Complete genome sequence of the dog commensal and human pathogen Capnocytophaga canimorsus strain 5

Capnocytophaga canimorsus is a commensal Gram-negative bacterium, originally isolated from a dog's mouth, that causes septicemia in humans. C. canimorsus has the unusual ability to feed on host cells, including phagocytes. This capacity depends on surface-exposed glycan-foraging systems. Here we present the first complete genome sequence of a C. canimorsus strain (Cc5).     

The genome and surface proteome of Capnocytophaga canimorsus reveal a key role of glycan foraging systems in host glycoproteins deglycosylation

Capnocytophaga canimorsus are commensal Gram-negative bacteria from dog's mouth that cause rare but dramatic septicaemia in humans. C. canimorsus have the unusual property to feed on cultured mammalian cells, including phagocytes, by harvesting the glycan moiety of cellular glycoproteins. To understand the mechanism behind this unusual property, the genome of strain Cc5 was sequenced and analysed. In addition, Cc5 bacteria were cultivated onto HEK 293 cells and the surface proteome was determined. The genome was found to encode many lipoproteins encoded within 13 polysaccharide utilization loci (PULs) typical of the Flavobacteria-Bacteroides group. PULs encode surface exposed feeding complexes resembling the archetypal starch utilization system (Sus). The products of at least nine PULs were detected among the surface proteome and eight of them represented more than half of the total peptides detected from the surface proteome. Systematic deletions of the 13 PULs revealed that half of these Sus-like complexes contributed to growth on animal cells. The complex encoded by PUL5, one of the most abundant ones, was involved in foraging glycans from glycoproteins. It was essential for growth on cells and contributed to survival in mice. It thus represents a fitness factor during infection.     

NMR-based Structural Analysis of the Complete Rough-type Lipopolysaccharide Isolated from Capnocytophaga canimorsus

We here describe the NMR analysis of an intact lipopolysaccharide (LPS, endotoxin) in water with 1,2-dihexanoyl-sn-glycero-3-phosphocholine as detergent. When HPLC-purified rough-type LPS of Capnocytophaga canimorsus was prepared, ¹³C,¹⁵N labeling could be avoided. The intact LPS was analyzed by homonuclear (¹H) and heteronuclear (¹H,¹³C, and ¹H,³¹P) correlated one- and two-dimensional NMR techniques as well as by mass spectrometry. It consists of a penta-acylated lipid A with an α-linked phosphoethanolamine attached to C-1 of GlcN (I) in the hybrid backbone, lacking the 4′-phosphate. The hydrophilic core oligosaccharide was found to be a complex hexasaccharide with two mannose (Man) and one each of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo), Gal, GalN, and l-rhamnose residues. Position 4 of Kdo is substituted by phosphoethanolamine, also present in position 6 of the branched Man^I residue. This rough-type LPS is exceptional in that all three negative phosphate residues are “masked” by positively charged ethanolamine substituents, leading to an overall zero net charge, which has so far not been observed for any other LPS. In biological assays, the corresponding isolated lipid A was found to be endotoxically almost inactive. By contrast, the intact rough-type LPS described here expressed a 20,000-fold increased endotoxicity, indicating that the core oligosaccharide significantly contributes to the endotoxic potency of the whole rough-type C. canimorsus LPS molecule. Based on these findings, the strict view that lipid A alone represents the toxic center of LPS needs to be reassessed.     

Prevalence of Capnocytophaga canimorsus in dogs and occurrence of potential virulence factors

Capnocytophaga canimorsus is a Gram-negative commensal of dog's mouth causing severe human infections. A strain isolated from a human fatal infection was recently shown to have a sialidase, to inhibit the bactericidal activity of macrophages and to block the release of nitric oxide by LPS-stimulated macrophages. The present study aimed at determining the prevalence of C. canimorsus in dogs and the occurrence of these hypothetical virulence factors. C. canimorsus could be retrieved from the saliva of 61 dogs out of 106 sampled. Like in clinical isolates, all dog strains had a sialidase and 60% blocked the killing of phagocytosed Escherichia coli by macrophages. In contrast, only 6.5% of dog strains blocked the release of nitric oxide by LPS-challenged macrophages, suggesting that this property might contribute to virulence. The comparative analysis of 69 16S rDNA sequences revealed the existence of C. canimorsus strains that could be misdiagnosed.     

