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The Poly-?-d-Glutamic Acid Capsule Surrogate of the Bacillus anthracis Capsule Is a Novel Toll-Like Receptor 2 Agonist.

ABSTRACT: Bacillus anthracis is a pathogenic Gram-positive bacterium that causes a highly lethal infectious disease, anthrax. The poly-?-d-glutamic acid (PGA) capsule is one of the major virulence factors of B. anthracis, along with exotoxins. PGA enables B. anthracis to escape phagocytosis and immune surveillance. Our previous study showed that PGA activates the human macrophage cell line THP-1 and human dendritic cells, resulting in the production of the proinflammatory cytokine interleukin-1? (IL-1?) (M. H. Cho et al., Infect Immun 78:387-392, 2010, http://dx.doi.org/10.1128/IAI.00956-09). Here, we investigated PGA-induced cytokine responses and related signaling pathways in mouse bone marrow-derived macrophages (BMDMs) using Bacillus licheniformis PGA as a surrogate for B. anthracis PGA. Upon exposure to PGA, BMDMs produced proinflammatory mediators, including tumor necrosis factor alpha (TNF-?), IL-6, IL-12p40, and monocyte chemoattractant protein 1 (MCP-1), in a concentration-dependent manner. PGA stimulated Toll-like receptor 2 (TLR2) but not TLR4 in Chinese hamster ovary cells expressing either TLR2 or TLR4. The ability of PGA to induce TNF-? and IL-6 was retained in TLR4(-/-) but not TLR2(-/-) BMDMs. Blocking experiments with specific neutralizing antibodies for TLR1, TLR6, and CD14 showed that TLR6 and CD14 also were necessary for PGA-induced inflammatory responses. Furthermore, PGA enhanced activation of mitogen-activated protein (MAP) kinases and nuclear factor-kappa B (NF-?B), which are responsible for expression of proinflammatory cytokines. Additionally, PGA-induced TNF-? production was abrogated not only in MyD88(-/-) BMDMs but also in BMDMs pretreated with inhibitors of MAP kinases and NF-?B. These results suggest that immune responses induced by PGA occur via TLR2, TLR6, CD14, and MyD88 through activation of MAP kinase and NF-?B pathways.

SUBMITTER: Jeon JH

PROVIDER: S-EPMC4567625 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

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The Poly-γ-d-Glutamic Acid Capsule Surrogate of the Bacillus anthracis Capsule Is a Novel Toll-Like Receptor 2 Agonist.

Jeon Jun Ho JH Lee Hae-Ri HR Cho Min-Hee MH Park Ok-Kyu OK Park Jungchan J Rhie Gi-eun GE

Infection and immunity 20150720 10

Bacillus anthracis is a pathogenic Gram-positive bacterium that causes a highly lethal infectious disease, anthrax. The poly-γ-d-glutamic acid (PGA) capsule is one of the major virulence factors of B. anthracis, along with exotoxins. PGA enables B. anthracis to escape phagocytosis and immune surveillance. Our previous study showed that PGA activates the human macrophage cell line THP-1 and human dendritic cells, resulting in the production of the proinflammatory cytokine interleukin-1β (IL-1β) ( ...[more]

PMID: 26195551

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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:BackgroundPoly-γ-glutamic acid (γ-PGA) is a valuable polymer with glutamate as its sole precursor. Enhancement of the intracellular glutamate synthesis is a very important strategy for the improvement of γ-PGA production, especially for those glutamate-independent γ-PGA producing strains. Corynebacterium glutamicum has long been used for industrial glutamate production and it exhibits some unique features for glutamate synthesis; therefore introduction of these metabolic characters into the γ-PGA producing strain might lead to increased intracellular glutamate availability, and thus ultimate γ-PGA production.ResultsIn this study, the unique glutamate synthesis features from C. glutamicum was introduced into the glutamate-independent γ-PGA producing Bacillus amyloliquefaciens NK-1 strain. After introducing the energy-saving NADPH-dependent glutamate dehydrogenase (NADPH-GDH) pathway, the NK-1 (pHT315-gdh) strain showed slightly increase (by 9.1%) in γ-PGA production. Moreover, an optimized metabolic toggle switch for controlling the expression of ɑ-oxoglutarate dehydrogenase complex (ODHC) was introduced into the NK-1 strain, because it was previously shown that the ODHC in C. glutamicum was completely inhibited when glutamate was actively produced. The obtained NK-PO1 (pHT01-xylR) strain showed 66.2% higher γ-PGA production than the NK-1 strain. However, the further combination of these two strategies (introducing both NADPH-GDH pathway and the metabolic toggle switch) did not lead to further increase of γ-PGA production but rather the resultant γ-PGA production was even lower than that in the NK-1 strain.ConclusionsWe proposed new metabolic engineering strategies to improve the γ-PGA production in B. amyloliquefaciens. The NK-1 (pHT315-gdh) strain with the introduction of NADPH-GDH pathway showed 9.1% improvement in γ-PGA production. The NK-PO1 (pHT01-xylR) strain with the introduction of a metabolic toggle switch for controlling the expression of ODHC showed 66.2% higher γ-PGA production than the NK-1 strain. This work proposed a new strategy for improving the target product in microbial cell factories.

Dataset Information

The Poly-?-d-Glutamic Acid Capsule Surrogate of the Bacillus anthracis Capsule Is a Novel Toll-Like Receptor 2 Agonist.

Publications

The Poly-γ-d-Glutamic Acid Capsule Surrogate of the Bacillus anthracis Capsule Is a Novel Toll-Like Receptor 2 Agonist.

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