Commensal and pathogenic biofilms differently modulate peri‐implant oral mucosa in an organotypic model

The impact of oral commensal and pathogenic bacteria on peri‐implant mucosa is not well understood, despite the high prevalence of peri‐implant infections. Hence, we investigated responses of the peri‐implant mucosa to Streptococcus oralis or Aggregatibacter actinomycetemcomitans biofilms using a novel in vitro peri‐implant mucosa‐biofilm model. Our 3D model combined three components, organotypic oral mucosa, implant material, and oral biofilm, with structural assembly close to native situation. S. oralis induced a protective stress response in the peri‐implant mucosa through upregulation of heat shock protein (HSP70) genes. Attenuated inflammatory response was indicated by reduced cytokine levels of interleukin‐6 (IL‐6), interleukin‐8 (CXCL8), and monocyte chemoattractant protein‐1 (CCL2). The inflammatory balance was preserved through increased levels of tumor necrosis factor‐alpha (TNF‐α). A. actinomycetemcomitans induced downregulation of genes important for cell survival and host inflammatory response. The reduced cytokine levels of chemokine ligand 1 (CXCL1), CXCL8, and CCL2 also indicated a diminished inflammatory response. The induced immune balance by S. oralis may support oral health, whereas the reduced inflammatory response to A. actinomycetemcomitans may provide colonisation advantage and facilitate later tissue invasion. The comprehensive characterisation of peri‐implant mucosa‐biofilm interactions using our 3D model can provide new knowledge to improve strategies for prevention and therapy of peri‐implant disease.     

sarA negatively regulates Staphylococcus epidermidis biofilm formation by modulating expression of 1 MDa extracellular matrix binding protein and autolysis‐dependent release of eDNA

Biofilm formation is essential for Staphylococcus epidermidis pathogenicity in implant‐associated infections. Nonetheless, large proportions of invasive Staphylococcus epidermidis isolates fail to form a biofilm in vitro. We here tested the hypothesis that this apparent paradox is related to the existence of superimposed regulatory systems suppressing a multicellular biofilm life style in vitro. Transposon mutagenesis of clinical significant but biofilm‐negative S. epidermidis 1585 was used to isolate a biofilm positive mutant carrying a Tn917 insertion in sarA, chief regulator of staphylococcal virulence. Genetic analysis revealed that inactivation of sarA induced biofilm formation via overexpression of the giant 1 MDa extracellular matrix binding protein (Embp), serving as an intercellular adhesin. In addition to Embp, increased extracellular DNA (eDNA) release significantly contributed to biofilm formation in mutant 1585ΔsarA. Increased eDNA amounts indirectly resulted from upregulation of metalloprotease SepA, leading to boosted processing of autolysin AtlE, in turn inducing augmented autolysis and release of eDNA. Hence, this study identifies sarA as a negative regulator of Embp‐ and eDNA‐dependent biofilm formation. Given the importance of SarA as a positive regulator of polysaccharide mediated cell aggregation, the regulator enables S. epidermidis to switch between mechanisms of biofilm formation, ensuring S. epidermidis adaptation to hostile environments.     

The green tea polyphenol EGCG inhibits E. coli biofilm formation by impairing amyloid curli fibre assembly and downregulating the biofilm regulator CsgD via the σE‐dependent sRNA RybB

In bacterial biofilms, which are often involved in chronic infections, cells are surrounded by a self‐produced extracellular matrix that contains amyloid fibres, exopolysaccharides and other biopolymers. The matrix contributes to the pronounced resistance of biofilms against antibiotics and host immune systems. Being highly inflammatory, matrix amyloids such as curli fibres of Escherichia coli can also play a role in pathogenicity. Using macrocolony biofilms of commensal and pathogenic E. coli as a model system, we demonstrate here that the green tea polyphenol epigallocatachin gallate (EGCG) is a potent antibiofilm agent. EGCG virtually eliminates the biofilm matrix by directly interfering with the assembly of curli subunits into amyloid fibres, and by triggering the σ^E cell envelope stress response and thereby reducing the expression of CsgD – a crucial activator of curli and cellulose biosynthesis – due to csgD mRNA targeting by the σ^E‐dependent sRNA RybB. These findings highlight EGCG as a potential adjuvant for antibiotic therapy of biofilm‐associated infections. Moreover, EGCG may support therapies against pathogenic E. coli that produce inflammatory curli fibres along with Shigatoxin.     

