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Lactic Acid Bacteria (LAB) are involved in the production of fermented foods whereby they improve the preservation properties as well as impart characteristic flavors and textures. Specific Lactobacillus strains are marketed as probiotics because of potential health benefits. However, the molecular mechanisms underlying much of these beneficial effects have yet to be established. In this thesis, a proteomic approach was developed to investigate the functionality of Lactobacillus plantarum WCFS1 both under laboratory conditions and the human intestinal tract. Proteomics was chosen as proteins present in bacteria isolated from fecal samples will likely reflect the proteins that are produced during transit of the colon. Initially, two-dimensional (2-DE) gel electrophoresis of L. plantarum WCFS1, whose genome has been sequenced, was used to investigate the dynamics of the proteome from the cytosolic fraction isolated from mid- and late-log, early- and late-stationary phase cells grown in a laboratory medium. Almost 200 protein spots were identified by MALDI-TOF mass spectrometry revealing differentially regulated proteins for each growth phase, and a proteome reference map was constructed to facilitate further studies. Parallel analysis by proteomics and transcriptomics was performed for various growth phases of L. plantarum WCFS1 and an isogenic ccpA (regulator of carbon catabolite repression) mutant, showing a considerable (70-80%) correlation throughout all growth phases. Genes with deviating transcript and protein levels mainly classified among purine and pyrimidines biosynthesis, energy metabolism, and stress proteins. Next, methods for specifically labeling L. plantarum and for sorting and isolation from human intestinal samples for proteomic analysis were investigated. An oligonucleotide probe was validated for application in Fluorescent In Situ Hybridization (FISH) for L. plantarum against numerous other LAB and 40 bacterial species commonly found in the human intestine. The FISH probe was successfully applied to label L. plantarum cells from ileal effluent and fecal samples. An innovative approach involving an extraction method based on immunomagnetic beads using polyclonal antibody against L. plantarum WCFS1 with subsequent proteomic analysis was subsequently developed. In the enriched fraction from a human intestinal sample, L. plantarum accounted for 86% of the total bacterial cells based on quantitative PCR using specific primers. The proteome of L. plantarum cells isolated by the extraction method from the ileal effluent of a subject carrying an ileostoma that had been fed with L. plantarum WCFS1 was visualized on a 2-DE gel. A number of proteins could be identified using the proteome reference map involved in carbohydrate metabolism, stress adaptation and translation, confirming the metabolic activity of the cell, and two of these proteins were confirmed by MALDI-TOF analysis. Glycolytic proteins previously shown to be involved in host-microbe interactions/adherence in other gram-positive bacteria were detected in the 2-DE gels, and these were also detected in cell membrane fractions. Overall, this thesis lays the foundation for application of proteomics to study the functionality of L. plantarum cells from human intestinal samples. |
