Phylogenetic lineage and pilus protein Spb1/SAN1518 affect opsonin-independent phagocytosis and intracellular survival of Group B Streptococcus

Opsonin-independent phagocytosis of Group B Streptococcus (GBS) is important in defense against neonatal GBS infections. A recent study indicated a role for GBS pilus in macrophage phagocytosis (Maisey et al Faseb J 22 2008 1715-24). We studied 163 isolates from different phylogenetic backgrounds and those possessing or lacking the gene encoding the pilus backbone protein, Spb1 (SAN1518, PI-2b) and spb1-deficient mutants of wild-type (WT) serotype III-3 GBS 874391 in non-opsonic phagocytosis assays using J774A.1 macrophages. Numbers of GBS phagocytosed differed up to 23-fold depending on phylogenetic background; isolates possessing spb1 were phagocytosed more than isolates lacking spb1. Comparing WT GBS and isogenic spb1-deficient mutants showed WT was phagocytosed better compared to mutants; Spb1 also enhanced intracellular survival as mutants were killed more efficiently. Complementation of mutants restored phagocytosis and resistance to killing in J774A.1 macrophages. Spb1 antiserum revealed surface expression in WT GBS and spatial distribution relative to capsular polysaccharide. spb1 did not affect macrophage nitric oxide and TNF-alpha responses; differences in phagocytosis did not correlate with N-acetyl d-glucosamine (from GBS cell-wall) according to enzyme-linked lectin-sorbent assay. Together, these findings support a role for phylogenetic lineage and Spb1 in opsonin-independent phagocytosis and intracellular survival of GBS in J774A.1 macrophages.

Food and Indoor Air Isolated Bacillus Non-Protein Toxins: : Structures, Physico-Chemical Properties and Mechanisms of Effects on Eukaryotic Cells

We report here the structures and properties of heat-stable, non-protein, and mammalian cell-toxic compounds produced by spore-forming bacilli isolated from indoor air of buildings and from food. Little information is available on the effects and occurrence of heat-stable non-protein toxins produced by bacilli in moisture-damaged buildings. Bacilli emit spores that move in the air and can serve as the carriers of toxins, in a manner similar to that of the spores of toxic fungi found in contaminated indoor air. Bacillus spores in food cause problems because they tolerate the temperatures applied in food manufacture and the spores later initiate growth when food storage conditions are more favorable. Detection of the toxic compounds in Bacillus is based on using the change in mobility of boar spermatozoa as an indicator of toxic exposure. GC, LC, MS, and nuclear magnetic resonance NMR spectroscopy were used for purification, detection, quantitation, and analysis of the properties and structures of the compounds. Toxicity and the mechanisms of toxicity of the compounds were studied using boar spermatozoa, feline lung cells, human neural cells, and mitochondria isolated from rat liver. The ionophoric properties were studied using the BLM (black-lipid membrane) method. One novel toxin, forming ion channels permeant to K+ > Na+ > Ca2+, was found and named amylosin. It is produced by B. amyloliquefaciens isolated from indoor air of moisture-damaged buildings. Amylosin was purified with an RP-HPLC and a monoisotopic mass of 1197 Da was determined with ESI-IT-MS. Furthermore, acid hydrolysis of amylosin followed by analysis of the amino acids with the GS-MS showed that it was a peptide. The presence of a chromophoric polyene group was found using a NMR spectroscopy. The quantification method developed for amylosin based on RP-HPLC-UV, using the macrolactone polyene, amphotericin B (MW 924), as a reference compound. The B. licheniformis strains isolated from a food poisoning case produced a lipopeptide, lichenysin A, that ruptured mammalian cell membranes and was purified with a LC. Lichenysin A was identified by its protonated molecules and sodium- and potassium- cationized molecules with MALDI-TOF-MS. Its protonated forms were observed at m/z 1007, 1021 and 1035. The amino acids of lichenysin A were analyzed with ESI-TQ-MS/MS and, after acid hydrolysis, the stereoisomeric forms of the amino acids with RP-HPLC. The indoor air isolates of the strain of B. amyloliquefaciens produced not only amylosin but also lipopeptides: the cell membrane-damaging surfactin and the fungicidal fengycin. They were identified with ESI-IT-MS observing their protonated molecules, the sodium- and potassium-cationized molecules and analysing the MS/MS spectra. The protonated molecules of surfactin and fengycin showed m/z values of 1009, 1023, and 1037 and 1450, 1463, 1493, and 1506, respectively. Cereulide (MW 1152) was purified with RP-HPLC from a food poisoning strain of B. cereus. Cereulide was identified with ESI-TQ-MS according to the protonated molecule observed at m/z 1154 and the ammonium-, sodium- and potassium-cationized molecules observed at m/z 1171, 1176, and 1192, respectively. The fragment ions of the MS/MS spectrum obtained from the protonated molecule of cereulide at m/z 1154 were also interpreted. We developed a quantification method for cereulide, using RP-HPLC-UV and valinomycin (MW 1110, which structurally resembles cereulide) as the reference compound. Furthermore, we showed empirically, using the BLM method, that the emetic toxin cereulide is a specific and effective potassium ionophore of whose toxicity target is especially the mitochondria.