Mucosal gene expression profiles following the colonization of immunocompetent defined-flora C3H mice with Helicobacter bilis: a prelude to typhlocolitis

An aberrant immune response to the commensal microbiota is widely hypothesized to contribute to the development of inflammatory bowel disease. Helicobacter bilis colonization of defined-flora mice has been shown to trigger host immune responses to the commensal flora. However, the magnitude of the effects on mucosal homeostasis following colonization with H. bilis has not been determined. Using microarray analysis, differential gene expression within the cecal mucosa was assessed at 15, 30, or 45 days following H. bilis colonization using Affymetrix Genechips. H. bilis colonization induced marked upregulation of genes associated with protein metabolism, immune responses, and downregulation of genes associated with fatty acid metabolism and detoxification which peaked at 15 days postinfection. A set of genes associated with glycoprotein synthesis and detoxification including Fut2, B3galt5, Ceacam12, Cyp4b1, and Ugt8a were uniquely identified and found to be similarly expressed following the induction of typhlocolitis by dextran sodium sulfate or Brachyspira hyodysenteriae. This study provides preliminary evidence as to the types of factors or changes in the intestinal mucosa that potentially predispose the host to the development of typhlocolitis.     

Dynamics of the Cag‐type IV secretion system of Helicobacter pylori as studied by bacterial co‐infections

Many pathogenic Gram‐negative bacteria possess type IV secretion systems (T4SS) to inject effector proteins directly into host cells to modulate cellular processes to their benefit. The human bacterial pathogen Helicobacter pylori, a major aetiological agent in the development of chronic gastritis, duodenal ulcer and gastric carcinoma, harbours the cag‐T4SS to inject the cytotoxin associated Antigen (CagA) into gastric epithelial cells. This results in deregulation of major signalling cascades, actin‐cytoskeletal rearrangements and eventually gastric cancer. We show here that a pre‐infection with live H. pylori has a dose‐dependent negative effect on the CagA translocation efficiency of a later infecting strain. This effect of the ‘first’ strain was independent of any of its T4SS, the vacuolating cytotoxin (VacA) or flagella. Other bacterial pathogens, e.g. pathogenic Escherichia coli, Campylobacter jejuni, Staphylococcus aureus, or commensal bacteria, such as lactobacilli, were unable to interfere with H. pylori's CagA translocation capacity in the same way. This interference was independent of the β1 integrin receptor availability for H. pylori, but certain H. pylori outer membrane proteins, such as HopI, HopQ or AlpAB, were essential for the effect. We suggest that the specific interference mechanism induced by H. pylori represents a cellularresponse to restrict and control CagA translocation into a host cell to control the cellular damage.     

Review: Pathogenesis of Helicobacter pylori infection

In this review, we shall focus on the last year progression understanding the pathogenesis of Helicobacter pylori infection in the light of recent data related to adaptation of H pylori to the harsh acidic environment in the stomach, colonization of gastric mucosa via interaction with mucin 5 (MUC5AC) and other host cell receptors, the ability to form biofilm, interference with the host metabolic pathways, and induction of neuroimmune cross‐talk as well as downregulation of gastric barrier homeostasis and its consequences for the disease development. The role of the membrane vesicles of these bacteria has been emphasized as an important source of virulence factors. Furthermore, we shall describe molecular and functional studies on new aspects of VacA and CagA virulence, including the role of urease in the upregulation of VacA toxicity, an epithelial‐mesenchymal transition mediated by CagA, and the role of interaction of HopQ adhesin with carcinoembryonic antigen‐related cell adhesion molecules (CEACAMs) in CagA translocation into the host cells by the type IV secretion system (T4SS). The role of molecular mimicry between a common sequence (ATVLA) of H pylori heat shock protein (Hsp) B and human Hsp60 in the induction of potentially autoreactive antibodies is discussed. All these new data illustrate further progress in understanding H pylori pathogenicity and facilitate the search for new therapeutic targets as well as development of immunoprophylaxis methods based on new chimeric UreB and HpA proteins.     

