Host Cell Adhesion Research Articles

Complement Regulator Factor H Mediates a Two-step Uptake of Streptococcus pneumoniae by Human Cells

Streptococcus pneumoniae, a human pathogen, recruits complement regulator factor H to its bacterial cell surface. The bacterial PspC protein binds Factor H via short consensus repeats (SCR) 8-11 and SCR19-20. In this study, we define how bacterially bound Factor H promotes pneumococcal adherence to and uptake by epithelial cells or human polymorphonuclear leukocytes (PMNs) via a two-step process. First, pneumococcal adherence to epithelial cells was significantly reduced by heparin and dermatan sulfate. However, none of the glycosaminoglycans affected binding of Factor H to pneumococci. Adherence of pneumococci to human epithelial cells was inhibited by monoclonal antibodies recognizing SCR19-20 of Factor H suggesting that the C-terminal glycosaminoglycan-binding region of Factor H mediates the contact between pneumococci and human cells. Blocking of the integrin CR3 receptor, i.e. CD11b and CD18, of PMNs or CR3-expressing epithelial cells reduced significantly the interaction of pneumococci with both cell types. Similarly, an additional CR3 ligand, Pra1, derived from Candida albicans, blocked the interaction of pneumococci with PMNs. Strikingly, Pra1 inhibited also pneumococcal uptake by lung epithelial cells but not adherence. In addition, invasion of Factor H-coated pneumococci required the dynamics of host-cell actin microfilaments and was affected by inhibitors of protein-tyrosine kinases and phosphatidylinositol 3-kinase. In conclusion, pneumococcal entry into host cells via Factor H is based on a two-step mechanism. The first and initial contact of Factor H-coated pneumococci is mediated by glycosaminoglycans expressed on the surface of human cells, and the second step, pneumococcal uptake, is integrin-mediated and depends on host signaling molecules such as phosphatidylinositol 3-kinase.

Downregulation of an Entamoeba histolytica Rhomboid Protease Reveals Roles in Regulating Parasite Adhesion and Phagocytosis

Entamoeba histolytica is a deep-branching eukaryotic pathogen. Rhomboid proteases are intramembrane serine proteases, which cleave transmembrane proteins in, or in close proximity to, their transmembrane domain. We have previously shown that E. histolytica contains a single functional rhomboid protease (EhROM1) and has unique substrate specificity. EhROM1 is present on the trophozoite surface and relocalizes to internal vesicles during erythrophagocytosis and to the base of the cap during surface receptor capping. In order to further examine the biological function of EhROM1 we downregulated EhROM1 expression by >95% by utilizing the epigenetic silencing mechanism of the G3 parasite strain. Despite the observation that EhROM1 relocalized to the cap during surface receptor capping, EhROM1 knockdown [ROM(KD)] parasites had no gross changes in cap formation or complement resistance. However, ROM(KD) parasites demonstrated decreased host cell adhesion, a result recapitulated by treatment of wild-type parasites with DCI, a serine protease inhibitor with activity against rhomboid proteases. The reduced adhesion phenotype of ROM(KD) parasites was noted exclusively with healthy cells, and not with apoptotic cells. Additionally, ROM(KD) parasites had decreased phagocytic ability with reduced ingestion of healthy cells, apoptotic cells, and rice starch. Decreased phagocytic ability is thus independent of the reduced adhesion phenotype, since phagocytosis of apoptotic cells was reduced despite normal adhesion levels. The defect in host cell adhesion was not explained by altered expression or localization of the heavy subunit of the Gal/GalNAc surface lectin. These results suggest no significant role of EhROM1 in complement resistance but unexpected roles in parasite adhesion and phagocytosis.