Protein Synthesis in Mycobacterium tuberculosis H37Rv and the Effect of Streptomycin in Streptomycin-Susceptible and -Resistant Strains

An efficient in vitro amino acid-incorporating system from Mycobacterium tuberculosis H37Rv was standardized. Ribonucleic acid (RNA) isolated from phage-infected M. smegmatis cells served as natural messenger RNA and directed the incorporation of 14C-amino acids into protein. The effects of various antitubercular drugs and “known inhibitors” of protein synthesis on amino acid incorporation were studied. Antibiotics like chloramphenicol and tetracycline inhibited mycobacterial protein synthesis, though they failed to prevent the growth of the organism. This failure was shown to be due to the impermeability of mycobacteria to these drugs by use of “membrane-active” agents along with the antibiotics in growth inhibition studies. Several independent streptomycin-resistant mutants of M. tuberculosis H37Rv were isolated. Streptomycin inhibited the incorporation of 14C-amino acids into proteins by whole cells of a streptomycin-susceptible strain by more than 90%, whereas very little or no inhibition was observed in either high-level or low-level streptomycin-resistant strains.

Purification, crystallization and partial characterization of the antitumour and insecticidal protein subunit from the delta-endotoxin of Bacillus thuringiensis var. thuringiensis

The antitumour protein from the α-endotoxin of Bacillus thuringiensis var. thuringiensis has been purified, crystallized and partially characterized. The same protein also shows the insecticidal activity. According to amino acid analysis it is an acidic protein with a molecular weight of approx. 13 000.

Effects of externally supplied protein on root morphology and biomass allocation in Arabidopsis

Growth, morphogenesis and function of roots are influenced by the concentration and form of nutrients present in soils, including low molecular mass inorganic N (IN, ammonium, nitrate) and organic N (ON, e.g. amino acids). Proteins, ON of high molecular mass, are prevalent in soils but their possible effects on roots have received little attention. Here, we investigated how externally supplied protein of a size typical of soluble soil proteins influences root development of axenically grown Arabidopsis. Addition of low to intermediate concentrations of protein (bovine serum albumen, BSA) to IN-replete growth medium increased root dry weight, root length and thickness, and root hair length. Supply of higher BSA concentrations inhibited root development. These effects were independent of total N concentrations in the growth medium. The possible involvement of phytohormones was investigated using Arabidopsis with defective auxin (tir1-1 and axr2-1) and ethylene (ein2-1) responses. That no phenotype was observed suggests a signalling pathway is operating independent of auxin and ethylene responses. This study expands the knowledge on N form-explicit responses to demonstrate that ON of high molecular mass elicits specific responses.

