Abstract
BackgroundMucosal surfaces are coated with layers of mucus gel that protect the underlying tissues and promote colonization by members of the commensal microflora. Lactobacillus fermentum is a common inhabitant of the oral cavity, gastrointestinal and reproductive tracts and is one of the most important lactic acid bacteria contributing to the formation of a healthy intestinal microflora. We have investigated the proteolytic activity in L. fermentum in response to interactions with the MUC5B mucin, which is a major component of mucus gels at sites colonized by this micro-organism.MethodsBiofilms of Lactobacillus fermentum were established in mini-flow cells in the presence or absence of human salivary MUC5B. The proteolytic activity of biofilm cells was examined in a confocal scanning laser microscope with a fluorescent protease substrate. Degradation of MUC5B by L. fermentum was analysed using SDS-PAGE followed by Western blotting with antisera raised against the MUC5B peptide. Cell surface proteins differentialy expressed in a MUC5B-rich environment were identified with the aid of comparative two-dimensional electrophoresis followed by LC-MS/MS.ResultsLactobacillus fermentum adhered well to surfaces coated with MUC5B mucin and in biofilms of L. fermentum formed in a MUC5B environment, the proportion of proteolytically-active cells (47 ± 0.6% of the population), as shown by cleavage of a fluorescent casein substrate, was significantly greater (p < 0.01) than that in biofilms formed in nutrient broth (0.4 ± 0.04% of the population). Thus, the presence of MUC5B mucins enhanced bacterial protease activity. This effect was mainly attributable to contact with surface-associated mucins rather than those present in the fluid phase. Biofilms of L. fermentum were capable of degrading MUC5B mucins suggesting that this complex glycoprotein can be exploited as a nutrient source by the bacteria.Comparison of the surface proteomes of biofilm cells of L. fermentum in a MUC5B environment with those in nutrient broth using two-dimensional electrophoresis and mass spectroscopy, showed that the enhanced proteolytic activity was associated with increased expression of a glycoprotease; O-sialoglycoprotein endopeptidase, as well as chaperone proteins such as DnaK and trigger factor.ConclusionsAdhesion to mucin-coated surfaces leads to a shift towards a more protease-active phenotype within L. fermentum biofilms and proteases produced within the biofilms can degrade MUC5B mucins. The enhanced proteolytic activity was associated with an increase in O-sialoglycoprotein endopeptidase on the cell surface. We propose that the upregulation of chaperone proteins in the mucin environment may contribute to the protease-active phenotype through activation of the glycopeptidase. This would represent one way for commensal lactobacilli e.g. L. fermentum to exploit complex substrates in their local environment in order to survive on mucosal surfaces.
Highlights
Mucosal surfaces are coated with layers of mucus gel that protect the underlying tissues and promote colonization by members of the commensal microflora
In the present study we investigate how proteolytic activity in biofilms of L. fermentum is related to the environment surrounding the bacteria; a mucin-rich environment or protein-rich nutrient broth medium, as well as whether large gelforming mucins can be degraded by L. fermentum as a potential source of nutrients
Comparison of proteolytic activity in planktonic and biofilm cells of L. fermentum To investigate the effect of MUC5B on proteolytic activity in planktonic cells of L. fermentum, cells were exposed to MUC5B in the fluid phase
Summary
Mucosal surfaces are coated with layers of mucus gel that protect the underlying tissues and promote colonization by members of the commensal microflora. Lactobacillus fermentum is a common inhabitant of the oral cavity, gastrointestinal and reproductive tracts and is one of the most important lactic acid bacteria contributing to the formation of a healthy intestinal microflora. The body’s mucosal surfaces are colonized by a large number of commensal bacterial species which co-exist in a mutualistic relationship with their human host These microbial communities, or biofilms, are believed to constitute the first line of defense against infection by competitively inhibiting non-indigenous organisms that may cause disease. The commensal flora has been proposed to have an important influence on development, immunity and nutrition (for reviews see [1,2,3]) Despite their importance, surprisingly little is known about how the members of these communities survive in the mucosal environment and interact with each other, and the host, to form a dynamic ecosystem. In contrast to the beneficial role in the intestine, Lactobacilli in the oral cavity are often associated with carious disease [6] and Lactobacillus fermentum is frequently isolated from dentine caries lesions in children, implying a role in the caries process [11]
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