MUC13 negatively regulates tight junction proteins and intestinal epithelial barrier integrity via protein kinase C.

Glycosylated mucin proteins contribute to the essential barrier function of the intestinal epithelium. The transmembrane mucin MUC13 is an abundant intestinal glycoprotein with important functions for mucosal maintenance that are not yet completely understood. We demonstrate that in human intestinal epithelial monolayers, MUC13 localized to both the apical surface and the tight junction (TJ) region on the lateral membrane. MUC13 deletion resulted in increased transepithelial resistance (TEER) and reduced translocation of small solutes. TEER buildup in ΔMUC13 cells could be prevented by addition of MLCK, ROCK or protein kinase C (PKC) inhibitors. The levels of TJ proteins including claudins and occludin were highly increased in membrane fractions of MUC13 knockout cells. Removal of the MUC13 cytoplasmic tail (CT) also altered TJ composition but did not affect TEER. The increased buildup of TJ complexes in ΔMUC13 and MUC13-ΔCT cells was dependent on PKC. The responsible PKC member might be PKCδ (or PRKCD) based on elevated protein levels in the absence of full-length MUC13. Our results demonstrate for the first time that a mucin protein can negatively regulate TJ function and stimulate intestinal barrier permeability.

Glycosylated extracellular mucin domains protect against SARS-CoV-2 infection at the respiratory surface.

Mucins play an essential role in protecting the respiratory tract against microbial infections while also acting as binding sites for bacterial and viral adhesins. The heavily O-glycosylated gel-forming mucins MUC5AC and MUC5B eliminate pathogens by mucociliary clearance. Transmembrane mucins MUC1, MUC4, and MUC16 can restrict microbial invasion at the apical surface of the epithelium. In this study, we determined the impact of host mucins and mucin glycans on epithelial entry of SARS-CoV-2. Human lung epithelial Calu-3 cells express the SARS-CoV-2 entry receptor ACE2 and high levels of glycosylated MUC1, but not MUC4 and MUC16, on their cell surface. The O-glycan-specific mucinase StcE specifically removed the glycosylated part of the MUC1 extracellular domain while leaving the underlying SEA domain and cytoplasmic tail intact. StcE treatment of Calu-3 cells significantly enhanced infection with SARS-CoV-2 pseudovirus and authentic virus, while removal of terminal mucin glycans sialic acid and fucose from the epithelial surface did not impact viral entry. In Calu-3 cells, the transmembrane mucin MUC1 and ACE2 are located to the apical surface in close proximity and StcE treatment results in enhanced binding of purified spike protein. Both MUC1 and MUC16 are expressed on the surface of human organoid-derived air-liquid interface (ALI) differentiated airway cultures and StcE treatment led to mucin removal and increased levels of SARS-CoV-2 replication. In these cultures, MUC1 was highly expressed in non-ciliated cells while MUC16 was enriched in goblet cells. In conclusion, the glycosylated extracellular domains of different transmembrane mucins might have similar protective functions in different respiratory cell types by restricting SARS-CoV-2 binding and entry.

The ALPK1 pathway drives the inflammatory response to Campylobacter jejuni in human intestinal epithelial cells.

The Gram-negative bacterium Campylobacter jejuni is a major cause of foodborne disease in humans. After infection, C. jejuni rapidly colonizes the mucus layer of the small and large intestine and induces a potent pro-inflammatory response characterized by the production of a large repertoire of cytokines, chemokines, and innate effector molecules, resulting in (bloody) diarrhea. The virulence mechanisms by which C. jejuni causes this intestinal response are still largely unknown. Here we show that C. jejuni releases a potent pro-inflammatory compound into its environment, which activates an NF-κB-mediated pro-inflammatory response including the induction of CXCL8, CXCL2, TNFAIP2 and PTGS2. This response was dependent on a functional ALPK1 receptor and independent of Toll-like Receptor and Nod-like Receptor signaling. Chemical characterization, inactivation of the heptose-biosynthesis pathway by the deletion of the hldE gene and in vitro engineering identified the released factor as the LOS-intermediate ADP-heptose and/or related heptose phosphates. During C. jejuni infection of intestinal cells, the ALPK1-NF-κB axis was potently activated by released heptose metabolites without the need for a type III or type IV injection machinery. Our results classify ADP-heptose and/or related heptose phosphates as a major virulence factor of C. jejuni that may play an important role during Campylobacter infection in humans.

MUC1 is a receptor for the Salmonella SiiE adhesin that enables apical invasion into enterocytes.

