Abstract
BackgroundDiabetic patients are at increased risk for bacterial infections; these studies provide new insight into the role of the host defense complement system in controlling bacterial pathogens in hyperglycemic environments.MethodsThe interactions of complement C3 with bacteria in elevated glucose were assayed for complement activation to opsonic forms, phagocytosis and bacterial killing. C3 was analyzed in euglycemic and hyperglycemic conditions by mass spectrometry to measure glycation and structural differences.ResultsElevated glucose inhibited S. aureus activation of C3 and deposition of C3b and iC3b on the bacterial surface. S. aureus-generated C5a and serum-mediated phagocytosis by neutrophils were both decreased in elevated glucose conditions. Interestingly, elevated glucose increased the binding of unactivated C3 to S. aureus, which was reversible on return to normal glucose concentrations. In a model of polymicrobial infection, S. aureus in elevated glucose conditions depleted C3 from serum resulting in decreased complement-mediated killing of E. coli. To investigate the effect of differing glucose concentration on C3 structure and glycation, purified C3 incubated with varying glucose concentrations was analyzed by mass spectrometry. Glycation was limited to the same three lysine residues in both euglycemic and hyperglycemic conditions over one hour, thus glycation could not account for observed changes between glucose conditions. However, surface labeling of C3 with sulfo-NHS-biotin showed significant changes in the surface availability of seven lysine residues in response to increasing glucose concentrations. These results suggest that the tertiary structure of C3 changes in response to hyperglycemic conditions leading to an altered interaction of C3 with bacterial pathogens.ConclusionsThese results demonstrate that hyperglycemic conditions inhibit C3-mediated complement effectors important in the immunological control of S. aureus. Mass spectrometric analysis reveals that the glycation state of C3 is the same regardless of glucose concentration over a one-hour time period. However, in conditions of elevated glucose C3 appears to undergo structural changes.
Highlights
Diabetic patients are at increased risk for bacterial infections; these studies provide new insight into the role of the host defense complement system in controlling bacterial pathogens in hyperglycemic environments
C3 is the central component of the complement system and its activation to C3b is critical for bacterial opsonization and subsequent phagocytosis, generation of the anaphylatoxin C5a [16], and terminal complement cascade activation leading to membrane attack complexes (MAC) that can lyse Gram-negative bacteria [17]
Glucose inhibition of C3-mediated opsonophagocytosis of S. aureus In order to test whether hyperglycemic conditions altered C3 activation and binding of activated C3-fragments (C3b and iC3b) on the S. aureus surface, we performed Western blot analysis of C3-fragments stripped from S. aureus incubated in serum in euglycemic, 3 mmol/l dextrose, and hyperglycemic, 17 mmol/l dextrose, conditions (Figure 1A)
Summary
Diabetic patients are at increased risk for bacterial infections; these studies provide new insight into the role of the host defense complement system in controlling bacterial pathogens in hyperglycemic environments. Limb-threatening infections are frequently polymicrobial with enteric Gram-negative bacteria and S. aureus present [7,8,9]. The human complement system is a major component of innate immunity and plays a vital role in the control of many bacterial pathogens [12] including S. aureus [13,14,15]. C3 is the central component of the complement system and its activation to C3b is critical for bacterial opsonization and subsequent phagocytosis, generation of the anaphylatoxin C5a [16], and terminal complement cascade activation leading to membrane attack complexes (MAC) that can lyse Gram-negative bacteria [17]. Normal C3 levels in human serum is typically about 1 mg/ml [18]
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