Ultrastructure of surface epithelial cells of the normal human rectum

Luminal surface epithelial cells, excluding a few endocrine cells of the normal human rectum, were studied electron microscopically and 5 types of cells were recognized with special reference to some structure containing mucous substances. Principal-1 cells showing few tiny vesicles and Principal-2 cells containing some tiny vesicles seemed to belong to the absorptive cell group. Vesicle cells having numerous tiny vesicles, and Columnar mucous cells accompanied by numerous tiny vesicles and some round or oval mucous vacuoles, seemed to be labelled as of the secretory cell group. The common features of the epithelial columnar cells, except for the Goblet cell, were columnar shape, microvilli whose length and density had considerable variation, glycocalyceal bodies around the microvilli, and thick surface coat. Goblet cells were characterized by a goblet shape which was expanded by numerous mucous droplets. It is of special interest that 4 different types of columnar epithelial cells are recognized on the luminal surface of the normal human rectum, and that Vesicle cells and Columnar mucous cells are first observed on the luminal surface of the large intestine. Similar epithelial cells have only been reported in the crypt of the large intestine and not on the luminal surface.     

Epithelial cells as phagocytes: apoptotic epithelial cells are engulfed by mammary alveolar epithelial cells and repress inflammatory mediator release

Clearance of apoptotic cells is critical to tissue homeostasis and resolution of inflammatory lesions. Macrophages are known to remove dying cells and release anti-inflammatory mediators in response; however, many cells traditionally thought of as poor phagocytes can mediate this function as well. In the lactating mammary gland following weaning, alveolar epithelial cell death is massive, yet the gland involutes rapidly, attaining its prepregnancy state in a matter of days. We found histologic evidence of apoptotic cell phagocytosis by viable mammary epithelial cells (MEC) in the involuting mouse mammary gland. Cultured MEC were able to engulf apoptotic cells in vitro, utilizing many of the same receptors used by macrophages, including the phosphatidylserine receptor (PSR), CD36, the vitronectin receptor alpha(v)beta3, and CD91. In addition, MEC, like macrophages, produced TGFbeta in response to stimulation of the PSR by apoptotic cells or the anti-PSR ab 217G8E9, and downregulated endotoxin-stimulated proinflammatory cytokine production. These data support the hypothesis that amateur phagocytes play a significant role in apoptotic cell clearance and its regulation of inflammation.     

The Lipopolysaccharide from Capnocytophaga canimorsus Reveals an Unexpected Role of the Core-Oligosaccharide in MD-2 Binding

Capnocytophaga canimorsus is a usual member of dog''s mouths flora that causes rare but dramatic human infections after dog bites. We determined the structure of C. canimorsus lipid A. The main features are that it is penta-acylated and composed of a “hybrid backbone” lacking the 4′ phosphate and having a 1 phosphoethanolamine (P-Etn) at 2-amino-2-deoxy-d-glucose (GlcN). C. canimorsus LPS was 100 fold less endotoxic than Escherichia coli LPS. Surprisingly, C. canimorsus lipid A was 20,000 fold less endotoxic than the C. canimorsus lipid A-core. This represents the first example in which the core-oligosaccharide dramatically increases endotoxicity of a low endotoxic lipid A. The binding to human myeloid differentiation factor 2 (MD-2) was dramatically increased upon presence of the LPS core on the lipid A, explaining the difference in endotoxicity. Interaction of MD-2, cluster of differentiation antigen 14 (CD14) or LPS-binding protein (LBP) with the negative charge in the 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) of the core might be needed to form the MD-2 – lipid A complex in case the 4′ phosphate is not present.     

Junctional gating: the achilles' heel of epithelial cells in pathogen infection

Mucosal epithelial cells are a major barrier restricting pathogen entry and, paradoxically, an important entry port for respiratory and enteric viruses. Elegant studies in this issue of Cell Host & Microbe describe how coxsackievirus B3 (related to human poliovirus) infects polarized epithelial cells by engaging two transmembrane proteins of the tight junctions, occludin and CAR. A distinctive endocytic mechanism opens the junctions and gates infectious virus entry.     

Immunogold visualization of actin near the coated pits at the apical surface of human mammary epithelial cells.

This ultrastructural study of both the normal human breast tissue and differentiated mammary carcinoma (NOS) epithelial cells has revealed pictures demonstrating luminal receptor-mediated endocytosis. By application of immunogold anti-actin labeling, actin surrounding the fusion ring of coated pits was visualized. However, the coated membrane was not actin labeled. We suggest that association of the actin with coated pits may evidence for its participation in pinching off of the coated vesicles.     