The Phosphoproteome of the Minimal Bacterium Mycoplasma pneumoniae: ANALYSIS OF THE COMPLETE KNOWN SER/THR KINOME SUGGESTS THE EXISTENCE OF NOVEL KINASES

Mycoplasma pneumoniae belongs to the Mollicutes, the group of organisms with the smallest genomes that are capable of host-independent life. These bacteria show little regulation in gene expression, suggesting an important role for the control of protein activities. We have studied protein phosphorylation in M. pneumoniae to identify phosphorylated proteins. Two-dimensional gel electrophoresis and mass spectrometry allowed the detection of 63 phosphorylated proteins, many of them enzymes of central carbon metabolism and proteins related to host cell adhesion. We identified 16 phosphorylation sites, among them 8 serine and 8 threonine residues, respectively. A phosphoproteome analysis with mutants affected in the two annotated protein kinase genes or in the single known protein phosphatase gene suggested that only one protein (HPr) is phosphorylated by the HPr kinase, HPrK, whereas four adhesion-related or surface proteins were targets of the protein kinase C, PrkC. A comparison with the phosphoproteomes of other bacteria revealed that protein phosphorylation is evolutionarily only poorly conserved. Only one single protein with an identified phosphorylation site, a phosphosugar mutase (ManB in M. pneumoniae), is phosphorylated on a conserved serine residue in all studied organisms from archaea and bacteria to man. We demonstrate that this protein undergoes autophosphorylation. This explains the strong conservation of this phosphorylation event. For most other proteins, even if they are phosphorylated in different species, the actual phosphorylation sites are different. This suggests that protein phosphorylation is a form of adaptation of the bacteria to the specific needs of their particular ecological niche.

Structural Characterization of Apical Membrane Antigen 1 (AMA1) from Toxoplasma gondii

Apical membrane antigen 1 (AMA1) is an essential component of the moving junction complex used by Apicomplexan parasites to invade host cells. We report the 2.0 A resolution x-ray crystal structure of the full ectodomain (domains I, II, and III) of AMA1 from the pervasive protozoan parasite Toxoplasma gondii. The structure of T. gondii AMA1 (TgAMA1) is the most complete of any AMA1 structure to date, with more than 97.5% of the ectodomain unambiguously modeled. Comparative sequence analysis reveals discrete segments of divergence in TgAMA1 that map to areas of established functional importance in AMA1 from Plasmodium vivax (PvAMA1) and Plasmodium falciparum (PfAMA1). Inspection of the TgAMA1 structure reveals a network of apical surface loops, reorganized in both size and chemistry relative to PvAMA1/PfAMA1, that appear to serve as structural filters restricting access to a central hydrophobic groove. The terminal portion of this groove is formed by an extended loop from DII that is 14 residues shorter in TgAMA1. A pair of tryptophan residues (Trp(353) and Trp(354)) anchor the DII loop in the hydrophobic groove and frame a conserved tyrosine (Tyr(230)), forming a contiguous surface that may be critical for moving junction assembly. The minimalist DIII structure folds into a cystine knot that probably stabilizes and orients the bulk of the ectodmain without providing excess surface area to which invasion-inhibitory antibodies can be generated. The detailed structural characterization of TgAMA1 provides valuable insight into the mechanism of host cell invasion by T. gondii.

A real-time quantitative PCR assay for evaluating Clostridium difficile adherence to differentiated intestinal Caco-2 cells

Herein we describe a real-time quantitative PCR assay for evaluating the adherence of Clostridium difficile to differentiated human intestinal Caco-2 cells. Our investigations demonstrated that the method, employing the C. difficile-specific triose-phosphate isomerase gene, is as reliable but less time-consuming than counting c.f.u. We conclude that the method will be useful for evaluating the role of host cell adherence in the pathogenesis of C. difficile infection.

