A novel role for IkappaB kinase (IKK) alpha and IKKbeta in ERK-dependent up-regulation of MUC5AC mucin transcription by Streptococcus pneumoniae

Epithelial cells represent the first line of host innate defense against invading microbes by elaborating a range of molecules involved in pathogen clearance. In particular, epithelial mucins facilitate the mucociliary clearance by physically trapping inhaled microbes. Up-regulation of mucin production thus represents an important host innate defense response against invading microbes. How mucin is induced in upper respiratory Streptococcus pneumoniae infections is unknown. In this study, we show that pneumolysin is required for up-regulation of MUC5AC mucin via TLR4-dependent activation of ERK in human epithelial cells in vitro and in mice in vivo. Interestingly, a "second wave" of ERK activation appears to be important in mediating MUC5AC induction. Moreover, IkappaB kinase (IKK) alpha and IKKbeta are distinctly involved in MUC5AC induction via an ERK1-dependent, but IkappaBalpha-p65- and p100-p52-independent, mechanism, thereby revealing novel roles for IKKs in mediating up-regulation of MUC5AC mucin by S. pneumoniae.     

N-Glycosylation of the MUC1 mucin in epithelial cells and secretions

The MUC1 mucin is an important tumor-associated antigen that shows extensive glycosylation in vivo. The O-glycosylation of this molecule, which has been well characterized in many cell types and tissues, is important in conferring the unusual biochemical and biophysical properties on a mucin. N-Glycosylation is crucial to the folding, sorting, membrane trafficking, and secretion of many proteins. Here, we evaluated the N-glycosylation of MUC1 derived from two sources: endogenous MUC1 isolated from human milk and a recombinant epitope-tagged MUC1F overexpressed in Caco2 colon carcinoma cells. N-Glycans on purified MUC1F/MUC1 were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), gas chromatography-mass spectrometry (GC-MS), and CAD-ESI-MS/MS. The spectra indicate that MUC1F N-glycans have compositions consistent with high-mannose structures (Hex(5-9)HexNAc(2)) and complex/hybrid-type glycans (NeuAc(0-3)Fuc(0-3)Hex(3-8)HexNAc(3-7)). Many of the N-glycan structures are identical on MUC1F and native MUC1; however, a marked difference is seen between the N-glycans on membrane-bound and secreted forms of the native molecule.     

A novel secreted metzincin metalloproteinase from Bacillus intermedius

The mprBi gene from Bacillus intermedius 3-19 encoding a novel secreted metalloproteinase was identified. The mpriBi gene was expressed in an extracellular proteinase-deficient Bacillus subtilis BG 2036 strain and the corresponding protein was characterized biochemically. The 19 kDa MprBi protein was purified to homogeneity and sequenced by mass spectroscopy and Edman degradation methods. Amino acid sequence analysis of MprBi identified an active site motif HEYGHNFGLPHD and a conserved structural component Met-turn, both of which are unique features of the metzincin clan. Furthermore, MprBi harbors a number of distinct sequence elements characteristic of proteinase domains in eukaryotic adamalysins. We conclude that MprBi and similar proteins from other Bacillus species form a novel group of metzincin metalloproteinases in prokaryotes.     

A study of the intracellular and secreted forms of the MUC2 mucin from the PC/AA intestinal cell line.

In this study we present data on the entire population of MUC2 molecules secreted from and within the cell layer of an intestinal cell line. The molecular size distribution of the extracted molecules and their reactivity with two different MUC2 polypeptide antibodies indicated the presence of precursor and mature forms of the mucin. Oligomerized forms of the mucin were found in both the cell layer and medium; however, precursor forms were confined to the cell layer. Isopycnic density gradient centrifugation gave good resolution of mature and precursor forms of MUC2 as assessed by agarose gel electrophoresis. Three different populations of MUC2 were identified: one at low density (>1.3 g/ml) containing the N-glycosylated, non-O-glycosylated polypeptide; a second at intermediate density (1.3-1.35 g/ml) which may represent partially O-glycosylated intermediates; and a third at high density (1.36-1.48 g/ml) containing the mature MUC2 mucins. Rate-zonal centrifugation and agarose electrophoretic analysis of the low-density fraction indicated that the N-glycosylated MUC2 polypeptide was present as putative monomer and dimer/oligomer species. The combination of isopycnic density gradient centrifugation with agarose electrophoresis provides a new and simple approach that allows us to follow the MUC2 gene product from polypeptide through to the mature glycosylated mucin.     