The cellular invasion machinery of the enteric pathogen Salmonella consists of a type III secretion system (T3SS) with injectable virulence factors that induce uptake by macropinocytosis. Salmonella invasion at the apical surface of intestinal epithelial cells is inefficient, presumably because of a glycosylated barrier formed by transmembrane mucins that prevents T3SS contact with host cells. We observed that Salmonella is capable of apical invasion of intestinal epithelial cells that express the transmembrane mucin MUC1. Knockout of MUC1 in HT29-MTX cells or removal of MUC1 sialic acids by neuraminidase treatment reduced Salmonella apical invasion but did not affect lateral invasion that is not hampered by a defensive barrier. A Salmonella deletion strain lacking the SiiE giant adhesin was unable to invade intestinal epithelial cells through MUC1. SiiE-positive Salmonella closely associated with the MUC1 layer at the apical surface, but invaded Salmonella were negative for the adhesin. Our findings uncover that the transmembrane mucin MUC1 is required for Salmonella SiiE-mediated entry of enterocytes via the apical route.

Duplicated TLR5 of zebrafish functions as a heterodimeric receptor.

Toll-like receptor 5 (TLR5) of mammals, birds, and reptiles detects bacterial flagellin and signals as a homodimeric complex. Structural studies using truncated TLR5b of zebrafish confirm the homodimeric TLR5-flagellin interaction. Here we provide evidence that zebrafish (Danio rerio) TLR5 unexpectedly signals as a heterodimer composed of the duplicated gene products drTLR5b and drTLR5a. Flagellin-induced signaling by the zebrafish TLR5 heterodimer increased in the presence of the TLR trafficking chaperone UNC93B1. Targeted exchange of drTLR5b and drTLR5a regions revealed that TLR5 activation needs a heterodimeric configuration of the receptor ectodomain and cytoplasmic domain, consistent with ligand-induced changes in receptor conformation. Structure-guided substitution of the presumed principal flagellin-binding site in human TLR5 with corresponding zebrafish TLR5 residues abrogated human TLR5 activation, indicating a species-specific TLR5-flagellin interaction. Our findings indicate that the duplicated TLR5 of zebrafish underwent subfunctionalization through concerted coevolution to form a unique heterodimeric flagellin receptor that operates fundamentally differently from TLR5 of other species.

Generation of the membrane potential and its impact on the motility, ATP production and growth in Campylobacter jejuni.

The generation of a membrane potential (Δψ), the major constituent of the proton motive force (pmf), is crucial for ATP synthesis, transport of nutrients and flagellar rotation. Campylobacter jejuni harbors a branched electron transport chain, enabling respiration with different electron donors and acceptors. Here, we demonstrate that a relatively high Δψ is only generated in the presence of either formate as electron donor or oxygen as electron acceptor, in combination with an acceptor/donor respectively. We show the necessity of the pmf for motility and growth of C. jejuni. ATP generation is not only accomplished by oxidative phosphorylation via the pmf, but also by substrate-level phosphorylation via the enzyme AckA. In response to a low oxygen tension, C. jejuni increases the transcription and activity of the donor complexes formate dehydrogenase (FdhABC) and hydrogenase (HydABCD) as well as the transcription of the alternative respiratory acceptor complexes. Our findings suggest that in the gut of warm-blooded animals, C. jejuni depends on at least formate or hydrogen as donor (in the anaerobic lumen) or oxygen as acceptor (near the epithelial cells) to generate a pmf that sustains efficient motility and growth for colonization and pathogenesis.

Catabolite repression in Campylobacter jejuni correlates with intracellular succinate levels.

Bacteria have evolved different mechanisms to catabolize carbon sources from nutrient mixtures. They first consume their preferred carbon source, before others are used. Regulatory mechanisms adapt the metabolism accordingly to maximize growth and to outcompete other organisms. The human pathogen Campylobacter jejuni is an asaccharolytic Gram-negative bacterium that catabolizes amino acids and organic acids for growth. It prefers serine and aspartate as carbon sources, however it lacks all regulators known to be involved in regulating carbon source utilization in other organisms. In which manner C. jejuni adapts its metabolism towards the presence or absence of preferred carbon sources is unknown. In this study, we show with transcriptomic analysis and enzyme assays how C. jejuni adapts its metabolism in response to its preferred carbon sources. In the presence of serine as well as lactate and pyruvate C. jejuni inhibits the utilization of other carbon sources, by repressing the expression of a number of central metabolic enzymes. The regulatory proteins RacR, Cj1000 and CsrA play a role in the regulation of these metabolic enzymes. This metabolism dependent transcriptional repression correlates with an accumulation of intracellular succinate. Hence, we propose a demand-based catabolite repression mechanism in C. jejuni, depended on intracellular succinate levels.

