Assessment of Adhesins as an Indicator of Pathovar-Associated Virulence Factors in Bovine Escherichia coli

The CS31A, F17, and F5 adhesins are usually targeted by serology-based methods to detect pathogenic Escherichia coli associated with calf enteritis. However, the virulence traits of the selected isolates are still poorly described. Here, from a set of 349 diarrheagenic E. coli isolates from cattle, we demonstrated a 70.8% concordance rate (Cohen''s kappa, 0.599) between serology- and PCR-based approaches for the detection of adhesins under field conditions. A 79% to 82.4% correspondence between the two methods was found for fimbrial adhesins, whereas major discrepancies (33%) were observed for CS31A-type antigens. Various F17A variants were found, such as F17Ac (20K) (50%), F17Aa (FY) (18.9%), F17Ab (8.1%), and F17Ad (111K) (5.4%), including a high proportion (17.6%) of new F17A internal combinations (F17Aab, F17Aac, and F17Abc) or untypeable variants. In addition, the highest proportion of pathovar-associated virulence factor (VF) genes was observed among E. coli isolates that produced F5/F41 adhesins. A specific link between the heat-stable toxins related to the enterotoxigenic E. coli (ETEC) pathovar and adhesins was identified. STa was significantly linked to F5/F41 and EAST1 to CS31A adhesins (P < 0.001), respectively, whereas NTEC was associated with F17 adhesin (P = 0.001). Clustering between phylogroups according to the adhesin types was also observed. Also, few Shiga toxin-producing E. coli (STEC) or enteropathogenic E. coli (EPEC) pathovars were identified. Finally, no statistically significant difference was observed in the occurrence of extended-spectrum beta lactamase (ESBL) production according to the adhesins expressed by the isolates (P = 0.09). Altogether, this study gives new insights into the relationship between adhesins, VF, and antimicrobial resistance in calf enteritis and supports the need for further standardization of methodologies for such approaches.     

Multiplex PCR Assay for Detection and Identification of Clostridium botulinum Types A, B, E, and F in Food and Fecal Material

Botulism is diagnosed by detecting botulinum neurotoxin and Clostridium botulinum cells in the patient and in suspected food samples. In this study, a multiplex PCR assay for the detection of Clostridium botulinum types A, B, E, and F in food and fecal material was developed. The method employs four new primer pairs with equal melting temperatures, each being specific to botulinum neurotoxin gene type A, B, E, or F, and enables a simultaneous detection of the four serotypes. A total of 43 C. botulinum strains and 18 strains of other bacterial species were tested. DNA amplification fragments of 782 bp for C. botulinum type A alone, 205 bp for type B alone, 389 bp for type E alone, and 543 bp for type F alone were obtained. Other bacterial species, including C. sporogenes and the nontoxigenic nonproteolytic C. botulinum-like organisms, did not yield a PCR product. Sensitivity of the PCR for types A, E, and F was 10² cells and for type B was 10 cells per reaction mixture. With a two-step enrichment, the detection limit in food and fecal samples varied from 10⁻² spore/g for types A, B, and F to 10⁻¹ spore/g of sample material for type E. Of 72 natural food samples investigated, two were shown to contain C. botulinum type A, two contained type B, and one contained type E. The assay is sensitive and specific and provides a marked improvement in the PCR diagnostics of C. botulinum.     

NT5E Mutations That Cause Human Disease Are Associated with Intracellular Mistrafficking of NT5E Protein

Ecto-5′-nucleotidase/CD73/NT5E, the product of the NT5E gene, is the dominant enzyme in the generation of adenosine from degradation of AMP in the extracellular environment. Nonsense (c.662C→A, p.S221X designated F1, c.1609dupA, p.V537fsX7 designated F3) and missense (c.1073G→A, p.C358Y designated F2) NT5E gene mutations in three distinct families have been shown recently to cause premature arterial calcification disease in human patients. However, the underlying mechanisms by which loss-of-function NT5E mutations cause human disease are unknown. We hypothesized that human NT5E gene mutations cause mistrafficking of the defective proteins within cells, ultimately blocking NT5E catalytic function. To test this hypothesis, plasmids encoding cDNAs of wild type and mutant human NT5E tagged with the fluorescent probe DsRed were generated and used for transfection and heterologous expression in immortalized monkey COS-7 kidney cells that lack native NT5E protein. Enzyme histochemistry and Malachite green assays were performed to assess the biochemical activities of wild type and mutant fusion NT5E proteins. Subcellular trafficking of fusion NT5E proteins was monitored by confocal microscopy and western blot analysis of fractionated cell constituents. All 3 F1, F2, and F3 mutations result in a protein with significantly reduced trafficking to the plasma membrane and reduced ER retention as compared to wild type protein. Confocal immunofluorescence demonstrates vesicles containing DsRed-tagged NT5E proteins (F1, F2 and F3) in the cell synthetic apparatus. All 3 mutations resulted in absent NT5E enzymatic activity at the cell surface. In conclusion, three familial NT5E mutations (F1, F2, F3) result in novel trafficking defects associated with human disease. These novel genetic causes of human disease suggest that the syndrome of premature arterial calcification due to NT5E mutations may also involve a novel “trafficking-opathy”.     

