Bacillus anthracis Poly-γ-D-Glutamate Capsule Inhibits Opsonic Phagocytosis by Impeding Complement Activation

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 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 Sharma et al. Bacillus anthracis Poly-γ-D-Glutamate Capsule 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.

A Stochastic Intracellular Model of Anthrax Infection With Spore Germination Heterogeneity

“…However, it can be generally agreed that its value has limitations when modelling disease ( 40 ). The capsule is known to protect extracellular bacteria from phagocytosis ( 14 , 41 ), and thus should be considered when modelling the extracellular dynamics of anthrax infection, which has not been explicitly modelled here. It is possible that the capsule also plays a role in protecting emerging intracellular bacteria from the antimicrobial environment of the host cell, since germinating spores are able to quickly produce and coat themselves in the capsule ( 42 , 43 ).…”

“…Together, the anthrax toxins cause suppression of the host’s immune system, often leading to death of the host. Another important factor for the survival of B. anthracis bacteria in the host is the antiphagocytic capsule, which allows extracellular, vegetative bacteria to avoid eradication by the immune system by preventing the bacteria being phagocytosed and destroyed ( 14 ).…”

ANTHRAX: LIFE CYCLE, MECHANISMS OF PATHOGENESIS AND PROSPECTS IN THE DEVELOPMENT OF VETERINARY VACCINES (review)

“…Образование компонентов капсулы кодирует плазмида pXO2. В результате возбудитель становится неуязвимым для иммунной системы хозяина (37,38). Токсины сибирской язвы представляют собой три белка, кодируемые плазмидой pXO1, -протективный антиген (PA), летальный фактор (LF) и отечный фактор (EF), которые объединяются в бинарные комплексы PA/LF и PA/EF с образованием соответственно летального токсина (LT) и отечного токсина (ET) (39).…”

The Bacillus anthracis Cell Envelope: Composition, Physiological Role, and Clinical Relevance

“…This study demonstrated that encapsulated virulent strain exhibit resistance toward complement-dependent and complement-independent bacterial phagocytosis by human macrophages. After incubation with normal human serum or heat-inactivated serum as well as serum-free media, the non-encapsulated strain became highly susceptible to phagocytosis by THP-1 macrophages [174]. In particular, an increased deposition of the derivate b of the complement component 3 (C3b) that serves as a potent opsonizing agent, was observed at the surface of cells lacking their capsule.…”