Genomic structure of capsular determinants.
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Bacterial Capsules↗
Biomedical subjects
I S Roberts
Publications and source records attributed to I S Roberts.
An investigation into the membrane-interactive potential of the Escherichia coli KpsE C-terminus.
Membrane binding via C-terminal amphiphilic alpha-helical structure is a novel anchoring mechanism, which has been characterised in a number of prokaryotic carboxypeptidases. Here, we have used graphical and DWIH analyses to ascertain if a similar anchoring mechanism may be utilised by the Escherichia coli KpsE protein in its binding to the periplasmic face of the inner membrane. The results of these analyses have been compared to those obtained for similar analyses of the C-terminal sequences of E. coli penicillin-binding proteins (PBPs) PBP5 and PBP6 which, are known to function as amphiphilic alpha-helical membrane anchors, and of melittin, a known membrane-interactive toxin. We have also used FTIR spectroscopy and lipid phase transition temperature analysis to investigate the interaction of a peptide homologue of KpsE C-terminal region with membrane lipid. Our results suggest that the KpsE C-terminal sequence has the potential to form an amphiphilic alpha-helix and that this alpha-helix could feature in the membrane binding of the protein.
Amino Acid Sequence↗
Vascular endothelial growth factor expression correlates with tumour grade and vascularity in gliomas.
AIMS: Tumour vascularity and vascular endothelial growth factor (VEGF) expression were studied in 41 primary brain tumours of astrocytic and oligodendroglial origin, in order to define the potential role of VEGF in the vascularization and growth of these tumours. METHODS AND RESULTS: Two commercial monoclonal antibodies to the VEGF protein (from R&D Systems and NeoMarkers), raised against different isoforms, were utilized. Each monoclonal antibody consistently detected the expression of VEGF in different cell types. The R&D Systems antibody only produced surface staining of endothelial cells in tumour capillaries, whereas staining with the Neomarkers antibody was largely confined to tumour cell cytoplasm. High levels of staining were seen with the R&D Systems and NeoMarkers antibodies in 13 and 14 of 15 glioblastomas, respectively, four and three of five oligodendrogliomas, four and seven of 10 anaplastic astrocytomas, one and three of six low-grade astrocytomas and none and none of five pilocytic astrocytomas. There was a close correlation between VEGF expression, tumour vascularity and grade. CONCLUSIONS: These findings support a role for VEGF in the angiogenesis of glioblastoma, anaplastic astrocytoma and oligodendroglioma. The distinct immunoreactivities of the two commercial monoclonal antibodies indicate either there is expression of different splice variants of VEGF or that the epitopes are differentially revealed during synthesis, secretion and receptor-binding of the growth factor. This highlights the importance of using more than one antibody in the evaluation of tissue VEGF expression.
Astrocytoma↗
Identification and characterization of the Cryptococcus neoformans phosphomannose isomerase-encoding gene, MAN1, and its impact on pathogenicity.
The polysaccharide capsule surrounding Cryptococcus neoformans comprises manose, xylose and glucuronic acid, of which mannose is the major constituent. The GDP-mannose biosynthesis pathway is highly conserved in fungi and consists of three key enzymes: phosphomannose isomerase (PMI), phosphomannomutase (PMM) and GDP-mannose pyrophosphorylase (GMP). The MAN1 gene, encoding for the PMI enzyme, was isolated and sequenced from C. neoformans, and a disruption of the MAN1 gene was generated. One MAN1 disruption mutant, man1, which showed poor capsule formation, reduced polysaccharide secretion and morphological abnormalities, was chosen for virulence studies. In both the rabbit and the mouse models of invasive cryptococcosis, man1 was shown to be severely impaired in its virulence, with complete elimination of the yeast from the host. A reconstituted strain of man1 was constructed using gene replacement at the native locus. The wild-type and reconstituted strains were significantly more virulent than the knock-out mutant in both animal models. Our findings reveal that PMI activity is essential for the survival of C. neoformans in the host. The fact that the man1 mutant was not pathogenic suggests that blocking mannose synthesis could be fungicidal in the mammalian host and thus an excellent target for antifungal drug development.
Amino Acid Sequence↗
Plasma transforming growth factor beta(1) and platelet activation: implications for studies in transplant recipients.
