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

Date Published: December 14, 2012

Publisher: Public Library of Science

Author(s): Michael E. Woodman, Randall G. Worth, R. Mark Wooten, Lisa A. Morici.


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.

Partial Text

The causative agent of melioidosis, Burkholderia pseudomallei, is a saprophytic bacterium that is endemic throughout Southeast Asia and northern Australia [1], [2], [3]. This organism is a leading cause of pneumonia and septicemia in endemic areas [1], [2], [4], [5], [6]. In the Western hemisphere, B. pseudomallei have been documented sporadically in northern South America, Central America, and certain Caribbean islands, including Puerto Rico [7], [8], [9], [10], [11], [12], [13], and melioidosis cases are becoming increasingly more widespread in these and other tropical/sub-tropical areas worldwide [14], [15]. While infection can be established in healthy individuals through skin abrasions, ingestion, or inhalation, the incidence of melioidosis is more common in individuals with certain predisposing conditions, the primary one being diabetes mellitus [4], [16], [17]. Infection with B. pseudomallei can produce widely varying clinical symptoms which often confounds accurate diagnosis. Acute melioidosis is a serious condition that can rapidly become fatal, and is commonly characterized by abscess formation in lungs, liver, and/or spleen, as well as bacteremia. Latent melioidosis is characterized by a persistent infection that can recrudesce at varying times after the initial infection to cause disease, with the longest confirmed report being 62 years post-infection [18]. Notably, B. pseudomallei are extremely virulent via aerosol exposure, with an estimated LD50 between 5–100 organisms depending on the model [19], [20], [21]. Because of these characteristics, B. pseudomallei has recently been elevated to Tier 1 status by the CDC and APHIS [22]. B. pseudomallei is inherently resistant to many classes of antibiotics, and even treatment with proven antibiotics is often unsuccessful, with mortality rates for acute melioidosis ranging from 40–90% [2], [4]. No vaccine is currently available for preventing melioidosis, and there is great interest in identifying immune mechanisms that can promote efficient clearance of these infections.

Melioidosis can be a highly lethal disease if not appropriately diagnosed and treated, particularly with the development of pneumonia and bacteremia that frequently leads to involvement of multiple organs. A major issue in controlling these infections is that B. pseudomallei are highly efficient at infecting and persisting within multiple non-immune and immune cell types. Within susceptible cell types, these bacteria can quickly escape endosomal compartments and subsequently utilize actin polymerization to efficiently invade adjacent cells without being exposed to the extracellular environment, thus limiting their exposure to antibodies and other soluble immune effectors. Therefore, it is important to identify immune cells involved in the cellular response that are best able to control these infections, as well as the mechanisms that promote bacterial clearance. Neutrophils are important for controlling systemic infections caused by numerous bacterial species, including many that are associated with pneumonia and bacteremia [70]. While neutrophils have been demonstrated to be critical for controlling melioidosis in vivo directly through depletion studies, it is still unclear if the neutrophils have a direct effect on B. pseudomallei clearance or the cells have an indirect role through modulation of other cell types [41]. A recent study conversely indicated that neutrophil recruitment during melioidosis may be detrimental in controlling bacterial numbers and host survival, and suggested monocytes may be important to limit B. pseudomallei infection [71]. There is indirect evidence that neutrophils may play a role in controlling melioidosis infection through correlative findings based on cellular recruitment to the infection site, predisposing conditions, and adjunctive therapies; however any protective properties have not been clearly confirmed to be attributable to neutrophils [44], [45], [46], [47], [48], [49], [50], [51], [52], [53], [54]. Notably, in vitro studies to delineate the relative abilities of neutrophils to kill B. pseudomallei have provided conflicting results [45], [55], [56], [57]. In our current study, we sought to determine the requirements that allow human neutrophils to kill B. pseudomallei, the mechanisms needed for this process, and whether there are differences in killing efficiencies between the relatively avirulent B. thailandensis and highly virulent B. pseudomallei that correlate with their contrasting pathogenesis in vivo.