Evasion of Complement-Mediated Lysis and Complement C3 Deposition Are Regulated by Francisella tularensis Lipopolysaccharide O Antigen

Abstract

The bacterium Francisella tularensis (Ft) is a potential weapon of bioterrorism when aerosolized. Macrophage infection is necessary for disease progression and efficient phagocytosis by human macrophages requires serum opsonization by complement. Microbial complement activation leads to surface deposition of a highly regulated protein complex resulting in opsonization or membrane lysis. The nature of complement component C3 deposition, i.e., C3b (opsonization and lysis) or C3bi (opsonization only) fragment deposition, is central to the outcome of activation. In this study, we examine the mechanisms of Ft resistance to complement-mediated lysis, C3 component deposition on the Ft surface, and complement activation. Upon incubation in fresh nonimmune human serum, Schu S4 (Ft subsp. tularensis), Fn (Ft subsp. novicida), and LVS (Ft subsp. holarctica live vaccine strain) were resistant to complement-mediated lysis, but LVSG and LVSR (LVS strains altered in surface carbohydrate structures) were susceptible. C3 deposition, however, occurred on all strains. Complement-susceptible strains had markedly increased C3 fragment deposition, including the persistent presence of C3b compared with C3bi, which indicates that C3b inactivation results in survival of complement-resistant strains. C1q, an essential component of the classical activation pathway, was necessary for lysis of complement-susceptible strains and optimal C3 deposition on all strains. Finally, use of Francisella LPS mutants confirmed O Ag as a major regulator of complement resistance. These data provide evidence that pathogenic Francisella activate complement, but are resistant to complement-mediated lysis in part due to limited C3 deposition, rapid conversion of surface-bound C3b to C3bi, and the presence of LPS O Ag.

Subversion of Host Recognition and Defense Systems by Francisella spp

“…Francisella's complement-inhibiting activity is highly dependent on the O-antigen oligosaccharide present on LPS on the surface of the bacterial cell (51,192,211). Mutants lacking O antigen, either by random selection for rough or gray variants or by targeting wbt locus genes necessary for O-antigen production, are significantly more sensitive to complement than their wild-type counterparts (51,192,211).…”

“…2) (28,29,51). Generation of these fragments prevents the ability of C3 to signal toward MAC formation.…”

“…Upon infection, Francisella initially comes into contact with extracellular defenses such as complement, antibody, and cationic antimicrobial peptides (28,29,51,190). Binding of these components to bacteria directly or indirectly leads to lysis and killing (189).…”

Molecular composition of the alveolar lining fluid in the aging lung

“…For human ALF, the following ABS w e r e u s e d : S P -A ( s c -1 6 6 9 1 4 , S a n t a C r u z Biotechnology, Inc.), SP-D (sc-59695, Santa Cruz Biotechnology, Inc.), C2 (sc-134639, Santa Cruz Biotechnology, Inc.), C3 (sc-28294/B9, Santa Cruz Biotechnology, Inc.), C5 (A220, Complement Technology, Inc., www.complementtech.com), and C7 (sc-160195, Santa Cruz Biotechnology, Inc.). Western blotting (WB) to detect SP-A, SP-D, and complement components and to determine the presence of nitrotyrosine and carbonyl residues were performed following the manufacturer's recommendations and established methods (Bolger et al 2007;Clay et al 2008). The carbonyl and nitrotyrosine presence was determined assessing the band density of all bands in the lane.…”

Francisella tularensis Suppresses the Proinflammatory Response of Endothelial Cells via the Endothelial Protein C Receptor

“…The LVS also evades killing by neutrophils, as it does not trigger a respiratory burst and inhibits assembly of NADPH oxidase on the phagosome (18). Both the LVS and the Schu S4 strain also resist complement-mediated lysis (19). Evasion of the host immune response, through these and other mechanisms, is likely key to the virulence of F. tularensis.…”