BACKGROUND: Evidence from animal models supports the hypothesis that dysregulated transforming growth factor beta(1) (TGF beta(1)) expression plays a role in chronic allograft rejection, the progression of diabetic nephropathy and fibrotic glomerulopathies. However, more evidence is required to support this hypothesis in man, and the current literature concerning blood TGF beta(1) levels in clinical studies is highly confused. We have investigated: (i) the hypothesis that the widespread practice of activating clinical samples prior to measurement of TGF beta(1) is detecting the platelet-released pool of TGF beta(1), artefactually generated on venepuncture and unrepresentative of the real circulating in vivo TGF beta(1) pool; and (ii) the effect of different immunosuppressive drugs on apparent TGF beta(1) plasma levels. METHODS: The effect of two different venepuncture procedures on plasma TGF beta(1) was compared in 10 healthy volunteers, one procedure designed to minimize platelet activation and the other representing standard venepuncture practice in a clinic situation. Blood samples from 52 renal transplant recipients on either cyclosporine or tacrolimus immunosuppression were taken by standard venepuncture to investigate the effect of immunosuppressive drugs on plasma TGF beta(1). Plasma TGF beta(1) and beta thromboglobulin were measured by ELISA. RESULTS: Among 10 healthy volunteers who underwent two different methods of venepuncture, eight of 10 had undetectable levels of TGF beta(1) (<100 pg/ml) under conditions that minimize platelet activation. In contrast, all 10 paired plasma samples collected by vacutainer had measurable TGF beta(1) (median 7.70 ng/ml, interquartile range 5.87-13.64 ng/ml) following acid/ urea activation. The median beta TG level (a measure of platelet degranulation) was 0.71 microg/ml (interquartile range 0.53-1.19 microg/ml) in the special collections compared with 3.39 microg/ml (interquartile range 2.27-4.33 microg/ml) in the vacutainer samples (P=0.0029). Among 52 allograft recipients there was a significantly higher mean TGF beta(1) level in plasma from patients on cyclosporine therapy compared with patients on tacrolimus (28,090+/-26,860 pg/ml vs 7173+/-10 610 pg/ml, respectively; P<0.002). Mean plasma beta TG levels were also significantly higher during cyclosporine therapy compared with tacrolimus (8.14+/-5.54 microg/ml vs 3.66+/-3.32 microg/ml, respectively; P<0.002). However, when TGF beta(1) values were corrected for the degree of platelet activation (by factoring with beta TG) there was no significant difference between TGF beta(1) levels on cyclosporine or tacrolimus (4117+/-2993 pg/microg beta TG vs 2971+/-658 pg/microg beta TG, respectively; P=0.294). CONCLUSIONS: To avoid erroneous hypotheses concerning TGF beta(1) and perpetuating confusion in the literature over levels in health and disease, it is imperative that proper internal controls for platelet activation are used. The effects of experimental treatments and drugs on platelet biology must be rigorously controlled when attempting to measure and interpret plasma levels of TGF beta(1) in clinical practice.
Adult↗
Clinicopathological correlation in biopsy-proven atherosclerotic nephropathy: implications for renal functional outcome in atherosclerotic renovascular disease.