Stability and Assembly of Pilus Subunits of Streptococcus pneumoniae

Pili are surface-exposed virulence factors involved in bacterial adhesion to host cells. The Streptococcus pneumoniae pilus is composed of three structural proteins, RrgA, RrgB, and RrgC and three transpeptidase enzymes, sortases SrtC-1, SrtC-2, and SrtC-3. To gain insights into the mechanism of pilus formation we have exploited biochemical approaches using recombinant proteins expressed in Escherichia coli. Using site-directed mutagenesis, mass spectrometry, limited proteolysis, and thermal stability measurements, we have identified isopeptide bonds in RrgB and RrgC and demonstrate their role in protein stabilization. Co-expression in E. coli of RrgB together with RrgC and SrtC-1 leads to the formation of a covalent RrgB-RrgC complex. Inactivation of SrtC-1 by mutation of the active site cysteine impairs RrgB-RrgC complex formation, indicating that the association between RrgB and RrgC is specifically catalyzed by SrtC-1. Mass spectrometry analyses performed on purified samples of the RrgB-RrgC complex show that the complex has 1:1 stoichiometry. The deletion of the IPQTG RrgB sorting signal, but not the corresponding sequence in RrgC, abolishes complex formation, indicating that SrtC-1 recognizes exclusively the sorting motif of RrgB. Finally, we show that the intramolecular bonds that stabilize RrgB may play a role in its efficient recognition by SrtC-1. The development of a methodology to generate covalent pilin complexes in vitro, facilitating the study of sortase specificity and the importance of isopeptide bond formation for pilus biogenesis, provide key information toward the understanding of this complex macromolecular process.

Structure and oligomerization of the PilC type IV pilus biogenesis protein from Thermus thermophilus

Type IV pili are expressed from a wide variety of Gram-negative bacteria and play a major role in host cell adhesion and bacterial motility. PilC is one of at least a dozen different proteins that are implicated in Type IV pilus assembly in Thermus thermophilus and a member of a conserved family of integral inner membrane proteins which are components of the Type II secretion system (GspF) and the archeal flagellum. PilC/GspF family members contain repeats of a conserved helix-rich domain of around 100 residues in length. Here, we describe the crystal structure of one of these domains, derived from the N-terminal domain of Thermus thermophilus PilC. The N-domain forms a dimer, adopting a six helix bundle structure with an up-down-up-down-up-down topology. The monomers are related by a rotation of 170 degrees , followed by a translation along the axis of the final alpha-helix of approximately one helical turn. This means that the regions of contact on helices 5 and 6 in each monomer are overlapping, but different. Contact between the two monomers is mediated by a network of hydrophobic residues which are highly conserved in PilC homologs from other Gram-negative bacteria. Site-directed mutagenesis of residues at the dimer interface resulted in a change in oligomeric state of PilC from tetramers to dimers, providing evidence that this interface is also found in the intact membrane protein and suggesting that it is important to its function.

The moonlighting protein fructose-1, 6-bisphosphate aldolase of Neisseria meningitidis: surface localization and role in host cell adhesion

Fructose-1, 6-bisphosphate aldolases (FBA) are cytoplasmic glycolytic enzymes, which despite lacking identifiable secretion signals, have also been found localized to the surface of several bacteria where they bind host molecules and exhibit non-glycolytic functions. Neisseria meningitidis is an obligate human nasopharyngeal commensal, which has the capacity to cause life-threatening meningitis and septicemia. Recombinant native N. meningitidis FBA was purified and used in a coupled enzymic assay confirming that it has fructose bisphosphate aldolase activity. Cell fractionation experiments showed that meningococcal FBA is localized both to the cytoplasm and the outer membrane. Flow cytometry demonstrated that outer membrane-localized FBA was surface-accessible to FBA-specific antibodies. Mutational analysis and functional complementation was used to identify additional functions of FBA. An FBA-deficient mutant was not affected in its ability to grow in vitro, but showed a significant reduction in adhesion to human brain microvascular endothelial and HEp-2 cells compared to its isogenic parent and its complemented derivative. In summary, FBA is a highly conserved, surface exposed protein that is required for optimal adhesion of meningococci to human cells.

