Search

Bacteria can exploit membrane receptor integrins for cellular invasion, either by direct binding of bacterial adhesins or utilizing extracellular matrix components. MUC1 is a large transmembrane glycoprotein expressed by most epithelial cells that can have direct defensive or receptor functions at the host-microbe interface and is involved in facilitating integrin clustering., At the intestinal host-microbe interface, the transmembrane mucin MUC1 can function as a physical barrier as well as a receptor for bacteria. MUC1 also influences epithelial cell morphology and receptor function. Various bacterial pathogens can exploit integrins to infect eukaryotic cells. It is yet unclear whether MUC1 influences the interaction of bacteria with integrins. We used Escherichia coli expressing the invasin (inv) protein of Yersinia pseudotuberculosis (E. coli inv) to assess the effects of MUC1 on β1 integrin (ITGB1)-mediated bacterial invasion. Our results show that expression of full-length MUC1 does not yield a physical barrier but slightly enhances E. coli inv uptake. Enzymatic removal of the MUC1 extracellular domain (ED) using a secreted protease of C1 esterase inhibitor (StcE) of pathogenic Escherichia coli had no additional effect on E. coli inv invasion. In contrast, expression of a truncated MUC1 that lacks the cytoplasmic tail (CT) reduced bacterial entry substantially. Substitution of tyrosine residues in the MUC1 CT also reduced bacterial uptake, while deletion of the C-terminal half of the cytoplasmic tail only had a minor effect, pointing to a regulatory role of tyrosine phosphorylation and the N-terminal region of the MUC1 CT in integrin-mediated uptake process. Unexpectedly, StcE removal of the ED in MUC1-ΔCT cells reversed the block in bacterial invasion. Together, these findings indicate that MUC1 can facilitate β1-integrin-mediated bacterial invasion by a concerted action of the large glycosylated extracellular domain and the membrane-juxtaposed cytoplasmic tail region.

The human gut microbiota plays a central role in intestinal health and disease. Yet, many of its bacterial constituents are functionally still largely unexplored. A crucial prerequisite for bacterial survival and proliferation is the creation and/or exploitation of an own niche. For many bacterial species that are linked to human disease, the inner mucus layer was found to be an important niche. Allobaculum mucolyticum is a newly identified, IBD-associated species that is thought be closely associated with the host epithelium. To explore how this bacterium is able to effectively colonize this niche, we screened its genome for factors that may contribute to mucosal colonization. Up to 60 genes encoding putative Carbohydrate Active Enzymes (CAZymes) were identified in the genome of A. mucolyticum. Mass spectrometry revealed 49 CAZymes of which 26 were significantly enriched in its secretome. Functional assays demonstrated the presence of CAZyme activity in A. mucolyticum conditioned medium, degradation of human mucin O-glycans, and utilization of liberated non-terminal monosaccharides for bacterial growth. The results support a model in which sialidases and fucosidases remove terminal O-glycan sugars enabling subsequent degradation and utilization of carbohydrates for A. mucolyticum growth. A. mucolyticum CAZyme secretion may thus facilitate bacterial colonization and degradation of the mucus layer and may pose an interesting target for future therapeutic intervention.

Respiratory infections caused by Bordetella pertussis are reemerging despite high pertussis vaccination coverage. Since the introduction of the acellular pertussis vaccine in the late twentieth century, circulating B. pertussis strains increasingly lack expression of the vaccine component pertactin (Prn). In some countries, up to 90% of the circulating B. pertussis strains are deficient in Prn. To better understand the resurgence of pertussis, we investigated the response of human monocyte-derived dendritic cells (moDCs) to naturally circulating Prn-expressing (Prn-Pos) and Prn-deficient (Prn-Neg) B. pertussis strains from 2016 in the Netherlands. Transcriptome analysis of moDC showed enriched IFNα response-associated gene expression after exposure to Prn-Pos B. pertussis strains, whereas the Prn-Neg strains induced enriched expression of interleukin- and TNF-signaling genes, as well as other genes involved in immune activation. Multiplex immune assays confirmed enhanced proinflammatory cytokine secretion by Prn-Neg stimulated moDC. Comparison of the proteomes from the Prn-Pos and Prn-Neg strains revealed, next to the difference in Prn, differential expression of a number of other proteins including several proteins involved in metabolic processes. Our findings indicate that Prn-deficient B. pertussis strains induce a distinct and stronger immune activation of moDCs than the Prn-Pos strains. These findings highlight the role of pathogen adaptation in the resurgence of pertussis as well as the effects that vaccine pressure can have on a bacterial population.

