Differential proteome and cellular adhesion analyses of the probiotic bacterium Lactobacillus acidophilus NCFM grown on raffinose – an emerging prebiotic

Abstract

Whole cell and surface proteomes were analyzed together with adhesive properties of the probiotic bacterium Lactobacillus acidophilus NCFM (NCFM) grown on the emerging prebiotic raffinose, exemplifying a synbiotic. Adhesion of NCFM to mucin and intestinal HT-29 cells increased three-fold after culture with raffinose versus glucose, as also visualized by scanning electron microscopy. Comparative proteomics using 2D-DIGE showed 43 unique proteins to change in relative abundance in whole cell lysates from NCFM grown on raffinose compared to glucose. Furthermore, 14 unique proteins in 18 spots of the surface subproteome underwent changes identified by differential 2DE, including elongation factor G, thermostable pullulanase, and phosphate starvation inducible stress-related protein increasing in a range of +2.1 - +4.7 fold. By contrast five known moonlighting proteins decreased in relative abundance by up to -2.4 fold. Enzymes involved in raffinose catabolism were elevated in the whole cell proteome; α-galactosidase (+13.9 fold); sucrose phosphorylase (+5.4 fold) together with metabolic enzymes from the Leloir pathway for galactose utilization and the glycolysis; β-galactosidase (+5.7 fold); galactose (+2.9/+3.1 fold) and fructose (+2.8 fold) kinases. The insights at the molecular and cellular levels contributed to the understanding of the interplay of a synbiotic composed of NCFM and raffinose with the host.

Plant Polyphenols Stimulate Adhesion to Intestinal Mucosa and Induce Proteome Changes in the Probiotic Lactobacillus acidophilus NCFM

“…Adhesion was measured as previously described with some modification . Briefly, freshly late‐log phase grown NCFM (20 h, OD 0.5 for tannic acid; 24 h, OD 1.0–1.1 for control and resveratrol; 24 h, OD 1.4–1.5 for caffeic and ferulic acids) was labeled with 100 μ m 5(6)‐carboxyfluorescein diacetate (Sigma‐Aldrich) in PBS (37 °C, 30 min), washed twice and resuspended in PBS to OD 600 0.5 ± 0.05.…”

Exo‐ and surface proteomes of the probiotic bacterium Lactobacillus acidophilus NCFM

“…Gels were stained by colloidal CBB , scanned (Microtek Scan maker 9800 XL; Microtek), and image‐analyzed (Progenesis SameSpots, version 3.3). In‐gel trypsin digestions of spots were performed as previously described . Aliquots (1 μL) of the tryptic digests of protein spots were applied onto an Anchor Chip target (Bruker‐Daltonics), covered by matrix solution (1 μL 0.5 mg/mL CHCA in 90% ACN, 0.1% TFA) and washed (2 μL 0.02% TFA).…”

“…Several exo‐ and surface proteome (Table ) proteins have been reported to be moonlighting proteins for other bacteria that may bind to ECM components (fibrinogen, collagen, plasminogen, mucin) and host epithelial cells . GAPDH is a universal moonlighting protein that has different roles in bacteria and eukaryotic cells .…”

“…Gels were stained by colloidal CBB [12], scanned (Microtek Scan maker 9800 XL; Microtek), and image-analyzed (Progenesis SameSpots, version 3.3). In-gel trypsin digestions of spots were performed as previously described [7,11].…”

“…Several exo-and surface proteome ( Table 1) proteins have been reported to be moonlighting proteins for other bacteria that may bind to ECM components (fibrinogen, collagen, plasminogen, mucin) and host epithelial cells [11,24,25]. GAPDH is a universal moonlighting protein that has dif- Signal Peptide, proteins that contain a signal peptide; Membrane, proteins predicted to be membrane proteins; Nonclassically secreted, proteins predicted to be secreted by nonclassical secretion systems [13,14] (B) Protein categories of exo-and surface proteomes of Lactobacillus acidophilus NCFM.…”