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Capsule anchoring in Bacillus anthracis occurs by a transpeptidation reaction that is inhibited by capsidin.

ABSTRACT: Bacillus anthracis, the causative agent of anthrax, is a dangerous biological weapon, as spores derived from drug-resistant strains cause infections for which antibiotic therapy is no longer effective. We sought to develop an anti-infective therapy for anthrax and targeted CapD, an enzyme that cleaves poly-gamma-D-glutamate capsule and generates amide bonds with peptidoglycan cross-bridges to deposit capsular material into the envelope of B. anthracis. In agreement with the model that capsule confers protection from phagocytic clearance, B. anthracis capD variants failed to deposit capsule into the envelope and displayed defects in anthrax pathogenesis. By screening chemical libraries, we identified the CapD inhibitor capsidin, 4-[(4-bromophenyl)thio]-3-(diacetylamino)benzoic acid), which covalently modifies the active-site threonine of the transpeptidase. Capsidin treatment blocked capsular assembly by B. anthracis and enabled phagocytic killing of non-encapsulated vegetative forms.

SUBMITTER: Richter S

PROVIDER: S-EPMC2770442 | biostudies-literature | 2009 Jan

REPOSITORIES: biostudies-literature

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Capsule anchoring in Bacillus anthracis occurs by a transpeptidation reaction that is inhibited by capsidin.

Richter Stefan S Anderson Valerie J VJ Garufi Gabriella G Lu Lianghua L Budzik Jonathan M JM Joachimiak Andrzej A He Chuan C Schneewind Olaf O Missiakas Dominique D

Molecular microbiology 20081111 2

Bacillus anthracis, the causative agent of anthrax, is a dangerous biological weapon, as spores derived from drug-resistant strains cause infections for which antibiotic therapy is no longer effective. We sought to develop an anti-infective therapy for anthrax and targeted CapD, an enzyme that cleaves poly-gamma-D-glutamate capsule and generates amide bonds with peptidoglycan cross-bridges to deposit capsular material into the envelope of B. anthracis. In agreement with the model that capsule co ...[more]

PMID: 19017271

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Project description:Bacillus anthracis poly-?-D-glutamic acid (PGA) capsule is an essential virulent factor that helps the bacterial pathogen to escape host immunity. Like other encapsulated bacterial species, the B. anthracis capsule may also inhibit complement-mediated clearance and ensure bacterial survival in the host. Previous reports suggest that B. anthracis spore proteins inhibit complement activation. However, the mechanism through which the B. anthracis capsule imparts a survival advantage to the active bacteria has not been demonstrated till date. Thus, to evaluate the role of the PGA capsule in evading host immunity, we have undertaken the present head-to-head comparative study of the phagocytosis and complement activation of non-encapsulated and encapsulated B. anthracis strains. The encapsulated virulent strain exhibited resistance toward complement-dependent and complement-independent bacterial phagocytosis by human macrophages. The non-encapsulated Sterne strain was highly susceptible to phagocytosis by THP-1 macrophages, after incubation with normal human serum (NHS), heat-inactivated serum, and serum-free media, thus indicating that the capsule inhibited both complement-dependent and complement-independent opsonic phagocytosis. An increased binding of C3b and its subsequent activation to C3c and C3dg, which functionally act as potent opsonins, were observed with the non-encapsulated Sterne strain compared with the encapsulated strain. Other known mediators of complement fixation, IgG, C-reactive protein (CRP), and serum amyloid P component (SAP), also bound more prominently with the non-encapsulated Sterne strain. Studies with complement pathway-specific, component-deficient serum demonstrated that the classical pathway was primarily involved in mediating C3b binding on the non-encapsulated bacteria. Both strains equally bound the complement regulatory proteins C4BP and factor H. Importantly, we demonstrated that the negative charge of the PGA capsule was responsible for the differential binding of the complement proteins between the non-encapsulated and encapsulated strains. At lower pH closer to the isoelectric point of PGA, the neutralization of the negative charge was associated with an increased binding of C3b and IgG with the encapsulated B. anthracis strain. Overall, our data have demonstrated that the B. anthracis capsule inhibits complement fixation and opsonization resulting in reduced phagocytosis by macrophages, thus allowing the bacterial pathogen to evade host immunity.

Project description:o-Succinylbenzoyl-CoA (OSB-CoA) synthetase (EC 6.2.1.26) catalyzes the ATP-dependent condensation of o-succinylbenzoate (OSB) and CoA to form OSB-CoA, the fourth step of the menaquinone biosynthetic pathway in Bacillus anthracis. Gene knockout studies have highlighted this enzyme as a potential target for the discovery of new antibiotics. Here we report the first studies on the kinetic mechanism of B. anthracis OSB-CoA synthetase, classifying it as an ordered bi uni uni bi ping-pong mechanism. Through a series of pre-steady-state and steady-state kinetic studies in conjunction with direct binding studies, it is demonstrated that CoA, the last substrate to bind, strongly activates the first half-reaction after the first round of turnover. The activation of the first half-reaction is most likely achieved by CoA stabilizing conformations of the enzyme in the "F" form, which slowly isomerize back to the E form. Thus, the kinetic mechanism of OSB-CoA synthetase may be more accurately described as an ordered bi uni uni bi iso ping-pong mechanism. The substrate specificity of OSB-CoA synthetase was probed using a series of OSB analogues with alterations in the carboxylate groups. OSB-CoA shows a strong preference for OSB over all of the analogues tested as none were active except 4-[2-(trifluoromethyl)phenyl]-4-oxobutyric acid which exhibited a 100-fold decrease in k(cat)/K(m). On the basis of an understanding of OSB-CoA synthetase's kinetic mechanism and substrate specificity, a reaction intermediate analogue of OSB-AMP, 5'-O-{N-[2-(trifluoromethyl)phenyl]-4-oxobutyl}adenosine sulfonamide (TFMP-butyl-AMS), was designed and synthesized. This inhibitor was found to be an uncompetitive inhibitor to CoA and a mixed-type inhibitor to ATP and OSB with low micromolar inhibition constants. Collectively, these results should serve as an important forerunner to more detailed and extensive inhibitor design studies aimed at developing lead compounds against the OSB-CoA synthetase class of enzymes.

Dataset Information

Capsule anchoring in Bacillus anthracis occurs by a transpeptidation reaction that is inhibited by capsidin.

Publications

Capsule anchoring in Bacillus anthracis occurs by a transpeptidation reaction that is inhibited by capsidin.

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