Host cell binding of the flagellar tip protein of Campylobacter jejuni.

Flagella are nanofibers that drive bacterial movement. The filaments are generally composed of thousands of tightly packed flagellin subunits with a terminal cap protein, named FliD. Here, we report that the FliD protein of the bacterial pathogen Campylobacter jejuni binds to host cells. Live-cell imaging and confocal microscopy showed initial contact of the bacteria with epithelial cells via the flagella tip. Recombinant FliD protein bound to the surface of intestinal epithelial cells in a dose-dependent fashion. Search for the FliD binding site on the host cell using cells with defined glycosylation defects indicated glycosaminoglycans as a putative target. Heparinase treatment of wild type cells and an excess of soluble heparin abolished FliD binding. Binding assays showed direct and specific binding of FliD to heparin. Addition of an excess of purified FliD or heparin reduced the attachment of viable C. jejuni to the host cells. The host cell binding domain of FliD was mapped to the central region of the protein. Overall, our results indicate that the C. jejuni flagellar tip protein FliD acts as an attachment factor that interacts with cell surface heparan sulfate glycosaminoglycan receptors.

Feedback control of Campylobacter jejuni flagellin levels through reciprocal binding of FliW to flagellin and the global regulator CsrA.

Bacterial flagella assembly is tightly regulated to ensure a timely and sequential production of the various flagellum constituents. In the pathogen Campylobacter jejuni the hierarchy in flagella biosynthesis is largely determined at the transcriptional level through the activity of the alternative sigma factors sigma54 and sigma28 . Here, we report that C. jejuni flagellin levels are also controlled at the post-transcriptional level via the thus far poorly-characterized flagellar assembly factor FliW. Analysis of flagellin synthesis in C. jejuni 81116 and a ΔfliW knock-out mutant showed reduced flagellin protein levels in the mutant strain while ectopic expression of FliW resulted in enhanced levels. Real-time RT-PCR revealed relatively minor changes in flaA and flaB mRNA levels for the recombinant and parent strain consistent with post-transcriptional regulation. Purified FliW was found to bind to FlaA and FlaB flagellin as well as to the global post-transcriptional regulator CsrA. Inactivation of CsrA resulted in increased levels of flagellin translation. An in vitro translation assay confirmed the regulatory role of CsrA in flagellin biosynthesis. We propose that competitive reciprocal binding of FliW to flagellins and the RNA binding protein CsrA serves as a feedback mechanism to control the number of cytosolic flagellin copies at the protein level.

Meningococcal Outer Membrane Vesicle Composition-Dependent Activation of the Innate Immune Response.

Meningococcal outer membrane vesicles (OMVs) have been extensively investigated and successfully implemented as vaccines. They contain pathogen-associated molecular patterns, including lipopolysaccharide (LPS), capable of triggering innate immunity. However, Neisseria meningitidis contains an extremely potent hexa-acylated LPS, leading to adverse effects when its OMVs are applied as vaccines. To create safe OMV vaccines, detergent treatment is generally used to reduce the LPS content. While effective, this method also leads to loss of protective antigens such as lipoproteins. Alternatively, genetic modification of LPS can reduce its toxicity. In the present study, we have compared the effects of standard OMV isolation methods using detergent or EDTA with those of genetic modifications of LPS to yield a penta-acylated lipid A (lpxL1 and pagL) on the in vitro induction of innate immune responses. The use of detergent decreased both Toll-like receptor 4 (TLR4) and TLR2 activation by OMVs, while the LPS modifications reduced only TLR4 activation. Mutational removal of PorB or lipoprotein factor H binding protein (fHbp), two proteins known to trigger TLR2 signaling, had no effect, indicating that multiple TLR2 ligands are removed by detergent treatment. Detergent-treated OMVs and lpxL1 OMVs showed similar reductions of cytokine profiles in the human monocytic cell line MM6 and human dendritic cells (DCs). OMVs with the alternative penta-acylated LPS structure obtained after PagL-mediated deacylation showed reduced induction of proinflammatory cytokines interleukin-6 (IL-6) and IL-1ß but not of IP-10, a typical TRIF-dependent chemokine. Taken together, these data show that lipid A modification can be used to obtain OMVs with reduced activation of innate immunity, similar to what is found after detergent treatment.