BACKGROUND: Atherosclerotic renovascular disease (ARVD) is commonly associated with renal failure. It is now recognized that intrarenal damage, (ischaemic or atherosclerotic nephropathy) is a major contributor to the renal impairment in these patients. In this study the impact of histological changes upon renal functional outcome was investigated in patients with atherosclerotic nephropathy. METHODS: The Hope Hospital renal biopsy database (1985-1998) was interrogated for patients with histology compatible with atherosclerotic nephropathy. Case-note review enabled the assessment of several clinical parameters and outcomes, including change in creatinine clearance per year (DeltaCrCl (ml/min/year)), blood pressure control, dialysis need, and death. Renal parenchymal damage was analysed by morphometric analysis (of interstitial fibrosis and glomerulosclerosis) and a semi-quantitative chronic damage score (score 0-3 (normal-severe) for each of glomerulosclerosis, interstitial fibrosis, tubular atrophy, and arteriolar hyalinosis; maximum=12). Patients were stratified into two groups who had either deteriorating (group 1) or stable (group 2) renal function during follow-up. RESULTS: Twenty-five patients (age 64.7+/-10.5, range 43-83 years; 17 male, eight female) were identified. Sixteen patients had undergone angiography; two had significant (>50%) renal artery stenosis. Mean follow-up was 25.6+/-14.8 (range 5-50) months. Group 1 patients had DeltaCrCl -7.4+/-6.8 ml/min/year, n=14 and group 2 patients had DeltaCrCl 4.8+/-7.0 ml/min/year, n=11. Four patients in group 1 developed end-stage renal disease and five patients died (three in group 1 and two in group 2). At study entry, group 1 patients had worse renal function (CrCl 27.6+/-17.6 vs 36.0+/-33.9, NS), greater proteinuria (1.2 vs 0.5 g/24 h, NS), and higher systolic blood pressure (167.1+/-30.8 mmHg vs 150.6+/-37.8, NS) compared with group 2 patients. Group 1 patients showed more glomerulosclerosis (51.6 vs 24.9%, P:<0.01), greater proportional interstitial volume (44.9 vs 33.9%, P:<0.02), and higher overall chronic damage score (P:<0.05) than those in group 2. There was a significant correlation between renal functional outcome and chronic damage score, glomerulosclerosis and proportional interstitial volume for the entire patient cohort. CONCLUSION: In patients with atherosclerotic nephropathy the severity of histopathological damage is an important determinant and predictor of renal functional outcome.
Adult↗
Handling of renal biopsies: different approaches reflect a lack of evidence for what constitutes "best practice".
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Biopsy↗
Apoptosis in podocytes induced by TGF-beta and Smad7.
Primary and secondary forms of focal segmental glomerulosclerosis (FSGS) are characterized by depletion of podocytes and constitute a central manifestation of chronic progressive glomerular diseases. Here we report that podocytes undergo apoptosis at early stages in the course of progressive glomerulosclerosis in TGF-beta1 transgenic mice. Apoptosis is associated with progressive depletion of podocytes and precedes mesangial expansion. Smad7 protein expression is strongly induced specifically in damaged podocytes of transgenic mice and in cultured murine podocytes treated with TGF-beta. TGF-beta1 and Smad7 each induce apoptosis in podocytes, and their coexpression has an additive effect. Activation of p38 MAP kinase and caspase-3 is required for TGF-beta-mediated apoptosis, but not for apoptosis induced by Smad7. Unlike TGF-beta, Smad7 inhibits nuclear translocation and transcriptional activity of the cell survival factor NF-kappaB. Our results suggest a novel functional role for Smad7 as amplifier of TGF-beta-induced apoptosis in podocytes and a new pathomechanism for podocyte depletion in progressive glomerulosclerosis.
Animals↗
The transport of group 2 capsular polysaccharides across the periplasmic space in Escherichia coli. Roles for the KpsE and KpsD proteins.
The cell surface expression of group 2 capsular polysaccharides involves the translocation of the polysaccharide from its site of synthesis on the inner face of the cytoplasmic membrane onto the cell surface. The transport process is independent of the repeat structure of the polysaccharide, and translocation across the periplasm requires the cytoplasmic membrane-anchored protein KpsE and the periplasmic protein KpsD. In this paper we establish the topology of the KpsE protein and demonstrate that the C terminus interacts with the periplasmic face of the cytoplasmic membrane. By chemical cross-linking we show that KpsE is likely to exist as a dimer and that dimerization is independent of the other Kps proteins or the synthesis of capsular polysaccharide. No interaction between KpsD and KpsE could be demonstrated by chemical cross-linking, although in the presence of both KpsE and Lpp, KpsD could be cross-linked to a 7-kDa protein of unknown identity. In addition, we demonstrate that KpsD is present not only within the periplasm but is also in both the cytoplasmic and outer membrane fractions and that the correct membrane association of KpsD was dependent on KpsE, Lpp, and the secreted polysaccharide molecule. Both KpsD and KpsE showed increased proteinase K sensitivity in the different mutant backgrounds, reflecting conformational changes in the KpsD and KpsE proteins as a result of the disruption of the transport process. Collectively the data suggest that the trans-periplasmic export involves KpsD acting as the link between the cytoplasmic membrane transporter and the outer membrane with KpsE acting to facilitate this transport process.
