Biosynthesis and ultrasonic degradation of bacterial poly(γ‐glutamic acid)

Pérez‐Camero, Graciela; Congregado, Francisco; Bou, Jordi J.; Muñoz‐Guerra, Sebastián

doi:10.1002/(sici)1097-0290(19990405)63:1<110::aid-bit11>3.3.co;2-k

Cited by 23 publications

(33 citation statements)

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“…1B) yielded powder purity of 96%. Purified PGGA was soluble in water at 3-5 mg/ml, showing a concentration-dependent viscosity that could be significantly decreased by raising the ionic strength of the medium as described previously for polyglutamate polymers (28,29). The complete analytical profile for two representative preparations is summarized in Table 1.…”

Section: Resultsmentioning

confidence: 95%

“…It is well documented that polyglutamic acids can exist in defined structural conformations (36,37,42,43) and that these conformations are sensitive to the effects of pH and ionic strength (28,29,36). It is likely that such extended and regularly repeating structures could be disrupted by random cleavage of polymer chains, particularly when the molecular mass is reduced below a certain critical degree of polymerization.…”

Section: Discussionmentioning

confidence: 99%

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Immunogenicity and Protective Efficacy of Bacillus anthracis Poly-γ-d-glutamic Acid Capsule Covalently Coupled to a Protein Carrier Using a Novel Triazine-based Conjugation Strategy

Joyce

Cook

Chabot

et al. 2006

Journal of Biological Chemistry

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The capsular polypeptide of Bacillus anthracis is composed of a unique polyglutamic acid polymer in which D-glutamate monomers are joined by ␥-peptidyl bonds. The capsule is poorly immunogenic, and efforts at exploiting the polymer for vaccine development have focused on increasing its inherent immunogenicity through chemical coupling to immune-stimulating protein carriers. The usual strategy has employed carbodiimide-based condensing reagents for activation of free ␣-carboxyl groups, despite reports that this chemistry may lead to chain scission. We have purified the high molecular mass capsule to >95% homogeneity and have demonstrated that the polymer contains >99% poly-␥-D-glutamic acid. The predominant structure of the polymer as assessed by circular dichroism and multiangle laser light scattering was unordered at near-neutral pH. We investigated the effects of various activation chemistries, and we demonstrated that carbodiimide treatment under aqueous conditions results in significant cleavage of the ␥-peptidyl bond, whereas scission is significantly reduced in nonaqueous polar solvents, although undesired side chain modification was still observed. An activation chemistry was developed using the triazine-based reagent 4-(4,6-dimethoxy (1,3,5)triazin-2-yl)-4-methylmorpholinium chloride, which allowed for controlled and reproducible derivatization of ␣-carbonyls. In a two-pot reaction scheme, activated capsule was derivatized with a sulfhydryl-reactive heterobifunctional moiety and was subsequently coupled to thiolated carrier protein. This conjugate elicited very high capsule-specific immune titers in mice. More importantly, mice immunized with conjugated capsule exhibited good protection against lethal challenge from a virulent B. anthracis strain in two models of infection. We also showed, for the first time, that treatment of capsule with carbodiimide significantly reduced recognition by capsule-specific antisera concurrent with the reagent-induced reduction of polymer mass. The data suggested that for vaccine development, maintenance of the high mass of the polymer may be important.

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Section: Resultsmentioning

confidence: 95%

Section: Discussionmentioning

confidence: 99%

Immunogenicity and Protective Efficacy of Bacillus anthracis Poly-γ-d-glutamic Acid Capsule Covalently Coupled to a Protein Carrier Using a Novel Triazine-based Conjugation Strategy

Joyce

Cook

Chabot

et al. 2006

Journal of Biological Chemistry

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“…3 C and D). Whereas capsular PGA adheres to B. anthracis, B. licheniformis sheds significant amounts of its capsule (4,14). Therefore, future experiments will need to analyze the effects of PGA-specific antibodies on B. anthracis directly.…”

Section: Resultsmentioning

confidence: 99%

“…Because of the large size of PGA, direct conjugation to PA would result in an insoluble gel, which is undesirable for vaccine formulation. Therefore, we degraded PGA by using an ultrasonic processor (Tekmar, Cincinnati) operating at 20 kHz (14). PGA solution (6.5 mg⅐ml Ϫ1 in PBS) was placed in an ice bath to maintain the system at room temperature.…”

Section: Methodsmentioning

confidence: 99%

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A dually active anthrax vaccine that confers protection against both bacilli and toxins

Rhie

Roehrl

Mourez

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

103

113

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Systemic anthrax is caused by unimpeded bacillar replication and toxin secretion. We developed a dually active anthrax vaccine (DAAV) that confers simultaneous protection against both bacilli and toxins. DAAV was constructed by conjugating capsular poly-␥-D-glutamic acid (PGA) to protective antigen (PA), converting the weakly immunogenic PGA to a potent immunogen, and synergistically enhancing the humoral response to PA. PGA-specific antibodies bound to encapsulated bacilli and promoted the killing of bacilli by complement. PA-specific antibodies neutralized toxin activity and protected immunized mice against lethal challenge with anthrax toxin. Thus, DAAV combines both antibacterial and antitoxic components in a single vaccine against anthrax. DAAV introduces a vaccine design that may be widely applicable against infectious diseases and provides additional tools in medicine and biodefense.

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Mark-Houwink Parameters of Biosynthetic Poly(γ-glutamic acid) in Aqueous Solution

Irurzun

Bou

Pérez‐Camero

et al. 2001

Macromol. Chem. Phys.

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A combined viscosity–light scattering–gel permeation chromatography (GPC) study was carried out on bacterially produced poly(γ‐glutamic acid) (PGGA). PGGA samples with weight‐average molecular weights ranging from 8×104 up to 8×105 g·mol–1 dissolved in phosphate buffer at 0.13 M ionic strength were used. It was found that the Mark–Houwink relation is acceptably obeyed, giving K and a values of 1.84×10–6 dL·g–1 and 1.16, respectively. As expected, GPC analysis showed that PGGA does not follow the universal calibration plot and that deviations can not be avoided by modifying the ionic strength.

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Biosynthesis and ultrasonic degradation of bacterial poly(γ‐glutamic acid)

Cited by 23 publications

References 0 publications

Immunogenicity and Protective Efficacy of Bacillus anthracis Poly-γ-d-glutamic Acid Capsule Covalently Coupled to a Protein Carrier Using a Novel Triazine-based Conjugation Strategy

Immunogenicity and Protective Efficacy of Bacillus anthracis Poly-γ-d-glutamic Acid Capsule Covalently Coupled to a Protein Carrier Using a Novel Triazine-based Conjugation Strategy

A dually active anthrax vaccine that confers protection against both bacilli and toxins

Mark-Houwink Parameters of Biosynthetic Poly(γ-glutamic acid) in Aqueous Solution

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