Mechanical contributions to astrocyte adhesion using a novel in vitro model of catheter obstruction

Drainage and diversion of cerebrospinal fluid (CSF) through shunt systems is the most common treatment for hydrocephalus, but complications due to tissue obstruction of the catheter occur in up to 61% of patients. Although shunt systems have undergone limited technological advancements to resist mammalian cell adhesion, there is a need to further reduce adhesion that can exacerbate obstruction. The high intrinsic variability in clinical studies and an inability to predict chronic adhesion of host cells in vitro while maintaining the environmental conditions observed in hydrocephalus have impeded progress. We designed the hydrocephalus shunt catheter bioreactor (HSCB) to measure inflammatory cell adhesion under experimentally manipulated conditions of CSF pressure, pulsation rate, and flow rates. For a 20-h period, astrocytes were perfused through the pulsatile flow system, and adhesion on silicone catheters was recorded. These results were compared with those obtained under static cell culture conditions. Astrocyte adhesion was significantly increased under conditions of increased flow rate (0.25 and 0.30 mL/min), and a trend toward increased adhesion was observed under conditions of elevated pressure and pulsation rate. Because the HSCB represents physiologic conditions more accurately than static cell culture, our results suggest that standard static cell culturing techniques are insufficient to model inflammatory cell adhesion on catheters used in the treatment of hydrocephalus and that changes to the ventricular microenvironment can alter the mechanisms of cellular adhesion. The HSCB represents a relevant test system and is an effective model system for the analysis of cellular adhesion and occlusion of shunt catheters.

Characterisation of NcGRA7 and NcSAG4 proteins: Immunolocalisation and their role in the host cell invasion by Neospora caninum tachyzoites

AbstractNeospora caninum negatively impacts bovine reproductive performance around the world. Addressing this problem requires a greater understanding of the parasite’s molecular biology. In this study, monoclonal antibodies against recombinant proteins were successfully developed and employed to characterise two different proteins of N. caninum: the acute phase-associated NcGRA7 and the chronic phase-associated NcSAG4. Immunofluorescence with the anti-rNcGRA7 monoclonal antibody suggested that NcGRA7 trafficks from tachyzoite dense granules to the matrix of the parasitophorous vacuole and parasite’s surroundings. Furthermore, NcGRA7 is also expressed in the bradyzoite stage and localised on the matrix of bradyzoite-positive vacuoles. NcGRA7 appears to be partially involved in the tachyzoite-invasion mechanisms, as an anti-rNcGRA7 monoclonal antibody partially inhibited in vitro tachyzoite-invasion. A monoclonal antibody specific for NcSAG4 confirmed this protein’s bradyzoitespecific expression both by western blot and immunofluorescence. However, some bradyzoite-positive vacuoles only weakly expressed NcSAG4, if it was expressed at all. The specificity of the anti-rNcSAG4 monoclonal antibody was confirmed by the recognition of the NcSAG4 in the membrane surface of Nc-1SAG4c transgenic tachyzoites, which constitutively expresses NcSAG4. Blocking NcSAG4 of Nc-1SAG4c tachyzoites with the monoclonal antibody did not affect host cell invasion. However, its implication on the host cell adhesion or host immune evasion should not be discarded.

Structural Basis of Host Cell Recognition by the Pilus Adhesin from Streptococcus pneumoniae

Pili are fibrous virulence factors associated directly to the bacterial surface that play critical roles in adhesion and recognition of host cell receptors. The human pathogen Streptococcus pneumoniae carries a single pilus-related adhesin (RrgA) that is key for infection establishment and provides protection from bacterial challenge in animal infection models, but details of these roles remain unclear. Here we report the high-resolution crystal structure of RrgA, a 893-residue elongated macromolecule whose fold contains four domains presenting both eukaryotic and prokaryotic origins. RrgA harbors an integrin I collagen-recognition domain decorated with two inserted "arms" that fold into a positively charged cradle, as well as three "stalk-forming" domains. We show by site-specific mutagenesis, mass spectrometry, and thermal shift assays that intradomain isopeptide bonds play key roles in stabilizing RrgA's stalk. The high sequence similarity between RrgA and its homologs in other Gram-positive microorganisms suggests common strategies for ECM recognition and immune evasion.

