Growth Mode and Carbon Source Impact the Surfaceome Dynamics of Lactobacillus rhamnosus GG

Abstract

Bacterial biofilms have clear implications in disease and in food applications involving probiotics. Here, we show that switching the carbohydrate source from glucose to fructose increased the biofilm formation and the total surface-antigenicity of a well-known probiotic, Lactobacillus rhamnosus GG. Surfaceomes (all cell surface-associated proteins) of GG cells grown with glucose and fructose in planktonic and biofilm cultures were identified and compared, which indicated carbohydrate source-dependent variations, especially during biofilm growth. The most distinctive differences under these conditions were detected with several surface adhesins (e.g., MBF, SpaC pilus protein and penicillin-binding proteins), enzymes (glycoside hydrolases, PrsA, PrtP, PrtR, and HtrA) and moonlighting proteins (glycolytic, transcription/translation and stress-associated proteins, r-proteins, tRNA synthetases, Clp family proteins, PepC, PepN, and PepA). The abundance of several known adhesins and candidate moonlighters, including enzymes acting on casein-derived peptides (ClpP, PepC, and PepN), increased in the biofilm cells grown on fructose, from which the surface-associated aminopeptidase activity mediated by PepC and PepN was further confirmed by an enzymatic assay. The mucus binding factor (MBF) was found most abundant in fructose grown biofilm cells whereas SpaC adhesin was identified specifically from planktonic cells growing on fructose. An additional indirect ELISA indicated both growth mode- and carbohydrate-dependent differences in abundance of SpaC, whereas the overall adherence of GG assessed with porcine mucus indicated that the carbon source and the growth mode affected mucus adhesion. The adherence of GG cells to mucus was almost completely inhibited by anti-SpaC antibodies regardless of growth mode and/or carbohydrate source, indicating the key role of the SpaCBA pilus in adherence under the tested conditions. Altogether, our results suggest that carbon source and growth mode coordinate mechanisms shaping the proteinaceous composition of GG cell surface, which potentially contributes to resistance, nutrient acquisition and cell-cell interactions under different conditions. In conclusion, the present study shows that different growth regimes and conditions can have a profound impact on the adherent and antigenic features of GG, thereby providing new information on how to gain additional benefits from this probiotic.

Surface-Shaving Proteomics of Mycobacterium marinum Identifies Biofilm Subtype-Specific Changes Affecting Virulence, Tolerance, and Persistence

“…These enzymes have been shown to protect Mtb against oxidative stress by the reduction of superoxide radicals into less toxic intermediates for inhibiting autophagy, apoptosis, and cellular damage ( 78 ). Iron-storing proteins ferritin (BfrB) and bacterioferritin (BfrA) can confer increased redox resistance to Mtb and protect the cells against oxidative stress and hypoxia, respectively ( 79 ). Here, these iron-storing proteins displayed biofilm subtype-specific abundance changes, implying that SBFs could rely on BfrB to cope with hypoxia and PBFs could rely on BrfA to help cells grow at the air-liquid interface.…”

Growth Mode and Physiological State of Cells Prior to Biofilm Formation Affect Immune Evasion and Persistence of Staphylococcus aureus

“…In many bacteria, the (p)ppGpp levels are regulated by an alarmone synthetase/hydrolase Rel in response to amino acid starvation, in which the branched-chain amino acids, such as valine and isoleucine, play an important role [78]. Previously, we demonstrated that in Lactobacillus rhamnosus, the surface-associated aminopeptidases (PepN/PepC) degrade oligopeptides while bound to the biofilm matrix, indicating that the cytoplasmic enzymes can also confer a similar function at the cell surface [79]. While the peptidolytic enzymes were less abundant on biofilm cells, an oligopeptide transporter protein, OppA, was notably more strongly produced on fully mature biofilm cells (SDBF_24 h) compared to planktonic cells or biofilm cells derived from exponential-phase cells (EDBF_24 h).…”

Structural and Functional Dynamics of Staphylococcus aureus Biofilms and Biofilm Matrix Proteins on Different Clinical Materials

“…Before protein identification, biofilm viability with and without the trypsin treatment was first assessed to exclude possible effects of trypsin on biofilm integrity. Biofilms were grown on G coupons in 12-WPs and then transferred into Falcon tubes (50 mL) containing 1 mL of 100 mM acetate buffer (pH 4.7), a condition preventing the release of the adhesive moonlighting proteins [56]. The tubes were sonicated (25 • C, 35 kHz, 5 min) and detached bacterial cells were collected by centrifugation (4 • C, 4000× g, 2 min).…”