Biofilm of pathogenic bacteria analyzed by MALDI-TOF imaging mass spectrometry (IMS) and top-down proteomic identification

Authors: Fagerquist, Clifton - Keith; Shi, Yanlin

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 3/6/2023
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: Biofilms are extracellular matrices composed of polysaccharide and proteins generated by bacteria under certain environmental conditions. Bacteria inhabiting these biofilm structures are often resistant to antibiotic treatment making decontamination of surfaces more difficult. The genes responsible for biofilm formation have also been linked to genes that produce a bacterial amyloid: curli. Curli are thin fiber-like projections (fimbriae) on the external envelope of bacteria that allow initial attachment to surfaces. The major component of curli is the CsgA protein. We have analyzed the biofilm produced by an E. coli O157:H7 strain linked to a major foodborne outbreak using MALDI-TOF-IMS and top-down proteomic analysis using MALDI-TOF-TOF-MS/MS. A number of proteins, including CsgA, were identified. E. coli O157:H7 strain was cultured at 28 °C for 2 to 10 days in 22 mL of low salt Luria-Bertani broth in a 50 mL tube. An indium tin oxide-coated glass slide was inserted into the tube which was half-submerged creating an air/liquid (A/L) interface. After culturing, the slide was washed and imaged. Caffeic acid matrix was applied to the conductive side of the slide using a TM sprayer. The slide was analyzed by MALDI-IMS using an Ultraflex MALDI-TOF-TOF mass spectrometer. For top-down protein identification, the biofilm was dissolved in hexafluoro-2-propanol (HFIPA) and, after evaporation, reconstituted in a saturated solution of caffeic acid and analyzed using a 4800 MALDI-TOF-TOF mass spectrometer. MALDI-TOF-IMS was performed on a rectangular area (13.5 mm x 23.5 mm) of the slide that includes the A/L interface as well as regions above and below the interface. Data was analyzed/viewed using flexImaging software. MS linear mode data (2-20 kDa mass range) was collected at 8149 positions. 1600 laser shots were acquired for each position using a random walk for each position. The raster width was 200 micrometers. A number of protein ion peaks were detected by IMS and later identified by top-down proteomic analysis including: CsgA; cold-shock proteins: CspC, CspE and CsbD; DNA-binding proteins: HU-alpha and HU-beta. The 2-D spatial distribution of CsgA (13.1 kDa) on the slide was primarily at the A/L interface with lesser amounts detected on the liquid side. As the culturing was conducted under aerobic conditions, it is possible that curli formation is induced by exposure to oxygen or lack of nutrients at the liquid surface. Spatial distribution of CspC, CspE and CsbD were also found primarily at the A/L interface. HFIPA was highly effective at dissolving biofilm and curli and found to be superior to the more commonly used a formic acid treatment as it does not result in formylation reactions that can complicate MS and MS/MS analysis. The mature, intact CsgA (signal peptide removed) fragmented poorly by MS/MS-post-source decay (PSD) due to its highly folded compact structure. However, along with its measured mass and a few sequence-specific fragment ions from backbone cleavage on the C-terminal side aspartic acid residues, CsgA was identified. Both CspC and CspE fragmented efficiently by MS/MS-PSD making confirmatory identification straightforward. CsbD fragmented poorly due in part to its lower abundance as well as multiple dissociative losses of ammonia from its four arginine residues.