Alterations of serum tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and sialic acids in malignant ovine theileriosis

The study was designed to evaluate the effect of the severity of Theileria lestoquardi (hirci) infection on tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and sialic acid concentration in blood sera in a naturally infected sheep. Infected animals (diseased group) comprised 50 Iranian fat-tailed sheep, about 1–2 years old, naturally infected with Theileria lestoquardi (hirci), were divided into three subgroups according to parasitemia rates ( 4%). As a control group, ten noninfected sheep were also selected. Hematological parameters and levels of TNF-α, IFN-γ, and sialic acid (TSA, LBSA, and PBSA) concentrations were measured in both groups. The results showed significant increases in the level of TNF-α, IFN-γ, and sialic acid concentrations in infected sheep compared with noninfected ones (P < 0.05). The increased level of TNF-α and IFN-γ in infected sheep may indicate the role of these cytokines to inhibit the growth and development of the parasite in infected host cells. Also, it is speculated that preventing the establishment of parasites by inhibiting the differentiation of trophozoites to schizonts is another aspect of the enhanced activity of these substances. In addition, increased levels of sialic acid concentration during parasitemia presumably stimulate the host immune response and influence the parasite–host cell adhesion. Clearly, further molecular and biochemical investigations are needed to elucidate the exact mechanisms involved in immunopathogenesis of malignant ovine theileriosis.

Review article: anti‐inflammatory mechanisms of action of Saccharomyces boulardii

Saccharomyces boulardii, a well-studied probiotic, can be effective in inflammatory gastrointestinal diseases with diverse pathophysiology, such as inflammatory bowel disease (IBD), and bacterially mediated or enterotoxin-mediated diarrhoea and inflammation. To discuss the mechanisms of action involved in the intestinal anti-inflammatory action of S. boulardii. Review of the literature related to the anti-inflammatory effects of this probiotic. Several mechanisms of action have been identified directed against the host and pathogenic microorganisms. S. boulardii and S. boulardii secreted-protein(s) inhibit production of proinflammatory cytokines by interfering with the global mediator of inflammation nuclear factor kappaB, and modulating the activity of the mitogen-activated protein kinases ERK1/2 and p38. S. boulardii activates expression of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) that protects from gut inflammation and IBD. S. boulardii also suppresses 'bacteria overgrowth' and host cell adherence, releases a protease that cleaves C. difficile toxin A and its intestinal receptor and stimulates antibody production against toxin A. Recent results indicate that S. boulardii may interfere with IBD pathogenesis by trapping T cells in mesenteric lymph nodes. The multiple anti-inflammatory mechanisms exerted by S. boulardii provide molecular explanations supporting its effectiveness in intestinal inflammatory states.

Bacterial Type III Effectors Inhibit Cell Lifting by Targeting Integrin-Linked Kinase

Many pathogenic bacteria are able to surmount exfoliation and colonize the host epithelium efficiently. In a recent article in Nature, Kim et al. (2009) show that Shigella flexneri OspE, a conserved type III effector, reinforces host cell adherence and prevents detachment of the infected cells by interacting with integrin-linked kinase.

Proteomic Analysis of Detergent-Solubilized Membrane Proteins from Insect-Developmental Forms of Trypanosoma cruzi

The cell surface of Trypanosoma cruzi, the etiologic agent of Chagas disease, is covered by a dense layer of glycosylphosphatidylinositol (GPI)-anchored molecules. These molecules are involved in a variety of interactions between this parasite and its mammalian and insect hosts. Here, using the neutral detergent Triton X-114, we obtained fractions rich in GPI-anchored and other membrane proteins from insect developmental stages of T. cruzi. These fractions were analyzed by two-dimensional liquid chromatography coupled to tandem mass spectrometry (2D-LC-MS/MS), resulting in the identification of 98 proteins of metacyclic trypomastigotes and 280 of epimastigotes. Of those, approximately 65% (n=245) had predicted lipid post-translational modification sites (i.e., GPI-anchor, myristoylation, or prenylation), signal-anchor sequence, or transmembrane domains that could explain their solubility in detergent solution. The identification of some of these modified proteins was also validated by immunoblotting. We also present evidence that, in contrast to the noninfective proliferative epimastigote forms, the infective nonproliferative metacyclic trypomastigote forms express a large repertoire of surface glycoproteins, such as GP90 and GP82, which are involved in adhesion and invasion of host cells. Taken together, our results unequivocally show stage-specific protein profiles that appear to be related to the biology of each T. cruzi insect-derived developmental form.