Inflammasome activation by Campylobacter jejuni.

The Gram-negative pathogen Campylobacter jejuni is the most common cause of bacterial foodborne disease worldwide. The mechanisms that lead to bacterial invasion of eukaryotic cells and massive intestinal inflammation are still unknown. In this study, we report that C. jejuni infection of mouse macrophages induces upregulation of pro-IL-1ß transcript and secretion of IL-1ß without eliciting cell death. Immunoblotting indicated cleavage of caspase-1 and IL-1ß in infected cells. In bone marrow-derived macrophages from different knockout mice, IL-1ß secretion was found to require NLRP3, ASC, and caspase-1/11 but not NLRC4. In contrast to NLRP3 activation by ATP, C. jejuni activation did not require priming of these macrophages. C. jejuni also activated the NLRP3 inflammasome in human macrophages as indicated by the presence of ASC foci and caspase-1-positive cells. Analysis of a vast array of C. jejuni mutants with defects in capsule formation, LPS biosynthesis, chemotaxis, flagella synthesis and flagellin (-like) secretion, type 6 secretion system needle protein, or cytolethal distending toxin revealed a direct correlation between the number of intracellular bacteria and NLRP3 inflammasome activation. The C. jejuni invasion-related activation of the NLRP3 inflammasome without cytotoxicity and even in nonprimed cells extends the known repertoire of bacterial inflammasome activation and likely contributes to C. jejuni-induced intestinal inflammation.

The Campylobacter jejuni†RacRS system regulates fumarate utilization in a low oxygen environment.

The natural environment of the human pathogen Campylobacter jejuni is the gastrointestinal tract of warm-blooded animals. In the gut, the availability of oxygen is limited; therefore, less efficient electron acceptors such as nitrate or fumarate are used by C. jejuni. The molecular mechanisms that regulate the activity of the highly branched respiratory chain of C. jejuni are still a mystery mainly because C. jejuni lacks homologues of transcription factors known to regulate energy metabolism in other bacteria. Here we demonstrate that dependent on the available electron acceptors the two-component system RacRS controls the production of fumarate from aspartate, as well as its transport and reduction to succinate. Transcription profiling, DNAse protection and functional assays showed that phosphorylated RacR binds to and represses at least five promoter elements located in front of genes involved in the uptake and synthesis of fumarate. The RacRS system is active in the presence of nitrate and trimethyl-amine-N-oxide under oxygen-limited conditions when fumarate is less preferred as an alternative electron acceptor. In the inactive state, RacRS allows utilization of fumarate for respiration. The unique C. jejuni RacRS regulatory system illustrates the disparate evolution of Campylobacter and aids the survival of this pathogen.

Identification of a functional type VI secretion system in Campylobacter jejuni conferring capsule polysaccharide sensitive cytotoxicity.

The pathogen Campylobacter jejuni is the principal cause of bacterial food-borne infections. The mechanism(s) that contribute to bacterial survival and disease are still poorly understood. In other bacterial species, type VI secretion systems (T6SS) are increasingly recognized to contribute to bacterial pathogenesis by toxic effects on host cells or competing bacterial species. Here we report the presence of a functional Type VI secretion system in C. jejuni. Proteome and genetic analyses revealed that C. jejuni strain 108 contains a 17-kb T6SS gene cluster consisting of 13 T6SS-conserved genes, including the T6SS hallmark genes hcp and vgrG. The cluster lacks an ortholog of the ClpV ATPase considered important for T6SS function. The sequence and organization of the C. jejuni T6SS genes resemble those of the T6SS located on the HHGI1 pathogenicity island of Helicobacter hepaticus. The C. jejuni T6SS is integrated into the earlier acquired Campylobacter integrated element CJIE3 and is present in about 10% of C. jejuni isolates including several isolates derived from patients with the rare clinical feature of C. jejuni bacteremia. Targeted mutagenesis of C. jejuni T6SS genes revealed T6SS-dependent secretion of the Hcp needle protein into the culture supernatant. Infection assays provided evidence that the C. jejuni T6SS confers contact-dependent cytotoxicity towards red blood cells but not macrophages. This trait was observed only in a capsule-deficient bacterial phenotype. The unique C. jejuni T6SS phenotype of capsule-sensitive contact-mediated hemolysis represents a novel evolutionary pathway of T6SS in bacteria and expands the repertoire of virulence properties associated with T6SS.