Bacterial Proteins↗
Identification that KfiA, a protein essential for the biosynthesis of the Escherichia coli K5 capsular polysaccharide, is an alpha -UDP-GlcNAc glycosyltransferase. The formation of a membrane-associated K5 biosynthetic complex requires KfiA, KfiB, and KfiC.
The Escherichia coli K5 capsular polysaccharide consists of the repeat structure -4)GlcA-beta(1,4)-GlcNAc-alpha(1- and requires the KfiA, KfiB, KfiC, and KfiD proteins for its synthesis. Previously, the KfiC protein was shown to be a beta-UDP-GlcA glycosyltransferase, and KfiD was shown to be a UDP-Glc dehydrogenase. Here, we demonstrate that KfiA is an alpha-UDP-GlcNAc glycosyltransferase and that biosynthesis of the K5 polysaccharide involves the concerted action of the KfiA and KfiC proteins. By site-directed mutagenesis, we determined that the acidic motif of DDD, which is conserved between the C family of glycosyltransferases, is essential for the enzymatic activity of KfiA. In addition, by Western blot analysis, we determined that association of KfiA with the cytoplasmic membrane requires KfiC but not KfiB, whereas the interaction of KfiC with the cytoplasmic membrane was dependent on both KfiA and KfiB. Likewise, KfiB was only detectable in cytoplasmic membrane fractions when both KfiA and KfiC were present. These data suggest that the interaction between the KfiA, KfiB, and KfiC proteins is essential for the stable association of these proteins with the cytoplasmic membrane and the biosynthesis of the K5 polysaccharide.
Amino Acid Sequence↗
Transcriptional organisation and regulation of E. coli group 2 capsule expression.
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Antigens, Bacterial↗
The molecular cloning, nucleotide sequence and expression of an antigenic determinant from Porphyromonas gingivalis.
A genomic library generated in Escherichia coli was probed with a monoclonal antibody (mAb) LDS28, which reacts with a species-specific cell-surface antigen of Porphyromonas gingivalis. A clone designated pGPR2.1 was shown to express a 46-kDa protein reactive with mAb LDS28, which maps to a 1.7-kb HincII fragment. DNA sequence analysis revealed pGPR2.1 contains a 5653-bp insert with six open reading frames, one of which shows significant DNA homology with the rnhB gene of E. coli. Several subclones of pGPR2.1 were randomly generated in plasmid vector pTTQ18* using restriction enzyme Sau3a. Immunoblotting of subclones demonstrated that the LDS28-reactive antigen was coded for by an open reading frame predicted to specify a protein of 455 amino acids (50 kDa). This open reading frame was designated pgaA (Porphyromonas gingivalis antigen). The predicted amino acid sequence of PgaA contains a putative ABC signature for binding NTPs as well as a predicted transmembrane domain. Minicell labelling of pGPR2.1-encoded proteins and subclone derivatives revealed that pgaA directs expression of protein of multiple molecular weights (31-46 kDa) from its own promoter in E. coli, and that some of these forms may be caused by proteolysis of a 50-kDa precursor which itself shows a reduced apparent molecular weight (46 kDa) on sodium dodecyl sulphate-polyacrylamide gel electrophoresis.
Amino Acid Sequence↗
Lewis X structures in the O antigen side-chain promote adhesion of Helicobacter pylori to the gastric epithelium.