In vivo delivery of bovine viral diahorrea virus, E2 protein using hollow mesoporous silica nanoparticles

Our work focuses on the application of mesoporous silica nanoparticles as a combined delivery vehicle and adjuvant for vaccine applications. Here we present results using the viral protein, E2, from bovine viral diarrhoea virus (BVDV). BVDV infection occurs in the target species of cattle and sheep herds worldwide and is therefore of economic importance. E2 is a major immunogenic determinant of BVDV and is an ideal candidate for the development of a subunit based nanovaccine using mesoporous silica nanoparticles. Hollow type mesoporous silica nanoparticles with surface amino functionalisation (termed HMSA) were characterised and assessed for adsorption and desorption of E2. A codon-optimised version of the E2 protein (termed Opti-E2) was produced in Escherichia coli. HMSA (120 nm) had an adsorption capacity of 80 [small mu ]g Opti-E2 per mg HMSA and once bound E2 did not dissociate from the HMSA. Immunisation studies in mice with a 20 [small mu ]g dose of E2 adsorbed to 250 [small mu ]g HMSA was compared to immunisation with Opti-E2 (50 [small mu ]g) together with the traditional adjuvant Quillaja saponaria Molina tree saponins (QuilA, 10 [small mu ]g). The humoral responses with the Opti-E2/HMSA nanovaccine although slightly lower than those obtained for the Opti-E2 + QuilA group demonstrated that HMSA particles are an effective adjuvant that stimulated E2-specific antibody responses. Importantly the cell-mediated immune responses were consistently high in all mice immunised with Opti-E2/HMSA nanovaccine formulation. Therefore we have shown the Opti-E2/HMSA nanoformulation acts as an excellent adjuvant that gives both T-helper 1 and T-helper 2 mediated responses in a small animal model. This study has provided proof-of-concept towards the development of an E2 subunit nanoparticle based vaccine.

Enzyme molecular choreography : studies on soluble inorganic pyrophosphatases

Inorganic pyrophosphatases (PPases, EC 3.6.1.1) hydrolyse pyrophosphate in a reaction that provides the thermodynamic 'push' for many reactions in the cell, including DNA and protein synthesis. Soluble PPases can be classified into two families that differ completely in both sequence and structure. While Family I PPases are found in all kingdoms, family II PPases occur only in certain prokaryotes. The enzyme from baker's yeast (Saccharomyces cerevisiae) is very well characterised both kinetically and structurally, but the exact mechanism has remained elusive. The enzyme uses divalent cations as cofactors; in vivo the metal is magnesium. Two metals are permanently bound to the enzyme, while two come with the substrate. The reaction cycle involves the activation of the nucleophilic oxygen and allows different pathways for product release. In this thesis I have solved the crystal structures of wild type yeast PPase and seven active site variants in the presence of the native cofactor magnesium. These structures explain the effects of the mutations and have allowed me to describe each intermediate along the catalytic pathway with a structure. Although establishing the ʻchoreographyʼ of the heavy atoms is an important step in understanding the mechanism, hydrogen atoms are crucial for the mechanism. The most unambiguous method to determine the positions of these hydrogen atoms is neutron crystallography. In order to determine the neutron structure of yeast PPase I perdeuterated the enzyme and grew large crystals of it. Since the crystals were not stable at ambient temperature, a cooling device was developed to allow neutron data collection. In order to investigate the structural changes during the reaction in real time by time-resolved crystallography a photolysable substrate precursor is needed. I synthesised a candidate molecule and characterised its photolysis kinetics, but unfortunately it is hydrolysed by both yeast and Thermotoga maritima PPases. The mechanism of Family II PPases is subtly different from Family I. The native metal cofactor is manganese instead of magnesium, but the metal activation is more complex because the metal ions that arrive with the substrate are magnesium different from those permanently bound to the enzyme. I determined the crystal structures of wild type Bacillus subtilis PPase with the inhibitor imidodiphosphate and an inactive H98Q variant with the substrate pyrophosphate. These structures revealed a new trimetal site that activates the nucleophile. I also determined that the metal ion sites were partially occupied by manganese and iron using anomalous X- ray scattering.

