Prebiotic Oligosaccharides Potentiate Host Protective Responses against L. Monocytogenes Infection

Abstract

Prebiotic oligosaccharides are used to modulate enteric pathogens and reduce pathogen shedding. The interactions with prebiotics that alter Listeria monocytogenes infection are not yet clearly delineated. L. monocytogenes cellular invasion requires a concerted manipulation of host epithelial cell membrane receptors to initiate internalization and infection often via receptor glycosylation. Bacterial interactions with host glycans are intimately involved in modulating cellular responses through signaling cascades at the membrane and in intracellular compartments. Characterizing the mechanisms underpinning these modulations is essential for predictive use of dietary prebiotics to diminish pathogen association. We demonstrated that human milk oligosaccharide (HMO) pretreatment of colonic epithelial cells (Caco-2) led to a 50% decrease in Listeria association, while Biomos pretreatment increased host association by 150%. L. monocytogenes-induced gene expression changes due to oligosaccharide pretreatment revealed global alterations in host signaling pathways that resulted in differential subcellular localization of L. monocytogenes during early infection. Ultimately, HMO pretreatment led to bacterial clearance in Caco-2 cells via induction of the unfolded protein response and eIF2 signaling, while Biomos pretreatment resulted in the induction of host autophagy and L. monocytogenes vacuolar escape earlier in the infection progression. This study demonstrates the capacity of prebiotic oligosaccharides to minimize infection through induction of host-intrinsic protective responses.

Keywords: ER stress; autophagy; c-di-AMP; cell-mediated immunity (CMI); eIF2 signaling; human milk oligosaccharide; prebiotic oligosaccharide; unfolded protein response.

Figure 1

Effect of prebiotic blocking on L. monocytogenes association with colonic epithelial cells.

Figure 2

TEM time course of L. monocytogenes infection. An infection time course of L. monocytogenes with Caco-2 monolayers following 1% HMO and Biomos pretreatment was done. Cytosolic Listeria are denoted by “c,” vacuole associated Listeria are denoted by “v.” Mitochondria are labeled as “M”; microvilli are labeled as “MV,” actin filaments are labeled as “AF,” tight junctions are labeled as “TJ.” Borders between two cells are indicated by the curved, dotted lines. (A–C) Intracellular L. monocytogenes at 20 min post infection (p.i.). Black and white arrows indicate bacterial and vacuole membrane. (D–F) L. monocytogenes at 40 min p.i. Inserts indicate lateral L. monocytogenes infection of neighboring cells along with corresponding actin tails. (G–I) L. monocytogenes at 60 min p.i. Inserts indicate differential infection stages between oligosaccharide pretreatments. (J–L) L. monocytogenes at 120 min p.i. Differential host outcome between oligosaccharide pretreatments. Black arrows indicate L. monocytogenes cell membrane ruffling during bacterial clearance by the host.

Figure 3

Prebiotic oligosaccharide-dependent differential canonical pathway enrichment following 60 min L. monocytogenes infection.

Figure 4

Differential canonical pathway expression during infection in the presence of prebiotic oligosaccharides. Differential expression (log₂FC) of individual genes visualized by heat map. Bacterial differential expression ranges from −10 < log₂FC < 10; host differential expression ranges from −4 < log₂FC < 4. HMO/+Lm is indicated as “H”; Biomos/+Lm is indicated as “B.” HMO is indicated by squares, triangles and circles above the membrane; Biomos is indicated by the hexagons above the membrane. (A) Differential subcellular localization is indicated by the intact double circle (H, vacuole associated) versus the dotted line double circle (B, vacuolar escape). c-di-AMP synthesis, transport and degradation genes are listed with arrow weights indicating host vs. bacterial contribution to cytosolic c-di-AMP. (B) Differential expression of signaling pathways with prebiotic treatment. (C) Differential expression of transcription factors and pseudogenes with prebiotic treatment.

Figure 5

Model of prebiotic-dependent differential host gene expression following L. monocytogenes infection.