CD14 protein acts as an adaptor molecule for the immune recognition of Salmonella curli fibers.

Amyloids, protein aggregates with a cross ß-sheet structure, contribute to inflammation in debilitating disorders, including Alzheimer's disease. Enteric bacteria also produce amyloids, termed curli, contributing to inflammation during infection. It has been demonstrated that curli and ß-amyloid are recognized by the immune system via the Toll-like receptor (TLR) 2/TLR1 complex. Here we investigated the role of CD14 in the immune recognition of bacterial amyloids. We used HeLa 57A cells, a human cervical cancer cell line containing a luciferase reporter gene under the control of an NF-κB promoter. When HeLa 57A cells were transiently transfected with combinations of human expression vectors containing genes for TLR2, TLR1, and CD14, membrane-bound CD14 enhanced NF-κB activation through the TLR2/TLR1 complex stimulated with curli fibers or recombinant CsgA, the curli major subunit. Similarly, soluble CD14 augmented the TLR2/TLR1 response to curli fibers in the absence of membrane-bound CD14. We further revealed that IL-6 and nitric oxide production were significantly higher by wild-type (C57BL/6) bone marrow-derived macrophages compared with TLR2-deficient or CD14-deficient bone marrow-derived macrophages when stimulated with curli fibers, recombinant CsgA, or synthetic CsgA peptide, CsgA-R4-5. Binding assays demonstrated that recombinant TLR2, TLR1, and CD14 bound purified curli fibers. Interestingly, CD14-curli interaction was specific to the fibrillar form of the amyloid, as demonstrated by using synthetic CsgA peptides proficient and deficient in fiber formation, respectively. Activation of the TLR2/TLR1/CD14 trimolecular complex by amyloids provides novel insights for innate immunity with implications for amyloid-associated diseases.

Campylobacter fetus infections in humans: exposure and disease.

Campylobacter fetus can cause intestinal illness and, occasionally, severe systemic infections. Infections mainly affect persons at higher risk, including elderly and immunocompromised individuals and those with occupational exposure to infected animals. Outbreaks are infrequent but have provided insight into sources. Source attribution of sporadic cases through case-control interviews has not been reported. The reservoirs for C. fetus are mainly cattle and sheep. Products from these animals are suspected as sources for human infections. Campylobacter fetus is rarely isolated from food, albeit selective isolation methods used in food microbiology are not suited for its detection. We hypothesize that the general population is regularly exposed to C. fetus through foods of animal origin, cross-contaminated foodstuffs, and perhaps other, as yet unidentified, routes. Campylobacter fetus infection should be suspected particularly in patients with nonspecific febrile illness who are immunocompromised or who may have been occupationally exposed to ruminants.

Cleavage and activation of a Toll-like receptor by microbial proteases.

Toll-like receptors (TLRs) are innate receptors that show high conservation throughout the animal kingdom. Most TLRs can be clustered into phylogenetic groups that respond to similar types of ligands. One exception is avian TLR15. This receptor does not categorize into one of the existing groups of TLRs and its ligand is still unknown. Here we report that TLR15 is a sensor for secreted virulence-associated fungal and bacterial proteases. Activation of TLR15 involves proteolytic cleavage of the receptor ectodomain and stimulation of NF-κB-dependent gene transcription. Receptor activation can be mimicked by the expression of a truncated TLR15 of which the entire ectodomain is removed, suggesting that receptor cleavage alleviates receptor inhibition by the leucine-rich repeat domain. Our results indicate TLR15 as a unique type of innate immune receptor that combines TLR characteristics with an activation mechanism typical for the evolutionary distinct protease-activated receptors.

Biological functions of bacterial lysophospholipids.

Lysophospholipids (LPLs) are lipid-derived metabolic intermediates in the cell membrane. The biological functions of LPLs are distinct from their corresponding phospholipids. In eukaryotic cells LPLs are important bioactive signaling molecules that regulate many important biological processes, but in bacteria the function of LPLs is still not fully defined. Bacterial LPLs are usually present in cells in very small amounts, but can strongly increase under certain environmental conditions. In addition to their basic function as precursors in membrane lipid metabolism, the formation of distinct LPLs contributes to the proliferation of bacteria under harsh circumstances or may act as signaling molecules in bacterial pathogenesis. This review provides an overview of the current knowledge of the biological functions of bacterial LPLs including lysoPE, lysoPA, lysoPC, lysoPG, lysoPS and lysoPI in bacterial adaptation, survival, and host-microbe interactions.

