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.
Highlights
Probiotic bacteria, applied in various aspects of the food and pharmaceutical industries (Sanders et al, 2013; Goldenberg et al, 2017), exploit sophisticated strategies to enable the utilization of many nutrients, maintain viability and adherence to mucosal surfaces, and modulate host immunity in a beneficial way during industrial processes and upon their consumption (Siezen and Wilson, 2010)
Biofilm formation of GG was first tested at three time points (24, 48, and 72 h after inoculation, hpi) in MRS-G and MRS-F (2% glc and 2% frc) and MRS without carbohydrate, which indicated that frc stimulated biofilm formation by approximately 2-fold compared to glc at each time point tested (Figure 1A)
The cell viability analysis of biofilm cells grown on glass-bottom dishes with both carbon sources revealed that biofilms formed on frc at 48 hpi contained proportionally more living cells than those grown on glc (Figure 1B)
Summary
Probiotic bacteria, applied in various aspects of the food and pharmaceutical industries (Sanders et al, 2013; Goldenberg et al, 2017), exploit sophisticated strategies to enable the utilization of many nutrients, maintain viability and adherence to mucosal surfaces, and modulate host immunity in a beneficial way during industrial processes and upon their consumption (Siezen and Wilson, 2010). Switching from the planktonic to the biofilm mode of growth can provide bacteria, including probiotics, with means to overcome lethal conditions, including antimicrobial treatments, host immune defenses, and changes in pH, salt, temperature and nutrients (Flemming et al, 2016). Reduced EPS synthesis has been linked to the increased adherence and biofilm formation of GG, which by uncovering specific adhesins necessary for biofilm formation, enabled the enhanced biofilm growth of this strain (Lebeer et al, 2009). The SpaCBA-pilus adhesin, which mediates the direct interaction with the host and abiotic surfaces, and MabA (the modulator of adhesion and biofilm), which strengthens the biofilm structure, are considered the central factors that mediate the biofilm formation of GG in vitro (Lebeer et al, 2007b; Velez et al, 2010). Systematic studies aiming to uncover all surface-bound proteins on GG biofilms have not been conducted
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