Access to nectin favors herpes simplex virus infection at the apical surface of polarized human epithelial cells

Viral entry may preferentially occur at the apical or the basolateral surfaces of polarized cells, and differences may impact pathogenesis, preventative strategies, and successful implementation of viral vectors for gene therapy. The objective of these studies was to examine the polarity of herpes simplex virus (HSV) entry using several different human epithelial cell lines. Human uterine (ECC-1), colonic (CaCo-2), and retinal pigment (ARPE-19) epithelial cells were grown on collagen-coated inserts, and the polarity was monitored by measuring the transepithelial cell resistance. Controls were CaSki cells, a human cervical cell line that does not polarize in vitro. The polarized cells, but not CaSki cells, were 16- to 50-fold more susceptible to HSV infection at the apical surface than at the basolateral surface. Disruption of the tight junctions by treatment with EGTA overcame the restriction on basolateral infection but had no impact on apical infection. No differences in binding at the two surfaces were observed. Confocal microscopy demonstrated that nectin-1, the major coreceptor for HSV entry, sorted preferentially to the apical surface, overlapping with adherens and tight junction proteins. Transfection with small interfering RNA specific for nectin-1 resulted in a significant reduction in susceptibility to HSV at the apical surface but had little impact on basolateral infection. Infection from the apical but not the basolateral surface triggered focal adhesion kinase phosphorylation and led to nuclear transport of viral capsids and viral gene expression. These studies indicate that access to nectin-1 contributes to preferential apical infection of these human epithelial cells by HSV.     

In vitro three-dimensional modelling of human ovarian surface epithelial cells

Objectives:  Ninety percent of malignant ovarian cancers are epithelial and thought to arise from the ovarian surface epithelium (OSE). We hypothesized that biological characteristics of primary OSE cells would more closely resemble OSE in vivo if established as three-dimensional (3D) cultures.
Materials and methods:  OSE cells were cultured as multicellular spheroids (MCS) (i) in a rotary cell culture system (RCCS) and (ii) on polyHEMA-coated plastics. The MCSs were examined by electron microscopy and compared to OSE from primary tissues and cells grown in 2D. Annexin V FACS analysis was used to evaluate apoptosis and expression of extracellular matrix (ECM) proteins was analysed by immunohistochemical staining.
Results:  On polyHEMA-coated plates, OSE spheroids had defined internal architecture. RCCS MCSs had disorganized structure and higher proportion of apoptotic cells than polyHEMA MCSs and the same cells grown in 2D culture. In 2D, widespread expression of AE1/AE3, laminin and vimentin were undetectable by immunohistochemistry, whereas strong expression of these proteins was observed in the same cells grown in 3D culture and in OSE on primary tissues.
Conclusions:  Physiological and biological features of OSE cells grown in 3D culture more closely resemble characteristics of OSE cells in vivo than when grown by classical 2D approaches. It is likely that establishing in vitro 3D OSE models will lead to greater understanding of the mechanisms of neoplastic transformation in epithelial ovarian cancers.     

Cell surface proteoglycan associates with the cytoskeleton at the basolateral cell surface of mouse mammary epithelial cells

The cell surface proteoglycan on normal murine mammary gland mouse mammary epithelial cells consists of an ectodomain bearing heparan and chondroitin sulfate chains and a lipophilic domain that is presumed to be intercalated into the plasma membrane. Because the ectodomain binds to matrix components produced by stromal cells with specificity and high affinity, we have proposed that the cell surface proteoglycan is a matrix receptor that binds epithelial cells to their underlying basement membrane. We now show that the proteoglycan surrounds cells grown in subconfluent or newly confluent monolayers, but becomes restricted to the basolateral surface of cells that have been confluent for a week or more; Triton X-100 extraction distinguishes three fractions of cell surface proteoglycan: a fraction released by detergent and presumed to be free in the membrane, a fraction bound via a salt-labile linkage, and a nonextractable fraction; the latter two fractions co-localize with actin filament bundles at the basal cell surface; and when proteoglycans at the apical cell surface are cross- linked by antibodies, they initially assimilate into detergent- resistant, immobile clusters that are subsequently aggregated by the cytoskeleton. These findings suggest that the proteoglycan, initially present on the entire surface and free in the plane of the membrane, becomes sequestered at the basolateral cell surface and bound to the actin-rich cytoskeleton as the cells become polarized in vitro. Binding of matrix components may cross-link proteoglycans at the basal cell surface and cause them to associate with the actin cytoskeleton, providing a mechanism by which the cell surface proteoglycan acts as a matrix receptor to stabilize the morphology of epithelial sheets.     