Campylobacter jejuni benefits from the bile salt deoxycholate under low-oxygen condition in a PldA dependent manner.

Enteric bacteria need to adapt to endure the antibacterial activities of bile salts in the gut. Phospholipase A (PldA) is a key enzyme in the maintenance of bacterial membrane homeostasis. Bacteria respond to stress by modulating their membrane composition. Campylobacter jejuni is the most common cause of human worldwide. However, the mechanism by which C. jejuni adapts and survives in the gut environment is not fully understood. In this study, we investigated the roles of PldA, bile salt sodium deoxycholate (DOC), and oxygen availability in C. jejuni biology, mimicking an in vivo situation. Growth curves were used to determine the adaptation of C. jejuni to bile salts. RNA-seq and functional assays were employed to investigate the PldA-dependent and DOC-induced changes in gene expression that influence bacterial physiology. Survival studies were performed to address oxidative stress defense in C. jejuni. Here, we discovered that PldA of C. jejuni is required for optimal growth in the presence of bile salt DOC. Under high oxygen conditions, DOC is toxic to C. jejuni, but under low oxygen conditions, as is present in the lumen of the gut, C. jejuni benefits from DOC. C. jejuni PldA seems to enable the use of iron needed for optimal growth in the presence of DOC but makes the bacterium more vulnerable to oxidative stress. In conclusion, DOC stimulates C. jejuni growth under low oxygen conditions and alters colony morphology in a PldA-dependent manner. C. jejuni benefits from DOC by upregulating iron metabolism in a PldA-dependent manner.

Chicken TLR21 is an innate CpG DNA receptor distinct from mammalian TLR9.

TLRs comprise a family of evolutionary conserved sensory receptors that respond to distinct classes of ligands. For one major evolutionary branch of TLRs, the ligands are still largely unknown. Here we report the cloning and function of one member of this group, chicken TLR21 (chTLR21). This TLR is absent in the human species but has homologs in fish and frog and displays similarity with mouse TLR13. Expression of chTLR21 in HEK293 cells resulted in activation of NF-kappaB in response to unmethylated CpG DNA, typically recognized by mammalian TLR9. Silencing of chTLR21 (but not chTLR4) in chicken macrophages inhibited the response to CpG-DNA (but not to LPS), indicating similar functionality of the endogenous receptor. ChTLR21 responded to human- and murine-specific TLR9 ligands, as well as to bacterial genomic DNA isolated from Salmonella enterica serovar Enteritidis. Confocal microscopy located chTLR21 in the same intracellular compartments as human TLR9. Inhibition of the chTLR21 response by the endosomal maturation inhibitor chloroquine suggested that the receptor is functional in endolysosomes, as known for TLR9. The analogous localization and function of the phylogenetically only distantly related chTLR21 and mammalian TLR9 suggest that during evolution different classes of TLRs have emerged that recognize the same type of ligands.

Galacto-oligosaccharides as an anti-bacterial and anti-invasive agent in lung infections.

Emerging antimicrobial resistance in infections asks for novel intervention strategies. Galacto-oligosaccharides (GOS) might be attractive alternatives to antibiotics due to their anti-inflammatory and anti-adhesive properties. Mannheimia haemolytica is one of the major Pasteurellaceae associated with bovine lung infections. Using M. haemolytica, we demonstrated that GOS have the capacity to reduce bacterial viability and can be used as adjuvant to improve antibiotic efficacy. Using M. haemolytica-treated primary bronchial epithelial cells (PBECs) of calves, we identified the anti-adhesive and anti-invasive activities of GOS. The observed inhibition of cytokine/chemokine release and the prevention of airway epithelial barrier dysfunction in M. haemolytica-treated PBECs by GOS might be related to the downregulation of "toll-like receptor 4/nuclear factor-κB" pathway and the anti-invasive and anti-adhesive properties of GOS. Particularly, GOS lowered lipopolysaccharides- but not flagellin-induced cytokine/chemokine release in calf and human airway epithelial cells. Finally, we performed in vivo experiments in calves and demonstrated for the first time that intranasal application of GOS can relieve lung infections/inflammation and lower M. haemolytica positivity in the lungs without affecting clinical performance. These findings not only shed light on the anti-inflammatory mechanisms of GOS during lung infections, but GOS might also be a promising anti-bacterial agent for preventing (lung) infections.