Pertussis is a highly contagious respiratory infection caused by the bacterium Bordetella pertussis. Humans are the only known natural reservoir of B. pertussis. In mice, macrophages and NK cells have a key role in confining B. pertussis to the respiratory tract. However, the mechanisms underlying this process, particularly during human infections, remain unclear. Here we characterized the activation of human macrophages and NK cells in response to B. pertussis and unraveled the role of inflammasomes in this process. NLRP3 inflammasome activation by B. pertussis in human macrophage-like THP-1 cells and primary monocyte-derived macrophages (mo-MΦ) was shown by the visualization of ASC-speck formation, pyroptosis, and the secretion of caspase-mediated IL-1β and IL-18. In contrast to macrophages, stimulation of human CD56+CD3- NK cells by B. pertussis alone did not result in activation of these cells. However, co-culture of B. pertussis-stimulated mo-MΦ and autologous NK cells resulted in high amounts of IFNγ secretion and an increased frequency of IL-2Rα+ and HLA-DR+ NK cells, indicating NK cell activation. This activation was significantly reduced upon inhibition of inflammasome activity or blocking of IL-18 in the mo-MΦ/NK cell co-culture. Furthermore, we observed increased secretion of proinflammatory cytokines in the B. pertussis-stimulated mo-MΦ/NK co-culture compared to the mo-MΦ single culture. Our results demonstrate that B. pertussis induces inflammasome activation in human macrophages and that the IL-18 produced by these cells is required for the activation of human NK cells, which in turn enhances the pro-inflammatory response to this pathogen. Our data provides a better understanding of the underlying mechanisms involved in the induction of innate immune responses against B. pertussis. These findings contribute to the knowledge required for the development of improved intervention strategies to control this highly contagious disease.

Mucosal surfaces line our body cavities and provide the interaction surface between commensal and pathogenic microbiota and the host. The barrier function of the mucosal layer is largely maintained by gel-forming mucin proteins that are secreted by goblet cells. In addition, mucosal epithelial cells express cell-bound mucins that have both barrier and signaling functions. The family of transmembrane mucins consists of diverse members that share a few characteristics. The highly glycosylated extracellular mucin domains inhibit invasion by pathogenic bacteria and can form a tight mesh structure that protects cells in harmful conditions. The intracellular tails of transmembrane mucins can be phosphorylated and connect to signaling pathways that regulate inflammation, cell-cell interactions, differentiation, and apoptosis. Transmembrane mucins play important roles in preventing infection at mucosal surfaces, but are also renowned for their contributions to the development, progression, and metastasis of adenocarcinomas. In general, transmembrane mucins seem to have evolved to monitor and repair damaged epithelia, but these functions can be highjacked by cancer cells to yield a survival advantage. This review presents an overview of the current knowledge of the functions of transmembrane mucins in inflammatory processes and carcinogenesis in order to better understand the diverse functions of these multifunctional proteins.