The focal complex of epithelial cells provides a signalling platform for interleukin‐8 induction in response to bacterial pathogens

Bacterial pathogens can induce an inflammatory response from epithelial tissues due to secretion of the pro‐inflammatory chemokine interleukin‐8 (IL‐8). Many bacterial pathogens manipulate components of the focal complex (FC) to induce signalling events in host cells. We examined the interaction of several bacterial pathogens with host cells, including Campylobacter jejuni, to determine if the FC is required for induction of chemokine signalling in response to bacterial pathogens. Our data indicate that secretion of IL‐8 is triggered by C. jejuni, Helicobacter pylori and Salmonella enterica serovar Typhimurium in response to engagement of β₁ integrins. Additionally, we found that the secretion of IL‐8 from C. jejuni infected epithelial cells requires FAK, Src and paxillin, which in turn are necessary for Erk 1/2 recruitment and activation. Targeting the FC component paxillin with siRNA prevented IL‐8 secretion from cells infected with several bacterial pathogens, including C. jejuni, Helicobacter pylori, Salmonella enterica serovar Typhimurium, Staphylococcus aureus, Pseudomonas aeruginosa, and Vibrio parahaemolyticus. Our findings indicate that maximal IL‐8 secretion from epithelial cells in response to bacterial infection is dependent on the FC. Based on the commonality of the host response to bacterial pathogens, we propose that the FC is a signalling platform for an epithelial cell response to pathogenic organisms.     

Borrelia burgdorferi BbHtrA degrades host ECM proteins and stimulates release of inflammatory cytokines in vitro

The Lyme disease spirochaete, Borrelia burgdorferi, causes damage to diverse host tissues and induces inflammation but the mechanisms of injury are poorly understood. We recently reported that a surface‐exposed B. burgdorferi protease, which is expressed during human disease and is conserved within the major Lyme disease spirochaete species, degrades the extracellular matrix proteoglycan, aggrecan. Here we demonstrate that BbHtrA also degrades fibronectin and numerous proteoglycans found in skin, joints and neural tissues. BbHtrA degradation of fibronectin released known pro‐inflammatory fibronectin fragments FnIII_13–14 and Fnf‐29, which may amplify the inflammatory processes triggered by the presence of the bacteria. When this hypothesis was tested directly by exposing chondrocytes to BbHtrA in vitro, inflammatory cytokines (sICAM‐1 and IL‐6) and chemokines (CXCL1, CCL1, CCL2 and CCL5) that are hallmarks of Lyme disease were induced. These results provide the first evidence that, by utilizing BbHtrA, B. burgdorferi may actively participate in its dissemination and in the tissue damage and inflammation observed in Lyme disease.     

The biofilm mode of life boosts the anti‐inflammatory properties of Lactobacillus

The predominant form of life for microorganisms in their natural habitats is the biofilm mode of growth. The adherence and colonization of probiotic bacteria are considered as essential factors for their immunoregulatory function in the host. Here, we show that Lactobacillus casei ATCC334 adheres to and colonizes the gut of zebrafish larvae. The abundance of pro‐inflammatory cytokines and the recruitment of macrophages were low when inflammation was induced in probiotic‐fed animals, suggesting that these bacteria have anti‐inflammatory properties. We treated human macrophage‐differentiated monocytic THP‐1 cells with supernatants of L. casei ATCC334 grown in either biofilm or planktonic cultures. TNF‐α production was suppressed and the NF‐κB pathway was inhibited only in the presence of supernatants from biofilms. We identified GroEL as the biofilm supernatant compound responsible, at least partially, for this anti‐inflammatory effect. Gradual immunodepletion of GroEL demonstrated that the abundance of GroEL and TNF‐α were inversely correlated. We confirmed that biofilm development in other Lactobacillus species affects the immune response. The biofilms supernatants of these species also contained large amounts of GroEL. Thus, our results demonstrate that the biofilm enhances the immunomodulatory effects of Lactobacillus sp. and that secreted GroEL is involved in this beneficial effect.     