Helicobacter pylori NCTC11637 expresses a lipopolysaccharide (LPS) that comprises an O antigen side-chain with structural homology to the human blood group antigen Lewis X (Le(x)). The role of this molecule in adhesion of H. pylori to gastric epithelial cells was investigated. Mutants expressing truncated LPS structures were generated through insertional mutagenesis of rfbM and galE; genes encode GDP mannose pyrophosphorylase and galactose epimerase respectively. Compositional and structural analysis revealed that the galE mutant expressed a rough LPS that lacked an O antigen side-chain. In contrast, an O antigen side-chain was still synthesized by the rfbM mutant, but it lacked fucose and no longer reacted with anti-Le(x) monoclonal antibodies (Mabs). The ability of these mutants to bind to paraffin-embedded sections from the antrum region of a human stomach was assessed. Adhesion of the wild type was characterized by tropic binding to the apical surface of mucosal epithelial cells and cells lining gastric pits. In contrast, both the rfbM and galE mutants failed to demonstrate tropic binding and adhered to the tissue surface in a haphazard manner. These results indicate that LPS and, more specifically, Le(x) structures in the O antigen side-chain play an important role in targeting H. pylori to specific cell lineages within the gastric mucosa. The role of Le(x) in this interaction was confirmed by the tropic binding of synthetic Le(x), conjugated to latex beads, to gastric tissue. The observed pattern of adhesion was indistinguishable from that of wild-type H. pylori.
Bacterial Adhesion↗
Regulation of the Escherichia coli K5 capsule gene cluster: evidence for the roles of H-NS, BipA, and integration host factor in regulation of group 2 capsule gene clusters in pathogenic E. coli.
The expression of Escherichia coli group 2 capsules (K antigens) is temperature dependent, with capsules only being expressed at temperatures above 20 degrees C. Thermoregulation is at the level of transcription, with no detectable transcription at 20 degrees C. Using the E. coli K5 capsule gene cluster as a model system, we have shown that the nucleoid-associated protein H-NS plays a dual role in regulating transcription of group 2 capsule gene clusters at 37 and 20 degrees C. At 37 degrees C H-NS is required for maximal transcription of group 2 capsule gene clusters, whereas at 20 degrees C H-NS functions to repress transcription. The BipA protein, previously identified as a tyrosine-phosphorylated GTPase and essential for virulence in enteropathogenic E. coli, was shown to play a similar role to H-NS in regulating transcription at 37 and 20 degrees C. The binding of integration host factor (IHF) to the region 1 promoter was necessary to potentiate transcription at 37 degrees C and IHF binding demonstrated by bandshift assays. The IHF binding site was 3' to the site of transcription initiation, suggesting that sequences in the 5' end of the first gene (kpsF) in region 1 may play a role in regulating transcription from this promoter at 37 degrees C. Two additional cis-acting sequences, conserved in both the region 1 and 3 promoters, were identified, suggesting a role for these sequences in the coordinate regulation of transcription from these promoters. These results indicate that a complex regulatory network involving a number of global regulators exists for the control of expression of group 2 capsules in E. coli.
Bacterial Capsules↗
Cloning, expression, and purification of the K5 capsular polysaccharide lyase (KflA) from coliphage K5A: evidence for two distinct K5 lyase enzymes.
The Escherichia coli K5 capsular polysaccharide [-4)-betaGlcA-(1, 4)-alphaGlcNAc-(1-] is a receptor for the capsule-specific bacteriophage K5A. Associated with the structure of bacteriophage K5A is a polysaccharide lyase which degrades the K5 capsule to expose the underlying bacterial cell surface. The bacteriophage K5A lyase gene (kflA) was cloned and sequenced. The kflA gene encodes a polypeptide with a predicted molecular mass of 66.9 kDa and which exhibits amino acid homology with ElmA, a K5 polysaccharide lyase encoded on the chromosome of E. coli SEBR 3282. There was only limited nucleotide homology between the kflA and elmA genes, suggesting that these two genes are distinct and either have been derived from separate progenitors or have diverged from a common progenitor for a considerable length of time. Southern blot analysis revealed that kflA was not present on the chromosome of the E. coli strains examined. In contrast, elmA was present in a subset of E. coli strains. Homology was observed between DNA flanking the kflA gene of bacteriophage K5A and DNA flanking a small open reading frame (ORF(L)) located 5' of the endosialidase gene of the E. coli K1 capsule-specific bacteriophage K1E. The DNA homology between these noncoding sequences indicated that bacteriophages K5A and K1E were related. The deduced polypeptide sequence of ORF(L) in bacteriophage K1E exhibited homology to the N terminus of KflA from bacteriophage K5A, suggesting that ORF(L) is a truncated remnant of KflA. The presence of this truncated kflA gene implies that bacteriophage K1E has evolved from bacteriophage K5A by acquisition of the endosialidase gene and subsequent loss of functional kflA. A (His)(6)-KflA fusion protein was overexpressed in E. coli and purified to homogeneity with a yield of 4.8 mg per liter of bacterial culture. The recombinant enzyme was active over a broad pH range and NaCl concentration and was capable of degrading K5 polysaccharide into a low-molecular-weight product.
