Staphylococcal protein Ecb impairs complement receptor-1 mediated recognition of opsonized bacteria

Staphyloccus aureus is a major human pathogen leading frequently to sepsis and soft tissue infections with abscesses. Multiple virulence factors including several immune modulating molecules contribute to its survival in the host. When S. aureus invades the human body, one of the first line defenses is the complement system, which opsonizes the bacteria with C3b and attract neutrophils by release of chemotactic peptides. Neutrophils express Complement receptor-1 [CR1, CD35) that interacts with the C3b-opsonized particles and thereby plays an important role in pathogen recognition by phagocytic cells. In this study we observed that a fraction of S. aureus culture supernatant prevented binding of C3b to neutrophils. This fraction consisted of S. aureus leukocidins and Efb. The C-terminus of Efb is known to bind C3b and shares significant sequence homology to the extracellular complement binding protein [Ecb). Here we show that S. aureus Ecb displays various mechanisms to block bacterial recognition by neutrophils. The presence of Ecb blocked direct interaction between soluble CR1 and C3b and reduced the cofactor activity of CR1 in proteolytic inactivation of C3b. Furthermore, Ecb could dose-dependently prevent recognition of C3b by cell-bound CR1 that lead to impaired phagocytosis of NHS-opsonized S. aureus. Phagocytosis was furthermore reduced in the presence of soluble CR1 [sCR1). These data indicate that the staphylococcal protein Ecb prevents recognition of C3b opsonized bacteria by neutrophil CR1 leading to impaired killing by phagocytosis and thereby contribute to immune evasion of S. aureus.Peer reviewe

Automatic Reading of ANCA-Slides: Evaluation of the AKLIDES System

The ANCA consensus prescribes screening by indirect immunofluorescence on neutrophils. We evaluated the first automated ANCA-pattern recognition system. C-ANCA and P-ANCA samples were selected from patients with ANCA-associated vasculitis (AAV). Non-AAV controls included sera from healthy controls , sera with possible interfering antibodies , or miscellaneous ANCA reactivity . ANCA slides were analysed by AKLIDES and routine fluorescence microscopy. The C-ANCA pattern was recognized by routine microscopy in 92% and 97% on ethanol- and formalin-fixed slides, respectively. AKLIDES reported C-ANCA in 74% and 95%, respectively. P-ANCA was recognized by routine microscopy on ethanol-fixed neutrophils in 90%, while AKLIDES reported P-ANCA in 80%. Typically, only 65% and 33% of these samples showed the expected C-ANCA on formalin-fixed neutrophils by routine microscopy and AKLIDES, respectively. A C- or P-ANCA pattern was observed on ethanol-fixed neutrophils in 28% and 23% of the controls by routine microscopy and AKLIDES, respectively. Only 5% of the controls revealed C-ANCA on formalin-fixed neutrophils by routine microscopy and AKLIDES. Altogether, automated ANCA-pattern recognition by AKLIDES is promising. Distinction of C- and P-ANCA is good, but sensitivity on ethanol-fixed neutrophils needs improvement. When optimized, pattern recognition software may play an important role in AAV diagnostics