Transgenerational developmental effects of species‐specific,maternally transmitted microbiota in Onthophagus dung beetles

1. The significance of host–microbe interactions is increasingly appreciated across biological disciplines, yet to what extent these interactions influence developmental outcomes within and across generations remains poorly understood. 2. This study investigated the putative role of host–microbe interactions in the adaptive diversification of Onthophagus dung beetles, one of the most species‐rich and ecologically successful genera of insects. Onthophagus mothers vertically transmit growth‐ and fitness‐enhancing gut symbionts to their offspring through a faecal secretion known as the pedestal. 3. Pedestals were reciprocally exchanged between two ecologically similar congeneric Onthophagus species to assess the degree to which pedestal microbiota from one species can substitute for those of another. 4. It was found that the presence of a heterospecific pedestal delays development and increases mortality, and that the fitness costs of non‐host‐specific microbiota are maintained transgenerationally. 5. Collectively, these results support the hypothesis that Onthophagus beetles maintain, interact with, and are dependent upon host species‐specific microbial communities to support normal growth and development. The implications of these results are discussed in the context of host microbiota coevolution.     

Pseudomonas aeruginosa uses a cyclic‐di‐GMP‐regulated adhesin to reinforce the biofilm extracellular matrix

Pseudomonas aeruginosa, the principal pathogen of cystic fibrosis patients, forms antibiotic‐resistant biofilms promoting chronic colonization of the airways. The extracellular (EPS) matrix is a crucial component of biofilms that provides the community multiple benefits. Recent work suggests that the secondary messenger, cyclic‐di‐GMP, promotes biofilm formation. An analysis of factors specifically expressed in P. aeruginosa under conditions of elevated c‐di‐GMP, revealed functions involved in the production and maintenance of the biofilm extracellular matrix. We have characterized one of these components, encoded by the PA4625 gene, as a putative adhesin and designated it cdrA. CdrA shares structural similarities to extracellular adhesins that belong to two‐partner secretion systems. The cdrA gene is in a two gene operon that also encodes a putative outer membrane transporter, CdrB. The cdrA gene encodes a 220 KDa protein that is predicted to be rod‐shaped protein harbouring a β‐helix structural motif. Western analysis indicates that the CdrA is produced as a 220 kDa proprotein and processed to 150 kDa before secretion into the extracellular medium. We demonstrated that cdrAB expression is minimal in liquid culture, but is elevated in biofilm cultures. CdrAB expression was found to promote biofilm formation and auto‐aggregation in liquid culture. Aggregation mediated by CdrA is dependent on the Psl polysaccharide and can be disrupted by adding mannose, a key structural component of Psl. Immunoprecipitation of Psl present in culture supernatants resulted in co‐immunoprecipitation of CdrA, providing additional evidence that CdrA directly binds to Psl. A mutation in cdrA caused a decrease in biofilm biomass and resulted in the formation of biofilms exhibiting decreased structural integrity. Psl‐specific lectin staining suggests that CdrA either cross‐links Psl polysaccharide polymers and/or tethers Psl to the cells, resulting in increased biofilm structural stability. Thus, this study identifies a key protein structural component of the P. aeruginosa EPS matrix.     

A fibronectin‐binding protein (FbpA) of Weissella cibaria inhibits colonization and infection of Staphylococcus aureus in mammary glands

Staphylococcus aureus (S. aureus) is a frequent cause of infections in both humans and animals. Probiotics are known to inhibit colonization of pathogens on host tissues. However, mechanisms for the inhibition are still elusive due to complex host–microbe and microbe–microbe interactions. Here, we show that reduced abilities of S. aureus to infect mammary glands in the presence of Weissella cibaria (W. cibaria) were correlated with its poor adherence to mammary epithelial cells. Such inhibition by W. cibaria isolates was at least partially attributed to a fibronectin‐binding protein (FbpA) on this lactic acid bacterium. Three W. cibaria isolates containing fbpA had higher inhibitory abilities than other three LAB isolates without the gene. The fbpA‐deficient mutant of W. cibaria isolate LW1, LW1ΔfbpA, lost the inhibitory activity to reduce the adhesion of S. aureus to mammary epithelial cells and was less able to reduce the colonization of S. aureus in mammary glands. Expression of FbpA to the surface of LW1ΔfbpA reversed its inhibitory activities. Furthermore, addition of purified FbpA inhibited S. aureus biofilm formation. Our results suggest that W. cibaria FbpA hinders S. aureus colonization and infection through interfering with the S. aureus invasion pathway mediated by fibronectin‐binding proteins and inhibiting biofilm formation of S. aureus.     

