Background: Biofilm formation by microorganisms plays an important role during infection by increasing resistance to antimicrobials and host defences. The formation of an extracellular matrix composed of polysaccharides and other molecules plays an important part in mediating these properties of biofilms. Aspergillus fumigatus biofilm is dependent on the polysaccharide galactosaminogalactan (GAG), a cationic polymer of alpha 1,4-linked galactose and partially deacetylated N-acetylgalactosamine (GalNAc). Recent work from our group has shown that Sph3, a fungal glycoside hydrolase, and PelA, a bacterial glycoside hydrolase involved in the synthesis of the Pseudomonas aeruginosa Pel polysaccharide, can degrade GAG, disrupt Aspergillus fumigatus biofilms and attenuates the virulence in a mouse model of invasive aspergillosis. The molecular mechanisms by which these enzymes disrupt biofilms have not been defined.
Hypothesis: We hypothesized that Sph3 and PelA share structural and functional similarities and underlie their ability to degrade GAG and disrupt A. fumigatus biofilms.
Methods: Sph3 and PelA were recombinantly expressed and purified from E. coli cultures. Enzyme structures were solved by X-ray diffraction crystallography and enzymatic mechanism performed by Matrix Assisted Laser Desorption and Ionization - Time Of Flight Mass Spectrometry (MALDI-TOF MS).
Results: Superimposition of PelA with Sph3 aligned the active site of these two enzymes but revealed structural motifs unique to PelA that generate a deeper, more electronegative groove in this enzyme. Despite these differences, examination of the putative active sites revealed a high degree of conservation with residues D166, N202 and E222 of Sph3h that interact with GalNAc superimposing with residues D160, N199 and E218 of PelA, suggesting that both enzymes may have similar catalytic mechanisms.
To probe the mechanism of action of these enzymes, MALDI-TOF MS was used to analyze oligosaccharides released from Sph3 and PelA treatment of A. fumigatus biofilms. Sph3 and PelA were observed to cleave oligosaccharides composed of alpha-1,4 GalNAc. A technique was developed to generate and purify oligomers of alpha-1.4-GalNAc from A. fumigatus biofilms. Oligosaccharide hydrolysis studies using these oligosaccharides demonstrated that both enzymes function as endo-N-acetylgalactosaminidases with a minimal substrate size of 7 sugars.
Conclusion: These studies demonstrate that despite substantial structural differences, Sph3 and PelA share a similar catalytic mechanism and function as endo-N-acetylgalactosaminidases with activity against A. fumigatus GAG. These studies demonstrate the molecular mechanism of action of these therapeutic hydrolases against GAG and will be invaluable for the further optimization and development of these novel anti-biofilm therapeutic agents.
Full conference title:
- AAA 8th (2018)