Browsing by Author "Ahmed, Mohamed Adel"

Lactobacillus rhamnosus GG (GG) is one of the most studied probiotics worldwide and it has proved to confer health benefits to human. However, the exact mechanism of its probiotic action is still not fully understood. Some proteins secreted by probiotics, such as p75 (Msp1) and p40 (Msp2) in GG, are reported to play an important role in gut epithelial homeostasis. The aim of this work was to develop a static biofilm plate model for analyzing exoproteomes (all secreted proteins) produced by L. rhamnosus GG biofilms. First, different culture volumes (7 mL and 10 mL), incubation times and protein precipitation methods were tested to optimize conditions in order to maximize the protein yield. Next, the impact of different sugars on the exoproteome composition of the GG cells during planktonic and biofilm mode of growth was explored. Finally, the planktonic and biofilm exoproteins were subjected to antigen profiling and protein identification. The 6-well Polystyrene Microtiter plate was used as the static biofilm model for inducing the biofilm formation of GG. Biofilms were formed for 24 h and 48 h and the protein secretion from each time point was assessed by precipitating the supernatant proteins using 10% trichloroacetic acid (TCA)/acetone or 2-D Clean Up kit (GE Healthcare). The purified proteins were subjected to one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1-DE) and the proteins were visualized using Coomassie blue staining. 1-DE immunoblotting using antibodies raised against whole GG cells was used to analyze antigens produced by the GG biofilms under the optimized biofilm formation conditions. The antigens produced by the GG biofilm cells were compared to those produced by the GG cells during planktonic growth (from over- night cultures) and the cross-reacting proteins were visualized using an IR800-conjugated secondary antibody. The immunoblots were scanned using an Odyssey Infrared Imaging System (Licor). In addition, the impact of two different carbohydrate sources on the antigen profiles was also explored. Finally, in-liquid tryptic digestion coupled with Liquid Chromatography- tandem Mass Spectrometry analysis (LC-MS/MS) was used to identify and compare exoproteomes produced by the biofilm cells cultured in the presence of the tested carbohydrates. The results showed that the commercial 2-D Clean Up kit is better than 10% TCA protein precipitation/acetone washing, because it produced clear protein patterns with less background. However, the TCA/acetone–protocol resulted in detection of higher number of proteins in 1-DE gels. Comparative immunoblot analyses of the planktonic and biofilm exoproteins at 24 h and 48 h time points revealed a clear difference in antigen profiles between the two modes of growth. In addition, the utilized carbon source was found to have a great impact on the antigen abundances and/or export. Using LC-MS/MS, 36 exoproteins were identified from the GG biofilm cultures (identification score ≥ 40, p < 0.05). Most of the identified proteins were associated with cell wall/membrane biogenesis, peptide and sugar transporters and transcription.