Suppressive Effect on Activation of Macrophages by Lactobacillus casei Strain Shirota Genes Determining the Synthesis of Cell Wall-Associated Polysaccharides

Abstract

Although many Lactobacillus strains used as probiotics are believed to modulate host immune responses, the molecular natures of the components of such probiotic microorganisms directly involved in immune modulation process are largely unknown. We aimed to assess the function of polysaccharide moiety of the cell wall of Lactobacillus casei strain Shirota as a possible immune modulator which regulates cytokine production by macrophages. A gene survey of the genome sequence of L. casei Shirota hunted down a unique cluster of 10 genes, most of whose predicted amino acid sequences had similarities to various extents to known proteins involved in biosynthesis of extracellular or capsular polysaccharides from other lactic acid bacteria. Gene knockout mutants of eight genes from this cluster resulted in the loss of reactivity to L. casei Shirota-specific monoclonal antibody and extreme reduction of high-molecular-mass polysaccharides in the cell wall fraction, indicating that at least these genes are involved in biosynthesis of high-molecular-mass cell wall polysaccharides. By adding heat-killed mutant cells to mouse macrophage cell lines or to mouse spleen cells, the production of tumor necrosis factor alpha, interleukin-12 (IL-12), IL-10, and IL-6 was more stimulated than by wild-type cells. In addition, these mutants additively enhanced lipopolysaccharide-induced IL-6 production by RAW 264.7 mouse macrophage-like cells, while wild-type cells significantly suppressed the IL-6 production of RAW 264.7. Collectively, these results indicate that this cluster of genes of L. casei Shirota, which have been named cps1A, cps1B, cps1C, cps1D, cps1E, cps1F, cps1G, and cps1J, determine the synthesis of the highmolecular-mass polysaccharide moiety of the L. casei Shirota cell wall and that this polysaccharide moiety is the relevant immune modulator which may function to reduce excessive immune reactions during the activation of macrophages by L. casei Shirota.

The Surface-Associated Exopolysaccharide of Bifidobacterium longum 35624 Plays an Essential Role in Dampening Host Proinflammatory Responses and Repressing Local T H 17 Responses

“…As already mentioned, a strong modulator of intestinal immune responses is PSA from B. fragilis, which is well described to influence lymphocyte polarization and which suppresses IL-17 production by intestinal immune cells (41)(42)(43). In line with data presented here, exopolysaccharide gene knockout mutants of Lactobacillus casei Shirota induced significantly more proinflammatory cytokine secretion from a mouse macrophage cell line than did wildtype cells (44). In addition, the cytokine response of PBMCs to two isogenic strains of B. animalis subsp.…”

A Novel Rhamnose-Rich Hetero-exopolysaccharide Isolated from Lactobacillus paracasei DG Activates THP-1 Human Monocytic Cells

“…Several studies failed to assign evident immunostimulatory abilities to the EPSs, and it was proposed that the EPSs anchored on the bacterial outer surface are immunologically inert molecules that keep cell wall-associated MAMPs from direct contact with immune cell receptors (18,42,43). However, in our study, the purified EPS produced by L. paracasei DG displayed immunostimulatory activity, particularly in terms of chemokine expression.…”

Anti-Infective Activities of Lactobacillus Strains in the Human Intestinal Microbiota: from Probiotics to Gastrointestinal Anti-Infectious Biotherapeutic Agents

“…Genes of L. johnsonii NCC 533 code for the cell factors that account for the particular properties of adhesion/colonization (406,407). A cluster of genes in L. casei Shirota code for the synthesis of the high-molecular-mass components that can be relevant for the bacterial whole-cell-induced, cell contact-dependent immune modulation (408). Genes of L. rhamnosus GG code for the pili involved in adhesion (191)(192)(193)(194), for the formation of biofilm (409), and for enzymes involved in the biosynthesis of extracellular polysaccharides (410).…”