Triacylglycerol lipases of yeast and plants: more than just hydrolases

In the yeast, mobilization of triacylglycerols (TAG) is facilitated by TGL3, TGL4 and TGL5 gene products. Interestingly, experiments using [32P] orthophosphate as a precursor for complex glycerophospholipids revealed that tgl mutants had a lower steady-state level of these membrane lipids. To understand a possible link between TAG lipolysis and phospholipid metabolism, we performed overexpression studies with Tgl3p and Tgl5p which clearly demonstrated that these two enzymes enhanced the level of phospholipids. Domains and motifs search analyses indicated that yeast TAG hydrolases posses a GXSXG lipase motif but also a HX4D acyltransferase motif. Purified Tgl3p and Tgl5p did not only exhibit TAG lipase activity but also catalyzed acyl-CoA dependent acylation of lyso-phosphatidylethanolamine and lyso-phosphatidic acid (LPA), respectively. Search for lipase/hydrolase homologues in the Arabidopsis thaliana genome led to the identification of At4g24160 which possess three motifs that are conserved across the plant species such as GXSXG motif, a HX4D motif and a probable lipid binding motif V(X)3HGF. Characterization of At4g24160 expressed in bacteria revealed that the presence of an acyl-CoA dependent LPA acyltransferase activity. In addition, the purified recombinant At4g24160 protein hydrolyzed both TAG and phosphatidylcholine. We hypothesize that the plant enzyme may be involved in membrane repair. In summary, our results indicate that these TAG lipases play a dual role and thereby contribute to both anabolic and catabolic processes in yeast and plants.

Association between feline immunodeficiency virus (FIV) plasma viral RNA load, concentration of acute phase proteins and disease severity

Veterinarians have few tools to predict the rate of disease progression in FIV-infected cats. In contrast, in HIV infection, plasma viral RNA load and acute phase protein concentrations are commonly used as predictors of disease progression. This study evaluated these predictors in cats naturally infected with FIV. In older cats (>5 years), log10 FIV RNA load was higher in the terminal stages of disease compared to the asymptomatic stage. There was a significant association between log10 FIV RNA load and both log10 serum amyloid A concentration and age in unwell FIV-infected cats. This study suggests that viral RNA load and serum amyloid A warrant further investigation as predictors of disease status and prognosis in FIV-infected cats.

Analysis of Crude Protein and Allergen Abundance in Peanuts (Arachis hypogaea cv. Walter) from Three Growing Regions in Australia

The effects of plant growth conditions on concentrations of proteins, including allergens, in peanut (Arachis hypogaea L.) kernels are largely unknown. Peanuts (cv. Walter) were grown at five sites (Taabinga, Redvale, Childers, Bundaberg, and Kairi) covering three commercial growing regions in Queensland, Australia. Differences in temperature, rainfall, and solar radiation during the growing season were evaluated. Kernel yield varied from 2.3 t/ha (Kairi) to 3.9 t/ha (Childers), probably due to differences in solar radiation. Crude protein appeared to vary only between Kairi and Childers, whereas Ara h 1 and 2 concentrations were similar in all locations. 2D-DIGE revealed significant differences in spot volumes for only two minor protein spots from peanuts grown in the five locations. Western blotting using peanut-allergic serum revealed no qualitative differences in recognition of antigens. It was concluded that peanuts grown in different growing regions in Queensland, Australia, had similar protein compositions and therefore were unlikely to show differences in allergenicity.

