings.
