Dynamics of Human Complement–Mediated Killing of Klebsiella pneumoniae

Abstract

With an in vitro system that used a luminescent strain of Klebsiella pneumoniae to assess bacterial metabolic activity in near-real-time, we investigated the dynamics of complement-mediated attack in healthy individuals and in patients presenting to the emergency department with community-acquired severe sepsis. A novel mathematical/statistical model was developed to simplify light output trajectories over time into two fitted parameters, the rate of complement activation and the delay from activation to the onset of killing. Using Factor B-depleted serum, the alternative pathway was found to be the primary bactericidal effector: In the absence of B, C3 opsonization as measured by flow cytometry did not progress and bacteria proliferated near exponentially. Defects in bacterial killing were easily demonstrable in patients with severe sepsis compared with healthy volunteers. In most patients with sepsis, the rate of activation was higher than in normal subjects but was associated with a prolonged delay between activation and bacterial killing (P , 0.05 for both). Theoretical modeling suggested that this combination of accentuated but delayed function should allow successful bacterial killing but with significantly greater complement activation. The use of luminescent bacteria allowed for the development of a novel and powerful tool for assessing complement immunology for the purposes of mechanistic study and patient evaluation.Keywords: alternative complement pathway; theoretical models; nonlinear dynamics; sepsisThe complement cascade fills a crucial niche in early host defense against bacterial infection. Activating by fully innate means (i.e., through the alternative and mannose-binding lectin pathways) or in conjunction with acquired humoral immunity (via the classical pathway), the system contributes to defense by enzymatically labeling microorganisms for phagocytosis through C3 opsonization, by generating a series of anaphylatoxins that alert cellular defenses to the presence of a pathogen, and, in some instances, by directly killing the invading cell by permeabilizing its cell wall with membrane attack complex (C5b6789 n or MAC).During experimental or human sepsis, globally assessing the complement system is typically done in one of two ways. Most often, traces of previous activation are sought by measuring concentrations of complement proteins (such as C3, which falls with activation) or species elaborated during activation such as C3a, C5a, or soluble MAC (1). Less commonly, function is directly assessed. This is typically done by measuring complement hemolytic activity, with the canonical example being the CH 50 method. CH 50 is known to fall in severe human sepsis and multiorgan failure (2). How best to interpret this capacity (i.e., the complement-mediated cytolysis of an enucleate and otherspecies blood cell fully opsonized by a third species' IgG) in the setting of an overwhelming infection is not self-evident.A more relevant functional assay of the complement system's performance in the presence of...

Complement C5a Generation by Staphylococcal Biofilms

“…(10, 29) Bound C3b was quantified flow-cytometrically using a FITC-labeled anti-C3b antibody (Thermo-Scientific, Fremont, CA). Cells opsonized with heat-inactivated serum served as a negative control.…”

Intradermal Immunization of BALB/C Mice with Heat Killed Klebsiella pneumoniae ATCC BAA1706 Leads to the Production of both IgG1 and IgG2a

“…Previous studies have shown that some strains of K. pneumoniae, which possesses certain serotypes of LPS (such as K1, K10 and K16), exhibited resistance mechanisms against the recognition and effector functions of the complement system (Merino et al, 1992;Alberti et al, 1996). However, when analyzing the proliferation of the bacteria in the scenario of absence of Factor B, the lack of activation of the classical pathway of the complement system seemed to support the bacterial proliferation almost exponentially, this result as also observed in a C5 depletion setup (Nypaver et al, 2010). Additionally, the intraperitoneal adoptive transference of the sera of mice immunized with extracellular vesicles of K. pneumoniae, led to protection from the lethality induced by the intraperitoneal challenge of a high dose of K. pneumoniae (Lee et al, 2015).…”

“…The activation of complement by direct cleavage of C3 by bacterial surface structures, such as LPS and outer membrane proteins, initiates the complement cascade by the alternative pathway. This activation of the complement system alternative pathway is prevalent in early stages of infection, while the activation of the classical and lectin pathway is present for prolonged infections (Nypaver et al, 2010). Previous studies have shown that some strains of K. pneumoniae, which possesses certain serotypes of LPS (such as K1, K10 and K16), exhibited resistance mechanisms against the recognition and effector functions of the complement system (Merino et al, 1992;Alberti et al, 1996).…”

“…These molecules participate in various effector mechanisms in the clearance of pathogens, such as the activation of C1q enzyme fragment of the classical pathway of the complement system. Activation of the complement system is a participant in the clearance of pathogens by the pathogen opsonization with C3b protein and the formation of C5b6789 (attack complex membrane, MAC) (Nypaver et al, 2010). The activation of complement by direct cleavage of C3 by bacterial surface structures, such as LPS and outer membrane proteins, initiates the complement cascade by the alternative pathway.…”

Genetic characterization of the K2 serotype capsule of Klebsiella pneumoniae ATCC 43816 and the development of a bioluminescent strain.

“…The CPS of K. pneumoniae resists the nonspecific host defense mechanism such as the complement cascade, which plays a crucial role in the early host defense against invading pathogens [59]. In vitro studies have shown that CPS inhibits the deposit of the C3 complement component onto the bacteria's surface and reduces the formation of the membrane attack corr'lplex [60,61].…”