Autoinducer‐2 is produced in saliva‐fed flow conditions relevant to natural oral biofilms

Aims: We evaluated the ability of a dual‐species community of oral bacteria to produce the universal signalling molecule, autoinducer‐2 (AI‐2), in saliva‐fed biofilms. Methods and Results: Streptococcus oralis 34, S. oralis 34 luxS mutant and Actinomyces naeslundii T14V were grown as single‐ and dual‐species biofilms within sorbarods fed with 25% human saliva. AI‐2 concentration in biofilm effluents was determined by the Vibrio harveyi BB170 bioluminescence assay. After homogenizing the sorbarods to release biofilm cells, cell numbers were determined by fluorometric analysis of fluorescent antibody‐labelled cells. After 48 h, dual‐species biofilm communities of interdigitated S. oralis 34 and A. naeslundii T14V contained 3·2 × 10⁹ cells: fivefold more than single‐species biofilms. However, these 48‐h dual‐species biofilms exhibited the lowest concentration ratio of AI‐2 to cell density. Conclusions: Oral bacteria produce AI‐2 in saliva‐fed biofilms. The decrease of more than 10‐fold in concentration ratio seen between 1 and 48 h in S. oralis 34–A. naeslundii T14V biofilms suggests that peak production of AI‐2 occurs early and is followed by a very low steady‐state level. Significance and Impact of the Study: High oral bacterial biofilm densities may be achieved by inter‐species AI‐2 signalling. We propose that low concentrations of AI‐2 contribute to the establishment of oral commensal biofilm communities.     

A novel NOD1‐ and CagA‐independent pathway of interleukin‐8 induction mediated by the Helicobacter pylori type IV secretion system

The type IV secretion system (T4SS) of Helicobacter pylori triggers massive inflammatory responses during gastric infection by mechanisms that are poorly understood. Here we provide evidence for a novel pathway by which the T4SS structural component, CagL, induces secretion of interleukin‐8 (IL‐8) independently of CagA translocation and peptidoglycan‐sensing nucleotide‐binding oligomerization domain 1 (NOD1) signalling. Recombinant CagL was sufficient to trigger IL‐8 secretion, requiring activation of α₅β₁ integrin and the arginine–glycine–aspartate (RGD) motif in CagL. Mutation of the encoded RGD motif to arginine‐glycine‐alanine (RGA) in the cagL gene of H. pylori abrogated its ability to induce IL‐8. Comparison of IL‐8 induction between H. pylori ΔvirD4 strains bearing wild‐type or mutant cagL indicates that CagL‐dependent IL‐8 induction can occur independently of CagA translocation. In line with this notion, exogenous CagL complemented H. pylori ΔcagL mutant in activating NF‐κB and inducing IL‐8 without restoring CagA translocation. The CagA translocation‐independent, CagL‐dependent IL‐8induction involved host signalling via integrin α₅β₁, Src kinase, the mitogen‐activated protein kinase (MAPK) pathway and NF‐κB but was independent of NOD1. Our findings reveal a novel pathway whereby CagL, via interaction with host integrins, can trigger pro‐inflammatory responses independently of CagA translocation or NOD1 signalling.     

