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Surface proteome analysis of a natural isolate of Lactococcus lactis reveals the presence of pili able to bind human intestinal epithelial cells.

ABSTRACT: Surface proteins of Gram-positive bacteria play crucial roles in bacterial adhesion to host tissues. Regarding commensal or probiotic bacteria, adhesion to intestinal mucosa may promote their persistence in the gastro-intestinal tract and their beneficial effects to the host. In this study, seven Lactococcus lactis strains exhibiting variable surface physico-chemical properties were compared for their adhesion to Caco-2 intestinal epithelial cells. In this test, only one vegetal isolate TIL448 expressed a high-adhesion phenotype. A nonadhesive derivative was obtained by plasmid curing from TIL448, indicating that the adhesion determinants were plasmid-encoded. Surface-exposed proteins in TIL448 were analyzed by a proteomic approach consisting in shaving of the bacterial surface with trypsin and analysis of the released peptides by LC-MS/MS. As the TIL448 complete genome sequence was not available, the tryptic peptides were identified by a mass matching approach against a database including all Lactococcus protein sequences and the sequences deduced from partial DNA sequences of the TIL448 plasmids. Two surface proteins, encoded by plasmids in TIL448, were identified as candidate adhesins, the first one displaying pilin characteristics and the second one containing two mucus-binding domains. Inactivation of the pilin gene abolished adhesion to Caco-2 cells whereas inactivation of the mucus-binding protein gene had no effect on adhesion. The pilin gene is located inside a cluster of four genes encoding two other pilin-like proteins and one class-C sortase. Synthesis of pili was confirmed by immunoblotting detection of high molecular weight forms of pilins associated to the cell wall as well as by electron and atomic force microscopy observations. As a conclusion, surface proteome analysis allowed us to detect pilins at the surface of L. lactis TIL448. Moreover we showed that pili appendages are formed and involved in adhesion to Caco-2 intestinal epithelial cells.

SUBMITTER: Meyrand M

PROVIDER: S-EPMC3861735 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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Surface proteome analysis of a natural isolate of Lactococcus lactis reveals the presence of pili able to bind human intestinal epithelial cells.

Meyrand Mickael M Guillot Alain A Goin Mélodie M Furlan Sylviane S Armalyte Julija J Kulakauskas Saulius S Cortes-Perez Naima G NG Thomas Ginette G Chat Sophie S Péchoux Christine C Dupres Vincent V Hols Pascal P Dufrêne Yves F YF Trugnan Germain G Chapot-Chartier Marie-Pierre MP

Molecular & cellular proteomics : MCP 20130903 12

Surface proteins of Gram-positive bacteria play crucial roles in bacterial adhesion to host tissues. Regarding commensal or probiotic bacteria, adhesion to intestinal mucosa may promote their persistence in the gastro-intestinal tract and their beneficial effects to the host. In this study, seven Lactococcus lactis strains exhibiting variable surface physico-chemical properties were compared for their adhesion to Caco-2 intestinal epithelial cells. In this test, only one vegetal isolate TIL448 e ...[more]

PMID: 24002364

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Project description:Lactococcus lactis is one of the most commonly used lactic acid bacteria in the dairy industry. Activation of competence for natural DNA transformation in this species would greatly improve the selection of novel strains with desired genetic traits. Here, we investigated the activation of natural transformation in L. lactis subsp. cremoris KW2, a strain of plant origin whose genome encodes the master competence regulator ComX and the complete set of proteins usually required for natural transformation. In the absence of knowledge about competence regulation in this species, we constitutively overproduced ComX in a reporter strain of late competence phase activation and showed, by transcriptomic analyses, a ComX-dependent induction of all key competence genes. We further demonstrated that natural DNA transformation is functional in this strain and requires the competence DNA uptake machinery. Since constitutive ComX overproduction is unstable, we alternatively expressed comX under the control of an endogenous xylose-inducible promoter. This regulated system was used to successfully inactivate the adaptor protein MecA and subunits of the Clp proteolytic complex, which were previously shown to be involved in ComX degradation in streptococci. In the presence of a small amount of ComX, the deletion of mecA, clpC, or clpP genes markedly increased the activation of the late competence phase and transformability. Altogether, our results report the functionality of natural DNA transformation in L. lactis and pave the way for the identification of signaling mechanisms that trigger the competence state in this species.IMPORTANCELactococcus lactis is a lactic acid bacterium of major importance, which is used as a starter species for milk fermentation, a host for heterologous protein production, and a delivery platform for therapeutic molecules. Here, we report the functionality of natural transformation in L. lactis subsp. cremoris KW2 by the overproduction of the master competence regulator ComX. The developed procedure enables a flexible approach to modify the chromosome with single point mutation, sequence insertion, or sequence replacement. These results represent an important step for the genetic engineering of L. lactis that will facilitate the design of strains optimized for industrial applications. This will also help to discover natural regulatory mechanisms controlling competence in the genus Lactococcus.

Dataset Information

Surface proteome analysis of a natural isolate of Lactococcus lactis reveals the presence of pili able to bind human intestinal epithelial cells.

Publications

Surface proteome analysis of a natural isolate of Lactococcus lactis reveals the presence of pili able to bind human intestinal epithelial cells.

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