Biomedical subjects

Todd Becker

Publications and source records attributed to Todd Becker.

5 recordsLinked to original sources

PubMed2007

Oral vaccination with salmonella simultaneously expressing Yersinia pestis F1 and V antigens protects against bubonic and pneumonic plague.

The gut provides a large area for immunization enabling the development of mucosal and systemic Ab responses. To test whether the protective Ags to Yersinia pestis can be orally delivered, the Y. pestis caf1 operon, encoding the F1-Ag and virulence Ag (V-Ag) were cloned into attenuated Salmonella vaccine vectors. F1-Ag expression was controlled under a promoter from the caf1 operon; two different promoters (P), PtetA in pV3, PphoP in pV4, as well as a chimera of the two in pV55 were tested. F1-Ag was amply expressed; the chimera in the pV55 showed the best V-Ag expression. Oral immunization with Salmonella-F1 elicited elevated secretory (S)-IgA and serum IgG titers, and Salmonella-V-Ag(pV55) elicited much greater S-IgA and serum IgG Ab titers than Salmonella-V-Ag(pV3) or Salmonella-V-Ag(pV4). Hence, a new Salmonella vaccine, Salmonella-(F1+V)Ags, made with a single plasmid containing the caf1 operon and the chimeric promoter for V-Ag allowed the simultaneous expression of F1 capsule and V-Ag. Salmonella-(F1+V)Ags elicited elevated Ab titers similar to their monotypic derivatives. For bubonic plague, mice dosed with Salmonella-(F1+V)Ags and Salmonella-F1-Ag showed similar efficacy (>83% survival) against approximately 1000 LD(50) Y. pestis. For pneumonic plague, immunized mice required immunity to both F1- and V-Ags because the mice vaccinated with Salmonella-(F1+V)Ags protected against 100 LD(50) Y. pestis. These results show that a single Salmonella vaccine can deliver both F1- and V-Ags to effect both systemic and mucosal immune protection against Y. pestis.

Administration, Oral↗

PubMed2006

Deletion of znuA virulence factor attenuates Brucella abortus and confers protection against wild-type challenge.

znuA is known to be an important factor for survival and normal growth under low Zn(2+) concentrations for Escherichia coli, Haemophilus spp., Neisseria gonorrhoeae, and Pasteurella multocida. We hypothesized that the znuA gene present in Brucella melitensis 16 M would be similar to znuA in B. abortus and questioned whether it may also be an important factor for growth and virulence of Brucella abortus. Using the B. melitensis 16 M genome sequence, primers were designed to construct a B. abortus deletion mutant. A znuA knockout mutation in B. abortus 2308 (DeltaznuA) was constructed and found to be lethal in low-Zn(2+) medium. When used to infect macrophages, DeltaznuA B. abortus showed minimal growth. Further study with DeltaznuA B. abortus showed that its virulence in BALB/c mice was attenuated, and most of the bacteria were cleared from the spleen within 8 weeks. Protection studies confirmed the DeltaznuA mutant as a potential live vaccine, since protection against wild-type B. abortus 2308 challenge was as effective as that obtained with the RB51 or S19 vaccine strain.

Animals↗

PubMedDate not supplied

Spherical alginate granules formulated for quick-release active subtilisin.

Novel attrition-resistant and spherical enzyme granules encapsulating active subtilisin were formed by emulsification of 2% alginate sol loaded with active enzyme, instantaneous gelation triggered through in situ release of Ca(2+) (internal gelation), particle separation, and finally acetone extractive drying. Granular subtilisin was highly active, readily dispersible, and mechanically robust. This technique serves as a new and attractive alternative to established enzyme granulation processes, such as fluid bed coating, extrusion followed by marumerization, drum granulation, or prilling, for use in industrial enzyme applications such as detergents, textile manufacturing, and food processing. The formulation and encapsulation conditions were optimized to maximize the resistance of the granule to compression and impact forces, consistent with enzyme release and particle dispersion in detergent solutions. Well characterized alginates, with specified guluronic/mannuronic acid (G/M) content and molecular weight, were used in the formulation. The characteristics of the resulting microspheres, including their size and distribution, morphology, shrinkage, compression resistance, impact strength, solubility and encapsulation yield, were examined. Spherical dry granules were formulated with a mean diameter of 500 microm with particle sizes ranging from 300 to 800 microm. Dry alginate granules were discrete, spherical, and glossy white and exhibited impact strength, compression resistance, and solubility difference dependent on composition. Reduced starch levels, high alginate concentration, low alginate molecular weight, and use of high guluronate alginates resulted in the lowest dust level and highest compression resistance. Subtilisin mass yields were approximately 50%, and specific activity yields ranged from 60% to 100%. A formulation consisting of 3% SG150 alginate, 10% starch, 10% TiO(2), and 1% CaCO(3) provided granules appropriate for use in detergent application.

Alginates↗

PubMedDate not supplied

Surfactant-induced unfolding of cellulase: kinetic studies.

Surfactant-induced unfolding is a significant degradation pathway for detergent enzymes. This study examines the kinetics of surfactant-induced unfolding for endoglucanase III, a detergent cellulase, under conditions of varying pH, temperature, ionic strength, surfactant type, and surfactant concentration. Interactions between protein and surfactant monomer are shown to play a key role in determining the kinetics of the unfolding process. We demonstrate that the unfolding rate can be slowed by (1) modifying protein charge and/or pH conditions to create electrostatic repulsion of ionic surfactants and (2) reducing the amount of monomeric ionic surfactant available for interaction with the enzyme (i.e., by lowering the critical micelle concentration). Additionally, our results illustrate that there is a poor correlation between thermodynamic stability in buffer (DeltaG(unfolding)) and resistance to surfactant-induced unfolding.

Bacteria↗

PubMedDate not supplied

Ca2+-surfactant interactions affect enzyme stability in detergent solutions.

Detergent proteases and amylases generally bind Ca(2+) ions. These bound ions enhance enzyme stability, reducing the rates of degradative reactions such as unfolding and proteolysis. Thus, surfactant aggregates, such as micelles, affect protease and amylase stability indirectly, by competing with the enzymes for Ca(2+) ions. Dissociation constants for Ca(2+) interactions with anionic surfactant micelles are in the 10(-3) to 10(-2) M range. These interactions are weak relative to enzyme-Ca(2+) interactions (K(d) of order 10(-6) M). However, surfactant is typically present at much higher concentration than enzyme, and it is the Ca(2+)-micelle equilibrium that largely determines the amount of free Ca(2+) available for binding to enzymes. The problem of surfactant-mediated Ca(2+) removal from enzymes can be avoided by adding calcium to a detergent formulation in an amount such that the concentration of free Ca(2+) is around 10(-5)M.

Bacillus↗