Defining a pro‐inflammatory neutrophil phenotype in response to schistosome eggs

Neutrophils contribute to the pathological processes of a number of inflammatory disorders, including rheumatoid arthritis, sepsis and cystic fibrosis. Neutrophils also play prominent roles in schistosomiasis japonica liver fibrosis, being central mediators of inflammation following granuloma formation. In this study, we investigated the interaction between Schistosoma japonicum eggs and neutrophils, and the effect of eggs on the inflammatory phenotype of neutrophils. Our results showed significant upregulated expression of pro‐inflammatory cytokines (IL‐1α, IL‐1β and IL‐8) and chemokines (CCL3, CCL4 and CXCL2) in neutrophils after 4 h in vitro stimulation with S. japonicum eggs. Furthermore, mitochondrial DNA was released by stimulated neutrophils, and induced the production of matrix metalloproteinase 9 (MMP‐9), a protease involved in inflammation and associated tissue destruction. We also found that intact live eggs and isolated soluble egg antigen (SEA) triggered the release of neutrophil extracellular traps (NETs), but, unlike those reported in bacterial or fungal infection, NETs did not kill schistosome eggs in vitro. Together these show that S. japonicum eggs can induce the inflammatory phenotype of neutrophils, and further our understanding of the host–parasite interplay that takes place within the in vivo microenvironment of schistosome‐induced granuloma. These findings represent novel findings in a metazoan parasite, and confirm characteristics of NETs that have until now, only been observed in response to protozoan pathogens.     

Towards non‐invasive monitoring of pathogen–host interactions during Candida albicans biofilm formation using in vivo bioluminescence

Candida albicans is a major human fungal pathogen causing mucosal and deep tissue infections of which the majority is associated with biofilm formation on medical implants. Biofilms have a huge impact on public health, as fungal biofilms are highly resistant against most antimycotics. Animal models of biofilm formation are indispensable for improving our understanding of biofilm development inside the host, their antifungal resistance and their interaction with the host immune defence system. In currently used models, evaluation of biofilm development or the efficacy of antifungal treatment is limited to ex vivo analyses, requiring host sacrifice, which excludes longitudinal monitoring of dynamic processes during biofilm formation in the live host. In this study, we have demonstrated for the first time that non‐invasive, dynamic imaging and quantification of in vitro and in vivo C. albicans biofilm formation including morphogenesis from the yeast to hyphae state is feasible by using growth‐phase dependent bioluminescent C. albicans strains in a subcutaneous catheter model in rodents. We have shown the defect in biofilm formation of a bioluminescent bcr1 mutant strain. This approach has immediate applications for the screening and validation ofantimycotics under in vivo conditions, for studying host–biofilm interactions in different transgenic mouse models and for testing the virulence of luminescent C. albicans mutants, hereby contributing to a better understanding of the pathogenesis of biofilm‐associated yeast infections.     

House dust mite extract activates apical Cl− channels through protease‐activated receptor 2 in human airway epithelia

Adequate fluid secretion from airway mucosa is essential for maintaining mucociliary clearance, and fluid hypersecretion is a prominent feature of inflammatory airway diseases such as allergic rhinitis. House dust mite extract (HDM) has been reported to activate protease‐activated receptors (PARs), which play various roles in airway epithelia. However, the role of HDM in regulating ion transporters and fluid secretion has not been investigated. We examined the effect of HDM on ion transport in human primary nasal epithelial cells. The Ca²⁺‐sensitive dye Fura2‐AM was used to determine intracellular Ca²⁺ concentration ([Ca²⁺]_i) by means of spectrofluorometry in human normal nasal epithelial cells (NHNE). Short‐circuit current (Isc) was measured using Ussing chambers. Fluid secretion from porcine airway mucosa was observed by optical measurement. HDM extract (10 µg/Ml) effectively cleaved the PAR‐2 peptide and induced an increase of [Ca²⁺]_i that was abolished by desensitization with trypsin, but not with thrombin. Apical application of HDM‐induced Isc sensitive to both a cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor and a Ca²⁺‐activated Cl^? channel (CaCC) inhibitor. HDM extract also stimulated fluid secretion from porcine airway mucosa. HDM extract activated PAR‐2 and apical Cl^? secretion via CaCC and CFTR, and HDM‐induced fluid secretion in porcine airway mucosa. Our results suggest a role for PAR‐2 in mucociliary clearance and fluid hypersecretion of airway mucosa in response to air‐borne allergens such as HDM. J. Cell. Biochem. 109: 1254–1263, 2010. © 2010 Wiley‐Liss, Inc.