Cell Surface-Associated Elongation Factor Tu Mediates the Attachment of Lactobacillus johnsonii NCC533 (La1) to Human Intestinal Cells and Mucins

Abstract

The aim of this work was to identify Lactobacillus johnsonii NCC533 (La1) surface molecules mediating attachment to intestinal epithelial cells and mucins. Incubation of Caco-2 intestinal epithelial cells with an L. johnsonii La1 cell wall extract led to the recognition of elongation factor Tu (EF-Tu) as a novel La1 adhesin-like factor. The presence of EF-Tu at the surface of La1 was confirmed by analysis of purified outer surface protein extract by immunoblotting experiments, by electron microscopy, and by enzyme-linked immunosorbent assays of live bacteria. Furthermore, tandem mass spectrometry analysis proved that EF-TU was expressed at the La1 surface as an intact molecule. Using recombinant La1 EF-Tu protein, we were able to determine that its binding to intestinal cells and to mucins is pH dependent. Competition experiments suggested that EF-Tu has an important role in La1 mucin binding capacity. In addition, immunomodulation studies performed on HT29 cells showed that EF-Tu recombinant protein can induce a proinflammatory response in the presence of soluble CD14. Our in vitro results indicate that EF-Tu, through its binding to the intestinal mucosa, might participate in gut homeostasis.Probiotic bacteria, mainly lactic acid bacteria and bifidobacteria, have been shown to have beneficial effects on the immune defenses and to alleviate or prevent diverse intestinal disorders (3,4,16,25,27,30,39,42,47). Several in vitro studies have shown that one of them, Lactobacillus johnsonii NCC533 (La1), is able to bind to epithelial cell lines (5,8,9,21) and can induce the secretion of different cytokines in coculture systems (9, 24). Furthermore, human and animal studies have demonstrated that La1 has immune adjuvant effects (40, 44) and can also act as a modulator of nonspecific immune responses (6,14,29,48,53,62).The mechanisms underlying these beneficial effects are not completely understood, but it is believed that the maximum probiotic effects can be achieved if the organisms adhere to mucus and/or intestinal epithelial cells (31, 62). It has recently been shown that lipoteichoic acid (LTA), a molecule associated with the surface of La1 bacteria, participates in their adhesion to intestinal cells (21) and has an immunomodulatory effect on gut homeostasis (64). However, competition experiments indicated that LTA is not the only surface molecule mediating La1 binding to epithelial cells (21). Indeed, it had already been suggested by Bernet et al. (5) that proteinaceous compounds are involved in the attachment of bacteria to these cells. This observation is in accordance with recent studies showing that surface proteins of other lactobacilli participate in adhesion to epithelial cell lines, gastrointestinal mucins, or extracellular matrix proteins (1,26,58,60).In this work, therefore, we have investigated the ability of La1 surface proteins to attach to intestinal epithelial cells and mucoproteins. We have identified the elongation factor Tu as a novel surface protein possessing the characteristics of an adhesion ...

IgG and IgG2 antibodies from cattle naturally infected with Anaplasma marginale recognize the recombinant vaccine candidate antigens VirB9, VirB10, and elongation factor-Tu

“…The codon-anticodon pairing triggers GTP hydrolysis by EF-Tu causing it to dissociate from the aminoacyl-tRNA, which now enters the A-site and can participate in chain elongation (Nilsson & Nissen 2005). Nevertheless, EF-Tu can be located in the bacterial surface, mediating attachment to host cells (Granato et al 2004). Due to the crucial importance of VirBs and EF-Tu for bacteria, the genes for these three proteins are expected to be conserved.…”

Lactobacillus spp. belonging to the Casei group display a variety of adhesins

“…The role of the TUFM protein in promoting the adhesion of bacteria (e.g. Lactobacillus johnsonii NCC533, Staphylococcus aureus, Mycoplasma pneumoniae, bacteria from the genus Leptospira) to the epithelium has been confirmed (Granato et al, 2004;Widjaja et al, 2017;Wolff et al, 2013). Western-blot detection of GAPDH in the extracellular protein fraction of the tested isolates showed the presence of proteins with molecular masses around 22 kDa, corresponding to the molecular mass of this protein, in bacteria of the Lactobacillus spp.…”

“…The capacity to bind to the mucus is also observed in the case of the Tu factor, a protein involved in the process of translation, identified on the cell surface of Lactobacillus bacteria (Granato et al, 2004) as well as the GroEL chaperonin, belonging to the class of conservative heat shock proteins. Both these proteins may also have an immunomodulatory effect by stimulating the IL-8 synthesis in intestinal epithelial cells (Bergonzelli et al, 2006).…”

PCR for Screening Potential Probiotic Lactobacilli for Piglets

“…Adhering protein molecules characterized from Lactobacillus are Mucus-binding protein (Mub) of L. reuteri 1063 (Roos & Jonsson, 2002), the lectine-like mannose-specific adhesin (Msa) of L. plantarum WCFS1 (Pretzer et al, 2005), the mucus adhesion promoting protein (MAPP or MapA) from L. reuteri 104R reported by its ability to bind porcine mucus and mucin (Rojas et al, 2002) and Caco-2 cells (Miyoshi et al, 2006) and the Mub of L. acidophilus NCFM (Buck et al, 2005). Moreover, two proteins EF-Tu (Elongation Factor-Tu) and GroEL (a class of heat shock protein) of L. johnsonii La1 NCC533 showed abilities to adhere to mucins at specific conditions of pH (Granato et al, 2004;Bergonzelli et al, 2006). Recently a piglet mucus adhesion protein was completely characterized from the potential probiotic L. fermentum strain BCS87 (Macías-Rodríguez et al, 2009).…”