Phosphodiesterase 4B mediates extracellular signal-regulated kinase-dependent up-regulation of mucin MUC5AC protein by Streptococcus pneumoniae by inhibiting cAMP-protein kinase A-dependent MKP-1 phosphatase pathway

Otitis media (OM) is the most common childhood bacterial infection and the major cause of conductive hearing loss in children. Mucus overproduction is a hallmark of OM. Streptococcus pneumoniae is the most common gram-positive bacterial pathogen causing OM. Among many mucin genes, MUC5AC has been found to be greatly up-regulated in the middle ear mucosa of human patients with OM. We previously reported that S. pneumoniae up-regulates MUC5AC expression in a MAPK ERK-dependent manner. We also found that MAPK phosphatase-1 (MKP-1) negatively regulates S. pneumoniae-induced ERK-dependent MUC5AC up-regulation. Therapeutic strategies for up-regulating the expression of negative regulators such as MKP-1 may have significant therapeutic potential for treating mucus overproduction in OM. However, the underlying molecular mechanism by which MKP-1 expression is negatively regulated during S. pneumoniae infection is unknown. In this study we show that phosphodiesterase 4B (PDE4B) mediates S. pneumoniae-induced MUC5AC up-regulation by inhibiting the expression of a negative regulator MKP-1, which in turn leads to enhanced MAPK ERK activation and subsequent up-regulation of MUC5AC. PDE4B inhibits MKP-1 expression in a cAMP-PKA-dependent manner. PDE4-specific inhibitor rolipram inhibits S. pneumoniae-induced MUC5AC up-regulation both in vitro and in vivo. Moreover, we show that PDE4B plays a critical role in MUC5AC induction. Finally, topical and post-infection administration of rolipram into the middle ear potently inhibited S. pneumoniae-induced MUC5AC up-regulation. Collectively, these data demonstrate that PDE4B mediates ERK-dependent up-regulation of mucin MUC5AC by S. pneumoniae by inhibiting cAMP-PKA-dependent MKP-1 pathway. This study may lead to novel therapeutic strategy for inhibiting mucus overproduction.     

Alteration of Streptococcus pneumoniae membrane properties by the folate analog methotrexate

The antifolate compound methotrexate (MTX) is toxic to the gram-positive bacterium Streptococcus pneumoniae. Interaction of MTX with this bacterium resulted in an increase in the electric transmembrane potential (delta psi) and enhanced the delta psi-dependent uptake of isoleucine and MTX. In contrast, delta psi-independent uptake of glutamine was not changed. Folate, a nontoxic analog of MTX, did not exhibit these membrane effects, nor did it prevent the effect of MTX, suggesting that the NH2 in position 4 of the pteridine ring of the MTX molecule is involved in the MTX response. A strain bearing the nonsense mutation amiA9, selected for MTX resistance, did not exhibit increased membrane potential after MTX pretreatment. This suggests that MTX interacts with a specific membrane component in S. pneumoniae. A resulting change in ion permeability could lead to changes in the magnitude of the delta psi. The MTX-sensitive component is altered or absent in mutant amiA9.     

IL-22 promotes the formation of a MUC17 glycocalyx barrier in the postnatal small intestine during weaning

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Immunohistochemical study of the expression of MUC6 mucin and co-expression of other secreted mucins (MUC5AC and MUC2) in human gastric carcinomas.