Amino Acid Sequence↗
Mast cells: the forgotten cells of renal fibrosis.
BACKGROUND/AIMS: Mast cells, when activated, secrete a large number of fibrogenic factors and have been implicated in the development of fibrotic conditions of the liver, lung, and skin. There is evidence that renal fibrosis is closely linked with a chronic inflammatory cell infiltrate within the interstitium, but a potential role for mast cells in this process has yet to be defined. Therefore, the numbers of mast cells in normal and fibrotic kidneys with various pathologies were investigated. METHODS: Mast cells were quantified in renal transplants showing acute and chronic rejection and cyclosporin toxicity, kidneys removed for chronic pyelonephritis, and renal biopsies from patients with IgA nephropathy, membranous nephropathy, and diabetic nephropathy. Mast cells were stained using two methods: acid toluidine blue detected less than 30% of the mast cells revealed by immunohistochemistry for mast cell tryptase. RESULTS: Mast cells were scarce or absent in normal kidney (median, 1.6 mast cells/mm2) but numerous throughout the cortex and medulla in all specimens that showed fibrosis. They were almost entirely confined to the renal interstitium. Mast cells were present in large numbers in biopsies from patients with membranous nephropathy (median, 21.7 mast cells/mm2) and diabetic nephropathy (median, 29.2 mast cells/mm2), which were selected on the basis of showing chronic injury. In 24 unselected IgA nephropathy biopsies there was a close correlation between numbers of mast cells and the extent of interstitial fibrosis (r = 0.771; p < 0.0001). In renal transplant biopsies, mast cells were associated with allograft fibrosis in chronic rejection (median, 27.1 mast cells/mm2) and chronic cyclosporin toxicity (median, 10.6 mast cells/mm2) but not acute rejection (median, 2.7 mast cells/mm2) or acute cyclosporin toxicity (median, 2.0 mast cells/mm2). There was no detectable increase in mast cell numbers during acute rejection in those transplants that subsequently progressed to chronic rejection. In some biopsies the mast cells were largely intact, but in most cases some or all were degranulated. CONCLUSIONS: An increased number of mast cells is a consistent feature of renal fibrosis, whatever the underlying pathology, and the number of mast cells correlates with the extent of interstitial fibrosis. This suggests that mast cells might play a pathogenetic role in the fibrotic process.
Acute Disease↗
Postmortem findings after fatal anaphylactic reactions.
AIMS: To determine the frequency at which classic manifestations of anaphylaxis are present at necropsy after fatal anaphylactic reactions. METHODS: A register has been established of fatal anaphylactic reactions in the UK since 1992, traced from the certified cause of death and other sources. Details of the previous medical history and the reaction suggest anaphylaxis as the cause of death for 130 cases; a postmortem report was available for 56. RESULTS: The 56 deaths studied included 19 reactions to bee or wasp venom, 16 to foods, and 21 to drugs or contrast media. Death occurred within one hour of anaphylaxis in 39 cases. Macroscopic findings included signs of asthma (mucous plugging and/or hyper-inflated lungs) (15 of 56), petechial haemorrhages (10 of 56), pharyngeal/laryngeal oedema (23 of 56), but for 23 of 56 there was nothing indicative of an allergic death. Mast cell tryptase was raised in 14 of 16 cases tested; three of three tested had detectable IgE specific for the suspected allergen. CONCLUSIONS: In many cases of fatal anaphylaxis no specific macroscopic findings are present at postmortem examination. This reflects the rapidity and mode of death, which is often the result of shock rather than asphyxia. Investigations that might help determine whether anaphylaxis was the cause of death had rarely been performed. In the presence of a typical clinical history, absence of postmortem findings does not exclude the diagnosis of anaphylaxis.
Adolescent↗
When should a coroner's inquest be held? The Manchester guidelines for pathologists.
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Autopsy↗