Capsule Influences the Deposition of Critical Complement C3 Levels Required for the Killing of Burkholderia pseudomallei via NADPH-Oxidase Induction by Human Neutrophils

Abstract

Burkholderia pseudomallei is the causative agent of melioidosis and is a major mediator of sepsis in its endemic areas. Because of the low LD50 via aerosols and resistance to multiple antibiotics, it is considered a Tier 1 select agent by the CDC and APHIS. B. pseudomallei is an encapsulated bacterium that can infect, multiply, and persist within a variety of host cell types. In vivo studies suggest that macrophages and neutrophils are important for controlling B. pseudomallei infections, however few details are known regarding how neutrophils respond to these bacteria. Our goal is to describe the capacity of human neutrophils to control highly virulent B. pseudomallei compared to the relatively avirulent, acapsular B. thailandensis using in vitro analyses. B. thailandensis was more readily phagocytosed than B. pseudomallei, but both displayed similar rates of persistence within neutrophils, indicating they possess similar inherent abilities to escape neutrophil clearance. Serum opsonization studies showed that both were resistant to direct killing by complement, although B. thailandensis acquired significantly more C3 on its surface than B. pseudomallei, whose polysaccharide capsule significantly decreased the levels of complement deposition on the bacterial surface. Both Burkholderia species showed significantly enhanced uptake and killing by neutrophils after critical levels of C3 were deposited. Serum-opsonized Burkholderia induced a significant respiratory burst by neutrophils compared to unopsonized bacteria, and neutrophil killing was prevented by inhibiting NADPH-oxidase. In summary, neutrophils can efficiently kill B. pseudomallei and B. thailandensis that possess a critical threshold of complement deposition, and the relative differences in their ability to resist surface opsonization may contribute to the distinct virulence phenotypes observed in vivo.

Delineating the Importance of Serum Opsonins and the Bacterial Capsule in Affecting the Uptake and Killing of Burkholderia pseudomallei by Murine Neutrophils and Macrophages

“…Previous studies have reported mixed results as to the ability of neutrophils to kill Bp. However, a more recent study indicated that human neutrophils could efficiently clear Bp, but only if they displayed at least a minimal threshold of complement deposition on its surface [62]. Our findings are most similar to this study, although the kinetics of murine ROS appears to be faster, of shorter duration, and of lower magnitude compared to that exhibited by human neutrophils, based on the in vitro findings.…”

“…In certain bacteria it is suggested their LPS binds complement components associated with the membrane attack complex at a distance from the bacterial surface, thus not allowing insertion into the bacterial outer membrane [101], and such a mechanism could explain why both Bp and Bt are serum-resistant, as they produce similar type II O-antigenic polysaccharide [64], [86], [102]. It is possible that the most prominent effect mediated by capsule was the ability to reduce complement deposition on the outer surface, which has also been reported by others [62], [63]. The significance of this property is reflected in our finding that complement deposition is required for neutrophil-mediated killing of Bp, but only if a certain level of deposition is achieved (i.e.…”

“…Capsule does not confer protection from direct complement-mediated killing, as both Bp and the acapsular bacteria possess similar resistance to killing, even though the levels of complement deposition was significantly higher in the acapsular strains. However, a significant level of serum sensitivity was displayed by the ΔLPS mutant, indicating that LPS does provide protection from the direct killing mechanisms elicited by complement deposition [62]. In certain bacteria it is suggested their LPS binds complement components associated with the membrane attack complex at a distance from the bacterial surface, thus not allowing insertion into the bacterial outer membrane [101], and such a mechanism could explain why both Bp and Bt are serum-resistant, as they produce similar type II O-antigenic polysaccharide [64], [86], [102].…”

“…A recent study published from our lab showed that human neutrophils can efficiently kill Bp once it is opsonized with human serum [62]. However there are no reports comparing the relative importance of neutrophils and macrophages in clearance of Bp, including the effects of different soluble immune opsonins in promoting bacterial uptake and killing.…”

Migration of Dendritic Cells Facilitates Systemic Dissemination of Burkholderia pseudomallei

“…During the early phases of infection, macrophages and neutrophils provide essential innate defense against B. pseudomallei. However, macrophages also provide a potential intracellular niche for B. pseudomallei persistence and replication (5)(6)(7)(8)(9)(10)(11).…”