To investigate the expression of MUC6 mucin in gastric carcinomas, we generated a novel monoclonal antibody (MAb CLH5) using an MUC6 synthetic peptide. MAb CLH5 reacted exclusively with the MUC6 peptide and with native and deglycosylated mucin extracts from gastric tissues. MAb CLH5 immunoreactivity was observed in normal gastric mucosa restricted to pyloric glands of the antrum and mucopeptic cells of the neck zone of the body region. In a series of 104 gastric carcinomas, 31 (29.8%) were immunoreactive for MUC6. The expression of MUC6 was not associated with histomorphological type or with clinicopathological features of the carcinomas. Analysis of the co-expression of MUC6 with other secreted mucins (MUC5AC and MUC2) in 20 gastric carcinomas revealed that different mucin core proteins are co-expressed in 55% of the cases. MUC6 was co-expressed and co-localized with MUC5AC in 45% and with MUC2 in 5% of the cases. Expression of MUC2 alone was observed in 25% of the cases. All carcinomas expressing MUC2 mucin in more than 50% of the cells were of the mucinous type according to the WHO classification. The co-expression of mucins was independent of the histomorphological type and stage of the tumors. In conclusion, we observed, using a novel well-characterized MAb, that MUC6 is a good marker of mucopeptic cell differentiation and is expressed in 30% of gastric carcinomas, independent of the clinicopathological features of the cases. Furthermore, we found that co-expression and co-localization of mucins in gastric carcinomas is independent of histomorphology and staging. Finally, we observed that intestinal mucin MUC2 is expressed as the most prominent mucin of the mucins tested in mucinous-type gastric carcinomas.     

Complex structure of the membrane nuclease of Streptococcus pneumoniae revealed by two-dimensional electrophoresis

Close contacts between Escherichia coli RNA polymerase and specific purine residues in the tryptophan (trp) operon promoter of Salmonella typhimurium were revealed using the methylating agent dimethyl sulfate. RNA polymerase bound to trp promoter DNA caused alterations in the rate of methylation at seven specific sites; in the anti-sense strand, guanine residues at positions ?37, ?34 and ?2 showed enhanced methylation, while those at positions ?14, ?6 and +3 showed reduced methylation. In the sense strand, only the guanine residue at ?32 showed reduced methylation. No RNA polymerase contacts with adenine residues were observed. Using the same method, close interactions between E. coli trp repressor and purine residues in the trp operator of S. typhimurium were examined. Bound trp repressor alters the methylation rates of both guanine and adenine residues from positions ?25 to +3. The points of contact are distributed rather symmetrically on both DNA strands. Three points of close contact are shared by RNA polymerase and trp repressor, supporting previous models of trp repressor action.     

Induction of mucin and MUC5AC expression by the protease activity of Aspergillus fumigatus in airway epithelial cells

Allergic bronchopulmonary mycosis, characterized by excessive mucus secretion, airflow limitation, bronchiectasis, and peripheral blood eosinophilia, is predominantly caused by a fungal pathogen, Aspergillus fumigatus. Using DNA microarray analysis of NCI-H292 cells, a human bronchial epithelial cell line, stimulated with fungal extracts from A. fumigatus, Alternaria alternata, or Penicillium notatum, we identified a mucin-related MUC5AC as one of the genes, the expression of which was selectively induced by A. fumigatus. Quantitative RT-PCR, ELISA, and histochemical analyses confirmed an induction of mucin and MUC5AC expression by A. fumigatus extracts or the culture supernatant of live microorganisms in NCI-H292 cells and primary cultures of airway epithelial cells. The expression of MUC5AC induced by A. fumigatus extracts diminished in the presence of neutralizing Abs or of inhibitors of the epidermal growth factor receptor or its ligand, TGF-α. We also found that A. fumigatus extracts activated the TNF-α-converting enzyme (TACE), critical for the cleavage of membrane-bound pro-TGF-α, and its inhibition with low-molecular weight inhibitors or small interfering RNA suppressed the expression of MUC5AC. The protease activity of A. fumigatus extracts was greater than that of other fungal extracts, and treatment with a serine protease inhibitor, but not with a cysteine protease inhibitor, eliminated its ability to activate TACE or induce the expression of MUC5AC mRNA in NCI-H292. In conclusion, the prominent serine protease activity of A. fumigatus, which caused the overproduction of mucus by the bronchial epithelium via the activation of the TACE/TGF-α/epidermal growth factor receptor pathway, may be a pathogenetic mechanism of allergic bronchopulmonary mycosis.     