Factor VIII A3 domain residues 1793–1795 represent a factor IXa-interactive site in the tenase complex

BackgroundFactor (F)VIII functions as a cofactor in the tenase complex responsible for conversion of FX to FXa by FIXa. Earlier studies indicated that one of the FIXa-binding sites is located in residues 1811–1818 (crucially F1816) of the FVIII A3 domain. A putative, three-dimensional structure model of the FVIIIa molecule suggested that residues 1790–1798 form a V-shaped loop, and juxtapose residues 1811–1818 on the extended surface of FVIIIa.AimTo examine FIXa molecular interactions in the clustered acidic sites of FVIII including residues 1790–1798.Methods and resultsSpecific ELISA's demonstrated that the synthetic peptides, encompassing residues 1790–1798 and 1811–1818, competitively inhibited the binding of FVIII light chain to active-site-blocked Glu-Gly-Arg-FIXa (EGR-FIXa) (IC₅₀; 19.2 and 42.9 μM, respectively), in keeping with a possible role for the 1790–1798 in FIXa interactions. Surface plasmon resonance-based analyses demonstrated that variants of FVIII, in which the clustered acidic residues (E1793/E1794/D1793) or F1816 contained substituted alanine, bound to immobilized biotin labeled-Phe-Pro-Arg-FIXa (bFPR-FIXa) with a 1.5–2.2-fold greater K_D compared to wild-type FVIII (WT). Similarly, FXa generation assays indicated that E1793A/E1794A/D1795A and F1816A mutants increased the K_m by 1.6–2.8-fold relative to WT. Furthermore, E1793A/E1794A/D1795A/F1816A mutant showed that the K_m was increased by 3.4-fold and the V_max was decreased by 0.75-fold, compared to WT. Molecular dynamics simulation analyses revealed the subtle changes between WT and E1793A/E1794A/D1795A mutant, supportive of the contribution of these residues for FIXa interaction.ConclusionThe 1790–1798 region in the A3 domain, especially clustered acidic residues E1793/E1794/D1795, contains a FIXa-interactive site.     

Karyotypes of tragopogon (Compositae: Lactuceae)

Karyotypes of 24 diploid (2n=12)Tragopogon species are similar with one long pair of chromosomes (A), two medium-length pairs (B and C), and three short pairs (D, E, and F). These species may be divided into three karyotypic groups: 1) seven species with a satellite on A and on D; 2) 14 species with a satellite on A only; and 3) three species with a satellite on D only. Most species within karyotype groups may be separated from each other either by distinctive features of certain chromosomes or by statistical differences in length of chromosome arms or long arm: short arm ratios of chromosome A. Three tetraploid (2n=24) species had two long pairs (A,A′), four medium-length pairs (B,B′;C,C′), and six short pairs (D,D′;E,E′;F,F′). Suggestions are made as to the putative diploid parents of these presumed allotetraploids.     

Effect of Alanine Replacement of L17 and F19 on the Aggregation and Neurotoxicity of Arctic-Type Aβ40

Alzheimer’s disease is the most common form of neurodegenerative disease. Beta-amyloid peptides (Aβ) are responsible for neuronal death both in vitro and in vivo. Previously, L17 and F19 residues were identified as playing key roles in the stabilization of the Aβ₄₀ conformation and in the reduction of its neurotoxicity. In this study, the effects of L17A/F19A mutations on the neurotoxicity of Aβ genetic mutant Arctic-type Aβ₄₀(E22G) were tested. The results showed that compared to Aβ₄₀(E22G), Aβ₄₀(L17A/F19A/E22G) reduced the rate of conformation conversion, aggregation, and cytotoxicity, suggesting that L17 and F19 are critical residues responsible for conformational changes which may trigger the neurotoxic cascade of Aβ. Aβ₄₀(L17A/F19A/E22G) also had decreased damage due to reactive oxygen species. The results are consistent with the discordant helix hypothesis, and confirm that residues 17–25 are in the discordant helix region. Compared to Aβ₄₀(L17A/F19A), reduction in aggregation of Aβ₄₀(L17A/F19A/E22G) was less significantly decreased. This observation provides an explanation based on the discordant helix hypothesis that the mutation of E22 to G22 of Aβ₄₀(E22G) alters the propensity of the discordant helix. Arctic-type Aβ₄₀(E22G) aggregates more severely than wild-type Aβ₄₀, with a consequential increase in toxicity.     

Chloroplast biogenesis. XXIX. The occurrence of several novel chlorophyll a and b chromophores in higher plants

With the use of low temperature spectrofluorometry and matrix calculations it was demonstrated that the chlorophyll a pool of higher plants is made up of four different chlorophyll a chromophores. The latter were segregated by high pressure liquid chromatography on a silica column. They were designated Chl a (E432 F664), Chl a (E436 F670), Chl a (E443 F672) and Chl a (E446 F674), where E refers to the Soret excitation maximum and F to the fluorescence emission maximum at 77 K in ether. Likewise the Chl b pool was shown to consist of at least four different Chl b chromophores which were designated: Chl b (E465), Chl b (E470), Chl b (E475) and Chl b (E485). It was proposed that the various chlorophyll chromophores differed by the degree of oxidation of their side chains at the 2 and 4 positions of the macrocycle. It was also suggested that the chemical modifications at the 2 and 4 positions of the macrocycle may play an important role in positioning the different chlorophyll chromophores in the thylakoid membranes.