Bacterial flagellin is important for intestinal immune homeostasis. Flagellins from most species activate Toll-like receptor 5 (TLR5). The principal bacterial food-borne pathogen Campylobacter jejuni escapes TLR5 recognition, probably due to an alternate flagellin subunit structure. We investigated the molecular basis of TLR5 evasion by aiming to reconstitute TLR5 stimulating activity in live C. jejuni. Both native glycosylated C. jejuni flagellins (FlaA and FlaB) and recombinant proteins purified from Escherichia coli failed to activate NF-kappaB in HEK293 cells expressing TLR5. Introduction of multiple defined regions from Salmonella flagellin into C. jejuni FlaA via a recombinatorial approach revealed three regions critical for the activation of human and mouse TLR5, including a beta-hairpin structure not previously implicated in TLR5 recognition. Surprisingly, this domain was not required for the activation of chicken TLR5, indicating a selective requirement for the beta-hairpin in the recognition of mammalian TLR5. Expression of the active chimeric protein in C. jejuni resulted in secreted glycosylated flagellin that induced a potent TLR5 response. Overall, our results reveal a novel structural requirement for TLR5 recognition of bacterial flagellin and exclude flagellin glycosylation as an additional mechanism of bacterial evasion of the TLR5 response.

Flagellin of Campylobacter jejuni is extensively modified with (derivatives of) pseudaminic acid. The flagellar glycosylation locus contains several genes with homopolymeric G-tracts prone to slipped-strand mispairing, some of which belong to the maf gene family. We investigated the function of the putative phase-variable maf4 gene of C. jejuni strain 108. A constructed maf4 mutant displayed unaltered flagella assembly and bacterial motility. 2D-PAGE analysis revealed that the flagellin of strain 108 migrated at a more acidic pI than the protein of the Maf4 mutant. MS-MS in combination with high-resolution matrix-assisted laser desorption/ionization Fourier transform ion cyclotron MS (MALDI-FT-ICR-MS) on flagellin-derived glycopeptides showed that the flagellins of the mutant lacked two previously unidentified modifications of pseudaminic acid. These glycoforms carried additional CO(2) and C(2)H(2)O(2) groups, consistent with the more acidic pI of the wild-type flagellin. Phenotypically, the maf4 mutant displayed strongly delayed bacterial autoagglutination. Collectively, our results suggest that the presence of a functional Maf4 expands the flagellin glycan repertoire with novel glycoforms of pseudaminic acid and, in the event of phase variation, alters the population behaviour of C. 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-1β transcript and secretion of IL-1β without eliciting cell death. Immunoblotting indicated cleavage of caspase-1 and IL-1β in infected cells. In bone marrow–derived macrophages from different knockout mice, IL-1β 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 species Campylobacter jejuni is naturally competent for DNA uptake; nevertheless, nonnaturally transformable strains do exist. For a subset of strains we previously showed that a periplasmic DNase, encoded by dns , inhibits natural transformation in C. jejuni . In the present study, genetic factors coding for DNase activity in the absence of dns were identified. DNA arrays indicated that nonnaturally transformable dns -negative strains contain putative DNA/RNA nonspecific endonucleases encoded by CJE0566 and CJE1441 of strain RM1221. These genes are located on C. jejuni integrated elements 2 and 4. Expression of CJE0566 and CJE1441 from strain RM1221 and a homologous gene from strain 07479 in DNase-negative Escherichia coli and C. jejuni strains indicated that these genes code for DNases. Genetic transfer of the genes to a naturally transformable C. jejuni strain resulted in a decreased efficiency of natural transformation. Modeling suggests that the C. jejuni DNases belong to the Serratia nuclease family. Overall, the data indicate that the acquisition of prophage-encoded DNA/RNA nonspecific endonucleases inhibits the natural transformability of C. jejuni through hydrolysis of DNA.

Acquired immunity is an important factor in the epidemiology of campylobacteriosis in the developing world, apparently limiting symptomatic infection to children of less than two years. However, also in developed countries the highest incidence is observed in children under five years and the majority of Campylobacter infections are asymptomatic, which may be related to the effects of immunity and/or the ingested doses. Not accounting for immunity in epidemiological studies may lead to biased results due to the misclassification of Campylobacter-exposed but apparently healthy persons as unexposed. In risk assessment studies, health risks may be overestimated when immunity is neglected.