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 gastrointestinal 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 plasmidencoded. 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 pilinlike 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. Molecular & Cellular

Lactococcus lactis Metabolism and Gene Expression during Growth on Plant Tissues

“…Pili were only recently found for members of the Lactococcus genus; however, pilus production is not active during growth in standard laboratory culture medium (40). Pili were also shown to facilitate L. lactis attachment to intestinal epithelial Caco-2 cells (61), and this capacity might also be important for binding to plant tissues. One of the pilus-associated proteins possessing a lectin domain (open reading frame 4 [ORF4]) was putatively designated to be responsible for epithelial cell binding (61).…”

“…Pili were also shown to facilitate L. lactis attachment to intestinal epithelial Caco-2 cells (61), and this capacity might also be important for binding to plant tissues. One of the pilus-associated proteins possessing a lectin domain (open reading frame 4 [ORF4]) was putatively designated to be responsible for epithelial cell binding (61). In KF147, the ATL-inducible gene ywfG (llkf_2426) encodes a putative LPXTG-anchored mucus binding protein with a lectin domain that might provide a similar function for L. lactis on plants.…”

Structural basis for adaptation of lactobacilli to gastrointestinal mucus

“…The mucus-binding ability of L. rhamnosus GG is conferred by the pilus-associated SpaC, present along the length of the pilus shaft potentially resulting in the prolonged residency of L. rhamnosus GG in the GI tract (Kankainen et al, 2009;Reunanen et al, 2012). More recently pili have also been implicated in GI colonization and persistence of Bifidobacteria in the murine gut (O'Connell Motherway et al, 2011;Turroni et al, 2013) and in bacterial autoaggregation and biofilm formation of Lactococcus lactis in vitro (Oxaran et al, 2012;Meyrand et al, 2013). These appendages that decorate the bacterial surface are increasingly considered as key molecules in mediating bacterial adherence to the host epithelium and in influencing mucosal immune responses.…”

“…Although the adhesive abilities of enteropathogenic bacteria have been extensively studied, the systems responsible for intestinal adhesion of gut commensal and probiotic bacteria to mucus are poorly understood. High-throughput sequencing of Lactobacillus genomes provides a platform for functional analysis of genes that may contribute to adhesion and probiotic function (Pridmore et al, 2004;Pfeiler and Klaenhammer, 2007;Ventura et al, 2009;Turpin et al, 2012;Meyrand et al, 2013). A growing number of reports indicate that LPXTGanchored cell wall proteins with modular domain organization, such as L. reuteri MUB, function in the adherence of probiotic strains to the host intestinal mucosa (Kleerebezem et al, 2010;Walter et al, 2011;Call and Klaenhammer, 2013).…”

Metabiotics: The Functional Metabolic Signatures of Probiotics: Current State-of-Art and Future Research Priorities—Metabiotics: Probiotics Effector Molecules

“…Recently the key adhesion protein of the L. rhamnosus GG pilus, SpaC has been demonstrated to show strong binding with two extracellular proteins i.e .mucin and collagen as well as promote bacterial aggregation phenomenon [54]. Similarly, the strong adhesion ability has been shown by the pili of L. lactis to intestinal epithelial cells (Caco-2) [55]. Therefore, in a nut shell, as surface protruding molecules they play vital role in the adhesion of probiotic species to their respective host cells and promotes immuno-modulatory functions.…”