Polarized uterine epithelial cells preferentially present antigen at the basolateral surface: role of stromal cells in regulating class II-mediated epithelial cell antigen presentation

To study Ag presentation in the female reproductive tract, DO11.10 TCR transgenic mice specific for the class II MHC-restricted OVA(323-339) peptide and non-transgenic BALB/c mice were used. We report here that freshly isolated uterine epithelial cells, uterine stromal, and vaginal APCs present OVA and OVA(323-339) peptide to naive- and memory T cells, which is reduced when cells are incubated with Abs to CD80 and 86. To determine whether polarized primary epithelial cells present Ags, uterine epithelial cells were cultured on cell inserts in either the upright or inverted position. After reaching confluence, as indicated by high transepithelial resistance (>2000 ohms/well), Ag presentation by epithelial cells incubated with memory T cells and OVA(323-339) peptide placed on the basolateral surface (inverted) was 2- to 3-fold greater than that seen with epithelial cells in contact with T cells and peptide on the apical surface (upright). In contrast, whereas freshly isolated epithelial cells process OVA, polarized epithelial cells did not. When epithelial cells grown upright on inserts were incubated with T cells and OVA(323-339) peptide, coculture with either hepatocyte growth factor or conditioned stromal medium increased epithelial cell Ag presentation (approximately 90% higher than controls). These studies indicate that uterine stromal cells produce a soluble factor(s) in addition to a hepatocyte growth factor, which regulates epithelial cell Ag presentation. Overall, these results demonstrate that polarized epithelial cells are able to present Ags and suggest that uterine stromal cells communicate with epithelial cells via a soluble factor(s) to regulate Ag presentation in the uterus.     

Growth of normal human ovarian surface epithelial cells in reduced-serum and serum-free media

Summary The human ovarian surface epithelium (OSE) is believed responsible for over 85% of ovarian cancers, yet little is known about the normal biology of these cells. To date, culture of OSE has only been reported in media with high serum supplements. We have developed two media, one with less than 1% of serum (OSEM-1) and the other comprised of highly purified and defined materials (OSEM-2), which allow us to study OSE under relatively defined conditions. By substituting 0.05% of Pedersen’s fetuin for 15% fetal bovine serum (FBS) with Medium 199/MCDB105 basal medium, the cell numbers reached 50 to 60% of those in the presence of 15% FBS over 7 days. However, over several weeks, the total number of population doublings achieved were comparable to those in 15% FBS. Addition of insulin, transferrin, ethanolamine, lipoic acid, and phosphatidylcholine to the medium with Pedersen’s fetuin (OSEM-1) enhanced growth up to 20% more than in their absence. Supplementation of M199/105 with highly purified (>99%) fetuin, alpha₂-macroglobulin, and hydrocortisone resulted in a defined medium (OSEM-2) that permitted 1 to 2 doublings/7 days. In addition, cells maintained a more normal, epithelial-like morphology in culture for a longer period in the presence of Pedersen’s or purified fetuin than in M199/105/15% FBS, thus increasing the number of morphologically normal cells available for experimentation. Addition of 0.05% Pedersen’s fetuin to M199/105 in the presence of 6 to 8% FBS resulted in levels of growth equivalent to those in M199/105/15% FBS alone. We are now able to study the effects of various compounds on the growth and differentiation of OSE under defined conditions, and have reduced the requirement for FBS to produce large numbers of OSE cells.     

诺如病毒：“完美的人类病原”

诺如病毒是近来在我国北京、上海、浙江、广东等地导致病毒性胃肠炎暴发的病原体。本文对该病毒的生物学特征与流行病学进行简短综述,并对正在研发的疫苗作介绍。     

Erythromycin increases bactericidal activity of surface liquid in human airway epithelial cells

Macrolide antibiotics have clinical benefits in patients with diffuse panbronchiolitis and in patients with cystic fibrosis. Although many mechanisms have been proposed, the precise mechanisms are still uncertain. We examined the effects of erythromycin on bactericidal activity of airway surface liquid secreted by cultured human tracheal epithelial cells. Airway surface liquid was collected by washing the surface of human tracheal epithelial cells with a sodium solution (40 meq/l). Methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa were incubated with airway surface liquid, and the number of surviving bacteria was examined. The number of bacteria in airway surface liquid from the cells cultured in medium alone was significantly lower than that in the sodium solution. Furthermore, the number of bacteria in airway surface liquid from the cells treated with erythromycin was significantly lower than that in airway surface liquid from the cells treated with solvent alone. The production of mRNA and protein of human beta-defensin-1 and human beta-defensin-2 was significantly increased by erythromycin. Bactericidal activity of airway surface liquid was observed at low concentrations (40 meq/l) of sodium but not at higher concentrations (> or =80 meq/l). Airway surface liquid did not contain significant amounts of antibiotics supplemented in the culture medium. Erythromycin at the levels in airway surface liquid and in culture medium did not inhibit bacterial growth. These results suggest that erythromycin may increase bactericidal activity of airway surface liquid in human airway epithelial cells through human beta-defensins production and reduce susceptibility of the airway to bacterial infection.