Purification, crystallization and partial characterization of the antitumour and insecticidal protein subunit from the σ-endotoxin of Bacillus thuringiensis var. thuringiensisstar, open

The antitumour protein from the α-endotoxin of Bacillus thuringiensis var. thuringiensis has been purified, crystallized and partially characterized. The same protein also shows the insecticidal activity. According to amino acid analysis it is an acidic protein with a molecular weight of approx. 13 000.

Silica vesicles as nanocarriers and adjuvants for generating both antibody and T-cell mediated immune resposes to Bovine Viral Diarrhoea Virus E2 protein

Bovine Viral Diarrhoea Virus (BVDV) is widely distributed in cattle industries and causes significant economic losses worldwide annually. A limiting factor in the development of subunit vaccines for BVDV is the need to elicit both antibody and T-cell-mediated immunity as well as addressing the toxicity of adjuvants. In this study, we have prepared novel silica vesicles (SV) as the new generation antigen carriers and adjuvants. With small particle size of 50 nm, thin wall (similar to 6 nm), large cavity (similar to 40 nm) and large entrance size (5.9 nm for SV-100 and 16 nm for SV-140), the SV showed high loading capacity (similar to 250 mu g/mg) and controlled release of codon-optimised E2 (oE2) protein, a major immunogenic determinant of BVDV. The in vivo functionality of the system was validated in mice immunisation trials comparing oE2 plus Quil A (50 mu g of oE2 plus 10 mu g of Quil A, a conventional adjuvant) to the oE2/SV-140 (50 mu g of oE2 adsorbed to 250 mu g of SV-140) or oE2/SV-140 together with 10 mu g of Quil A. Compared to the oE2 plus Quil A, which generated BVDV specific antibody responses at a titre of 10(4), the oE2/SV-140 group induced a 10 times higher antibody response. In addition, the cell-mediated response, which is essential to recognise and eliminate the invading pathogens, was also found to be higher [1954-2628 spot forming units (SFU)/million cells] in mice immunised with oE2/SV-140 in comparison to oE2 plus Quil A (512-1369 SFU/million cells). Our study has demonstrated that SV can be used as the next-generation nanocarriers and adjuvants for enhanced veterinary vaccine delivery. (C) 2014 Elsevier Ltd. All rights reserved.

Digestion of forages in the rumen is increased by the amount but not the type of protein supplement

Three polyester bag experiments were conducted with fistulated Bos indicus steers to determine the effect of the amount and type of nitrogen (N) supplement on the digestion rate of forages different in quality. In Experiment 1, test substrates were incubated in polyester bags in the rumen of steers fed ryegrass, pangola grass, speargrass and Mitchell grass hays in a 4 by 4 Latin-square design. In Experiment 2, test substrates were incubated in polyester bags in the rumen of steers fed speargrass hay supplemented with urea and ammonium sulfate (US), branched-chain amino acids with US (USAA), casein, cottonseed meal, yeast and Chlorella algae in a 7 by 3 incomplete Latin-square design. In Experiment 3, test substrates were incubated in polyester bags in the rumen of steers fed Mitchell grass hay supplemented with increasing amounts of US or Spirulina algae (Spirulina platensis). The test substrates used in all experiments were speargrass, Mitchell grass, pangola grass or ryegrass hays. Digestion rate of the ryegrass substrate was higher than that of the speargrass substrate (P < 0.05) in Experiment 1. Supplementation with various N sources increased the degradation rate and effective degradability of all incubated substrates above that apparent in Control steers (P < 0.05; Experiment 2). Supplementation of US and Spirulina increased degradation rate and effective degradability of ryegrass, pangola grass and Mitchell grass substrates above that apparent in Control steers (P < 0.05; Experiment 3). However, there was no further response on digestion rate of the substrates in increasing supplementation levels either for US or Spirulina. In conclusion, rate of digestion was affected by forage physical and anatomical properties. Supplementation with various N sources increased rate of digestion when the Control forage ration was very low in N but once a minimum level of N supplementation was reached, irrespective of form of N or other potential growth factors, there was no further increase in rate of digestion.