Functional amyloids are important structural and functional components of many biofilms, yet our knowledge of these fascinating polymers is limited to a few examples for which the native amyloids have been isolated in pure form. Isolation of the functional amyloids from other cell components represents a major bottleneck in the search for new functional amyloid systems. However, by taking advantage of the structural stability and the polymeric nature of the amyloids, compared to most other proteins, a new method has been developed to screen for potential amyloids. The method has successfully been evaluated on two established Gram-negative amyloid systems. Proof of concept for the application to complex microbial biofilms was done using activated sludge, where amyloid producing E. coli was added and subsequently traced. In addition, another potential novel amyloid candidate was found in the activated sludge biofilm. METHODOLOGY Utilizing formic acid’s ability to depolymerize an amyloid fibril, lysed bacteria were freeze-dried and dissolved in increasing concentrations of formic acid (0-100%) and then analyzed for a characteristic increase in protein abundance at a higher percentage of formic acid, where they are dissolved. The proteins were identified using label-free quantitative LC-MS/MS using MaxQuant and the MaxLFQ algorithm. Eventually, positive amyloid candidates are identified based on their abundance profiles with respect to the formic acid concentration using an automated script. AIM The identification of novel amyloids is key to understand the many roles of amyloids in biofilm structure and function. This study aimed to develop a technique that allows the direct identification of functional amyloids in biofilms and eventually discover novel amyloid candidates. RESULTS Escherichia coli SM2258 producing curli functional amyloids A) TEM image of E. coli SM2258. B) Curly biosynthesis. C) Proteins with amyloid-specific abundance signatures in at least two biological replicates. Negative controls including the household proteins, Glyceraldehyde-3-phosphate dehydrogenase A (GapA) and 60 kDa chaperonin (GroEL); and β-barrel outer membrane proteins, like outer membrane protein A (OmpA) and antigen 43 (Ag43). Pseudomonas sp. UK4 producing Fap functional amyloids A) TEM image of Pseudomonas sp. UK4 . B) Fap biosynthesis. C) Proteins with amyloid-specific abundance signatures including the major functional amyloid subunit (FapC) and negative controls. Activated sludge spiked with 1% E. coli producing curli Cells from E. coli producing curli were mixed with activated sludge from a Danish WWTP. As expected, the profiles of the CsgA and CsgB monomers was found. Interestingly, another structurally confirmed functional amyloid was identified among the sludge proteins, namely the gas vesicle protein A, GvpA Danielsen et al., Biomolecules 2017, 7(3), 58 Susan H. Hansen, Heidi N. Danielsen, Florian A. Herbst, Per H. Nielsen, Morten S. Dueholm Centre for Microbial Communities | Department of Chemistry and Bioscience | Aalborg University | Denmark shh@bio.aau.dk @SusanHove FINDINGS • We have developed a technique that allows the direct identification of functional amyloids in cell lysates. • The technique is general applicable and do not require detailed knowledge about the bacterial composition. • The technique can be applied to complex samples e.g. clinical biofilms. It can therefore be used to identify amyloids produced by stimuli that can’t be mimicked in the laboratory. • We believe that the method will result in the identification of numerous new functional amyloids in the coming years. A B C
Biofilms are formed by a complex bacterial community encapsulated by a polymeric matrix, with strong adherent properties and persistent phenotype. Biofilms are considered one of the most challenging areas of modern medicine. Existing antibiotics have been developed against free-floating bacterial cells, and thus, many treatments of biofilm-related infection fail. In this study, we compared the effects of different media on biofilm growth of clinical reference strains of Staphylococci and Enterococci, including multi-drug resistant representatives. Further, we optimized the resazurin-based assay for determining the minimal biofilm inhibitory concentration (MBIC) of standard antibiotics, and evaluated its use for the determination of minimal biofilm eradication concentration (MBEC). We showed that tryptic soy broth supplemented with 1% glucose was an optimal media for maximum biofilm growth of all strains tested, with an extended incubation time for Enterococci. A range of parameters were tested for the resazurin assay, including concentration, temperature and time of incubation. Using quality parameters to analyze the assay’s performance, the conditions for the resazurin assay were set as follows: 4 μg/mL and 8 μg/mL, with incubation at 25 °C for 20 min and 40 min for Staphylococci and Enterococci, respectively. In summary, we defined conditions for optimal biofilm growth and for standardized resazurin assay for MBIC determination against six Gram-positive clinical reference strains. We also observed that MBEC determination by the resazurin-based assay is limited due to the poor detection limit of the assay. Complementary cell counting data is needed for precise determination of MBEC.