Analysis of cytoplasmic membrane proteome of Streptococcus pneumoniae by shotgun proteomic approach

In this study, cytoplasmic membrane proteins of S. pneumoniae strain R6 (ATCC BBA-255) were effectively separated from cell wall or extracellular proteins by sodium carbonate precipitation (SCP) and ultracentrifugation. Forty seven proteins were analyzed as cytoplasmic membrane proteins from the 260 proteins identified by the shotgun proteomic method using SDS-PAGE/LC/MS-MS. ABC transporters for metabolites such as metals, oligopeptides, phosphate, sugar, and amino acids, and membrane proteins involved in phosphotransferse systems, were identified as the predominant and abundant, cytoplasmic membrane proteins that would be essential for nutrient uptake, antibiotic resistance and virulence mechanisms. Our result supports that gel-based shotgun proteomics combined with sodium carbonate precipitation and ultracentrifugation is an effective method for analysis of cytoplasmic membrane proteins of S. pneumoniae.     

MUC16 is lost from the uterodome (pinopode) surface of the receptive human endometrium: in vitro evidence that MUC16 is a barrier to trophoblast adherence

In order for the preimplantation embryo to implant into the uterus, the trophoblast cells must initially adhere to the uterine epithelial surface. In preparation, the luminal secretory cells of the epithelium lose their nonadhesive character and their surface microvilli and bulge into the lumen, forming uterodomes (pinopodes; uterodome is used instead of pinopode, since in humans the surface membrane exocytoses rather than endocytoses (Murphy, Hum Reprod 2000; 15:2451-2454). Previous research has led to the hypothesis that loss of the nonadhesive membrane-spanning mucin MUC1 from the uterodome surface allows trophoblast adherence. Immunofluorescence microscopic assay of luminal epithelia on human uterine biopsies taken from LH+0 to LH+13 show that another membrane-spanning mucin, MUC16, was lost from uterodome surfaces in all samples taken during the receptive phase, LH+6 to LH+8 (n = 12), and that MUC1 was present on uterodomes in 4 of 12 samples and on all ciliated cells of the epithelium in the receptive phase. Short interfering RNA (siRNA) knockdown of MUC16 in a uterine epithelial cell line ECC-1 that, like uterine epithelium, expresses MUC16 and MUC1 allowed increased adherence of cells of a trophoblast cell line. In parallel experiments, siRNA knockdown of MUC1 did not affect trophoblast cell adherence. These data indicate that MUC16 is a membrane component of the nonreceptive luminal uterine surface, which prevents cell adhesion, and that its removal during uterodome formation facilitates adhesion of the trophoblast.     

Glycosyltransferase domain of penicillin-binding protein 2a from Streptococcus pneumoniae is membrane associated

Penicillin-binding proteins (PBPs) are bacterial cytoplasmic membrane proteins that catalyze the final steps of the peptidoglycan synthesis. Resistance to beta-lactams in Streptococcus pneumoniae is caused by low-affinity PBPs. S. pneumoniae PBP 2a belongs to the class A high-molecular-mass PBPs having both glycosyltransferase (GT) and transpeptide (TP) activities. Structural and functional studies of both domains are required to unravel the mechanisms of resistance, a prerequisite for the development of novel antibiotics. The extracellular region of S. pneumoniae PBP 2a has been expressed (PBP 2a*) in Escherichia coli as a glutathione S-transferase fusion protein. The acylation kinetic parameters of PBP 2a* for beta-lactams were determined by stopped-flow fluorometry. The acylation efficiency toward benzylpenicillin was much lower than that toward cefotaxime, a result suggesting that PBP 2a participates in resistance to cefotaxime and other beta-lactams, but not in resistance to benzylpenicillin. The TP domain was purified following limited proteolysis. PBP 2a* required detergents for solubility and interacted with lipid vesicles, while the TP domain was water soluble. We propose that PBP 2a* interacts with the cytoplasmic membrane in a region distinct from its transmembrane anchor region, which is located between Lys 78 and Ser 156 of the GT domain.     

