AUTHOR=Sharma Shikhar , Bhatnagar Rakesh , Gaur Deepak TITLE=Bacillus anthracis Poly-γ-D-Glutamate Capsule Inhibits Opsonic Phagocytosis by Impeding Complement Activation JOURNAL=Frontiers in Immunology VOLUME=Volume 11 - 2020 YEAR=2020 URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2020.00462 DOI=10.3389/fimmu.2020.00462 ISSN=1664-3224 ABSTRACT=Bacillus anthracis poly-γ-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 poly-γ-D-glutamic acid 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 towards complement dependent and 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 independent opsonic phagocytosis. An increased binding of C3b and its subsequent activation to C3c and C3dg, which functionally act as potent opsonins, was observed in the non-encapsulated Sterne strain compared to the encapsulated strain. Other known mediators of complement fixation, IgG, C-reactive protein (CRP) and Serum amyloid component P (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 poly-γ-D-glutamic acid 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 poly-γ-D-glutamic acid, 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 has 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.