Biofilm disruption: using their own hydrolases against them
Researchers from McGill University Health Center have demonstrated the cross-kingdom activity of the glycoside hydrolase family to treat biofilm infections.
- Researchers show activity of recombinant glycoside hydrolases against biofilms in Aspergillus fumigatus and Pseudomonas aeruginosa.
- The hydrolases were only effective against the organism from which they were isolated.
- Mouse models showed that these enzymes could be effective treatments in the future.
In a study published in PNAS, researchers developed recombinant glycoside hydrolases from both Aspergillus fumigatus and Pseudomonas aeruginosa. Both of these species had been shown to require glycoside hydrolases in the production of their respective extracellular matrices. Upon further inspection, they found that A. fumigatus and Ps. aeruginosa had some similarities when it came to the extracellular matrix. A. fumigatus produces a biofilm matrix of galactosaminogalactan (GAG) which is a heteroglycan composed of α1,4-linked galactose and N-acetyl-D-galactosamine. Ps. aeruginosa has the ability to produce 3 different types of ECM, but one of them named Pel is very similar to GAG.
The research team then produced recombinant versions of the glycoside hydrolases from A. fumigatus and Ps. aeruginosa and tested their ability to disrupt biofilms. They saw that in both cases, biofilm could be disrupted but only by the organisms own hydrolase enzyme.
In a test of treatment effectiveness, the researchers looked at the impact of the glycoside hydrolase from A. fumigatus (Sph3h) on the fungal burden in a mouse model of pulmonary aspergillosis. They found that not only were there no side effects on the mice, but that the fungal burden was quickly and significantly reduced. The team believes that this could be an effective treatment in the future.
The production of biofilms is an important strategy used by both bacteria and fungi to colonize surfaces and to enhance resistance to killing by immune cells and antimicrobial agents. We demonstrate that glycoside hydrolases derived from the opportunistic fungus Aspergillus fumigatus and Gram-negative bacterium Pseudomonas aeruginosa can be exploited to disrupt preformed fungal biofilms and reduce virulence. Additionally, these glycoside hydrolases can be used to potentiate antifungal drugs by increasing their hyphal penetration, to protect human cells from fungal-induced injury, and attenuate virulence of A. fumigatus in a mouse model of invasive aspergillosis. The findings of this study identify recombinant microbial glycoside hydrolases as promising therapeutics with the potential for antibiofilm activity against pathogens across different taxonomic kingdoms.
Galactosaminogalactan and Pel are cationic heteropolysaccharides produced by the opportunistic pathogens Aspergillus fumigatus and Pseudomonas aeruginosa, respectively. These exopolysaccharides both contain 1,4-linked N-acetyl-D-galactosamine and play an important role in biofilm formation by these organisms. Proteins containing glycoside hydrolase domains have recently been identified within the biosynthetic pathway of each exopolysaccharide. Recombinant hydrolase domains from these proteins (Sph3h from A. fumigatus and PelAh from P. aeruginosa) were found to degrade their respective polysaccharides in vitro. We therefore hypothesized that these glycoside hydrolases could exhibit antibiofilm activity and, further, given the chemical similarity between galactosaminogalactan and Pel, that they might display cross-species activity. Treatment of A. fumigatus with Sph3h disrupted A. fumigatus biofilms with an EC50 of 0.4 nM. PelAh treatment also disrupted preformed A. fumigatus biofilms with EC50 values similar to those obtained for Sph3h. In contrast, Sph3h was unable to disrupt P. aeruginosa Pel-based biofilms, despite being able to bind to the exopolysaccharide. Treatment of A. fumigatus hyphae with either Sph3h or PelAh significantly enhanced the activity of the antifungals posaconazole, amphotericin B, and caspofungin, likely through increasing antifungal penetration of hyphae. Both enzymes were noncytotoxic and protected A549 pulmonary epithelial cells from A. fumigatus-induced cell damage for up to 24 h. Intratracheal administration of Sph3h was well tolerated and reduced pulmonary fungal burden in a neutropenic mouse model of invasive aspergillosis. These findings suggest that glycoside hydrolases can exhibit activity against diverse microorganisms and may be useful as therapeutic agents by degrading biofilms and attenuating virulence.
Brendan D. Snarr, Perrin Baker, Natalie C. Bamford, Yukiko Sato, Hong Liu, Mélanie Lehoux, Fabrice N. Gravelat, Hanna Ostapska, Shane R. Baistrocchi, Robert P. Cerone, Elan E. Filler, Matthew R. Parsek, Scott G. Filler, P. Lynne Howell, and Donald C. Sheppard
Microbial glycoside hydrolases as antibiofilm agents with cross-kingdom activity
PNAS 2017 ; published ahead of print June 20, 2017, doi:10.1073/pnas.1702798114