Rhinovirus enhances various bacterial adhesions to nasal epithelial cells simultaneously

Viral upper respiratory tract infections are often followed by secondary bacterial infections in the form of acute rhinosinusitis. We investigate the effect of rhinovirus infection on the expression of cell adhesion molecules and bacterial adherence to primary human nasal epithelial cells. Cells were infected with rhinovirus serotype 16 (RV-16), and then Staphylococcus aureus, Streptococcus pneumoniae, or Hemophilus influenzae were added to the culture. Rhinovirus-induced expression of fibronectin, platelet-activating factor receptor, and carcinoembryonic antigen-related cell adhesion molecule, was assayed by confocal microscopy, real-time polymerase chain reaction, and Western blot analysis. Bacterial adhesion to cells was assessed by confocal microscopy and the fluorescence intensity of adherent bacteria was analyzed using Image-Pro Plus 5.1 (Media Cybernetics, Inc., Bethesda, MD). RV-16 infection significantly increased the gene and protein expression of fibronectin, platelet-activating factor receptor, and carcinoembryonic antigen-related cell adhesion molecule in nasal epithelial cells. Compared with rhinovirus-uninfected control cells, the adhesion of S. aureus, S. pneumoniae, and H. influenzae increased significantly to 2.53-fold, 1.51-fold, and 2.74-fold of control levels, respectively, in rhinovirus-infected nasal epithelial cells. These findings suggest that increased expression of host cell adhesion molecules may be the mechanism accounting for the increase in susceptibility to bacterial rhinosinusitis associated with rhinovirus-induced upper respiratory infections.

Bacteria hijack integrin-linked kinase to stabilize focal adhesions and block cell detachment

The rapid turnover and exfoliation of mucosal epithelial cells provides an innate defence system against bacterial infection. Nevertheless, many pathogenic bacteria, including Shigella, are able to surmount exfoliation and colonize the epithelium efficiently. Here we show that the Shigella flexneri effector OspE (consisting of OspE1 and OspE2 proteins), which is highly conserved among enteropathogenic Escherichia coli, enterohaemorrhagic E. coli, Citrobacter rodentium and Salmonella strains, reinforces host cell adherence to the basement membrane by interacting with integrin-linked kinase (ILK). The number of focal adhesions was augmented along with membrane fraction ILK by ILK-OspE binding. The interaction between ILK and OspE increased cell surface levels of 1 integrin and suppressed phosphorylation of focal adhesion kinase and paxillin, which are required for rapid turnover of focal adhesion in cell motility. Nocodazole-washout-induced focal adhesion disassembly was blocked by expression of OspE. Polarized epithelial cells infected with a Shigella mutant lacking the ospE gene underwent more rapid cell detachment than cells infected with wild-type Shigella. Infection of guinea pig colons with Shigella corroborated the pivotal role of the OspE-ILK interaction in suppressing epithelial detachment, increasing bacterial cell-to-cell spreading, and promoting bacterial colonization. These results indicate that Shigella sustain their infectious foothold by using special tactics to prevent detachment of infected cells.

Extreme Substrate Promiscuity of the Neisseria Oligosaccharyl Transferase Involved in Protein O-Glycosylation