Structural studies of an acidic polysaccharide from the mucin secreted by Drosera capensis

The polysaccharide of the mucin secreted by the leaves of Drosera capensis is composed of l-arabinose, d-xylose, d-galactose, d-mannose, and d-glucuronic acid in the molar ratio of 3.6:1.0:4.9:8.4:8.2. For structural elucidation, methylation analysis using g.l.c. and g.l.c.-m.s. was performed on the native, the carboxyl-reduced, and the degraded polysaccharides. Partial hydrolysis, periodate oxidation, chromium trioxide oxidation, and uronic acid degradation were also performed on the native and carboxyl-reduced polysaccharides. Partial hydrolysis of the native and carboxyl-reduced polysaccharides gave various oligosaccharides that were characterized and suggest a structure containing a d-glucurono-d-mannan backbone having a repeating unit → 4)-β-d-GlcpA-(1 → 2)-α-d-Manp-(1 →. l-Arabinose and d-xylose are present as nonreducing furanosyl and pyranosyl end-groups, respectively, both attached to O-3 of d-glucuronic acid residues of the backbone. d-Galactose is present as non-reducing pyranosyl end-group linked to O-3 of d-mannose residues.     

Outer membrane protein A from Klebsiella pneumoniae activates bronchial epithelial cells: implication in neutrophil recruitment

Aside from its mechanical barrier function, bronchial epithelium plays an important role both in the host defense and in the pathogenesis of inflammatory airway disorders. To investigate its role in lung defense, the effect of a bacterial cell wall protein, the outer membrane protein A from Klebsiella pneumoniae (kpOmpA) on bronchial epithelial cells (BEC) was evaluated on adhesion molecule expression and cytokine production. Moreover, the potential implication of this mechanism in kpOmpA-induced lung inflammation was also determined. Our in vitro studies demonstrated that kpOmpA strongly bound to BEAS-2B cells, a human BEC line, and to BEC primary cultures, resulting in NF-kappaB signaling pathway activation. Exposure to kpOmpA increased ICAM-1 mRNA and cell surface expression, as well as the secretion of IL-6, CXC chemokine ligand (CXCL)1, CXCL8, C-C chemokine ligand 2, CXCL10 by BEAS-2B cells, and BEC primary cultures (p < 0.005). We analyzed in vivo the consequences of intratracheal injection of kpOmpA to BALB/c mice. In kpOmpA-treated mice, a transient neutrophilia (with a maximum at 24 h) was observed in bronchoalveolar lavage and lung sections. In vivo kpOmpA priming induced bronchial epithelium activation as evaluated by ICAM-1 and CXCL1 expression, associated with the secretion of CXCL1 and CXCL5 in bronchoalveolar lavage fluids. In the lung, an increased level of the IL-6, CXCL1, CXCL5, CXCL10 mRNA was observed with a maximum at 6 h. These data showed that kpOmpA is involved in host defense mechanism by its ability to activate not only APC but also BEC, resulting in a lung neutrophilia.     

MKP1 regulates the induction of MCP1 by Streptococcus pneumoniae pneumolysin in human epithelial cells

Epithelial cells act as the first line of host defense against microbes by producing a range of different molecules for clearance. Chemokines facilitate the clearance of invaders through the recruitment of leukocytes. Thus, upregulation of chemokine expression represents an important innate host defense response against invading microbes such as Streptococcus pneumoniae. In this study, we report that the expression of Monocyte Chemotactic Protein 1 (MCP1) was highly induced in response to S. pneumoniae in vitro and in vivo. Among numerous virulence factors, pneumococcal pneumolysin was found to be the major factor responsible for this induction. Furthermore, MCP1 induction was mediated by the p38 mitogen-activated protein kinase (MAPK) whose activation was controlled by MAPK phosphatase 1 (MKP1). Therefore, this study reveals novel roles of pneumolysin in mediating MKP1 expression for the regulation of MCP1 expression in human epithelial cells.     

The immunoglobulin A1 proteinase from Streptococcus pneumoniae is inhibited by tetracycline compounds

Streptococcus pneumoniae produces a zinc-dependent proteinase that cleaves human immunoglobulin (Ig) A1 in the hinge region. This metalloproteinase is hypothesized to act as a virulence factor by allowing S. pneumoniae to evade the protection provided by IgA1, thus enhancing its ability to colonize the human nasopharyngeal region. No biologically compatible inhibitors of this enzyme have been identified. We determined that doxycycline and a chemically modified tetracycline inhibit this enzyme in vitro at low micromolar concentrations.