Neisseria meningitidis PglL belongs to a novel family of bacterial oligosaccharyltransferases (OTases) responsible for O-glycosylation of type IV pilins. Although members of this family are widespread among pathogenic bacteria, there is little known about their mechanism. Understanding the O-glycosylation process may uncover potential targets for therapeutic intervention, and can open new avenues for the exploitation of these pathways for biotechnological purposes. In this work, we demonstrate that PglL is able to transfer virtually any glycan from the undecaprenyl pyrophosphate (UndPP) carrier to pilin in engineered Escherichia coli and Salmonella cells. Surprisingly, PglL was also able to interfere with the peptidoglycan biosynthetic machinery and transfer peptidoglycan subunits to pilin. This represents a previously unknown post-translational modification in bacteria. Given the wide range of glycans transferred by PglL, we reasoned that substrate specificity of PglL lies in the lipid carrier. To test this hypothesis we developed an in vitro glycosylation system that employed purified PglL, pilin, and the lipid farnesyl pyrophosphate (FarPP) carrying a pentasaccharide that had been synthesized by successive chemical and enzymatic steps. Although FarPP has different stereochemistry and a significantly shorter aliphatic chain than the natural lipid substrate, the pentasaccharide was still transferred to pilin in our system. We propose that the primary roles of the lipid carrier during O-glycosylation are the translocation of the glycan into the periplasm, and the positioning of the pyrophosphate linker and glycan adjacent to PglL. The unique characteristics of PglL make this enzyme a promising tool for glycoengineering novel glycan-based vaccines and therapeutics.

The effect of adhesive ligands on bacterial and fibroblast adhesions to surfaces

The modification of medical device surface with adhesive ligands has been recently shown to be an effective means for making a bioselective surface which can inhibit bacterial adhesion while promoting host cell adhesion on device materials. Currently, the lack of quantitative correlation between the adhesion strength of bacteria, nature of adhesive ligand and adhesion kinetics of mammalian cells hinders the development of such device surface. In this study, the biophysical responses of bacteria and mammalian cells towards adhesive ligand on model device surfaces formed by the chemisorption of dopamine (a moderate antibiotic) on glass are elucidated. The effects of RGD, collagen and dopamine modification on the adhesion strength of two clinically significant bacteria including Escherichia coli ( E. coli) and Staphylococcus aureus ( S. aureus) were investigated by the determination of minimum lateral forces for bacterial detachment and the density of adhering bacteria. The result indicates that RGD has no apparent effect on E. coli and S. aureus adhesion, while collagen reduces E. coli but enhances S. aureus. In order to assess the degree of host cell integration, the adhesion kinetics of 3T3 fibroblasts on the four surfaces was examined by confocal reflectance interference contrast microscopy (C-RICM). In contrast to the difference found in bacterial adhesion, the result indicates that both collagen and RGD significantly enhance the initial rate of deformation and adhesion energy for fibroblasts compared to those on glass and dopamine–glass. Overall, it is demonstrated that the choice of adhesive ligand is critical for designing a device surface which simultaneously minimizes bacterial adhesion and enhances host cell integrations.

Identification of a novel spore wall protein (SWP26) from microsporidia Nosema bombycis

Microsporidia are obligate intracellular parasites related to fungi with resistant spores against various environmental stresses. The rigid spore walls of these organisms are composed of two major layers, which are the exospore and the endospore. Two spore wall proteins (the endosporal protein-SWP30 and the exosporal protein-SWP32) have been previously identified in Nosema bombycis. In this study, using the MALDI-TOF-MS technique, we have characterised a new 25.7-kDa spore wall protein (SWP26) recognised by monoclonal antibody 2G10. SWP26 is predicted to have a signal peptide, four potential N-glycosylation sites, and a C-terminal heparin-binding motif (HBM) which is known to interact with extracellular glycosaminoglycans. By using a host cell binding assay, recombinant SWP26 protein (rSWP26) can inhibit spore adherence by 10%, resulting in decreased host cell infection. In contrast, the mutant rSWP26 (rΔSWP26, without HBM) was not effective in inhibiting spore adherence. Immuno-electron microscopy revealed that this protein was expressed largely in endospore and plasma membrane during endospore development, but sparsely distributed in the exospore of mature spores. The present results suggest that SWP26 is a microsporidia cell wall protein that is involved in endospore formation, host cell adherence and infection in vitro. Moreover, SWP26 could be used as a good prospective target for diagnostic research and drug design in controlling the silkworm, Bombyx mori, pebrine disease in sericulture.