aureussurvival, which may result in increased bacterial fitness in the host. were recovered from blood and several organs using a murine intravenous infection model. Collectively, our findings reveal a new property of SdrD as an important key contributor toS. aureussurvival and the ability to escape the innate immune system in blood. KEYWORDS: SdrD, virulence, neutrophils, systemic infection, whole blood == INTRODUCTION == Staphylococcusaureusis a human commensal that persistently colonizes the anterior nares of healthy individuals but is also a leading cause of multiple community-acquired and nosocomial infections ranging from mild skin and soft tissue infections to life-threatening invasive diseases, including sepsis and endocarditis (1). The bacterium expresses various cell wall-anchored proteins, including the microbial surface components recognizing adhesive matrix molecules (MSCRAMMs). Among the MSCRAMMs are the clumping factor (Clf)serine-aspartate repeat (Sdr) group of proteins, including SdrD (2). SdrD promotes the adherence ofS. aureusto desquamated nasal epithelial cells (3) and to human keratinocytesin vitro(4) and contributes to Rabbit Polyclonal to CDH11 abscess formationin vivo(5). Neutrophils, the most abundant circulating phagocytes of the innate immune system, play an important role in protection against pathogen dissemination (6, 7). Decoration of bacteria with opsonins, i. e., specific immunoglobulins and components of the complement system, activates the phagocytic machinery through interaction with the corresponding neutrophil Fc receptors or complement receptors (reviewed in reference8). The generation of reactive oxygen species (ROS) through activation of NADPH oxidase and expression of antibacterial molecules in granules is a key mechanism for neutrophil-mediated killing of phagocytized bacteria, while DO-264 elaboration DO-264 of DNA-based neutrophil extracellular traps (NETs) promotes extracellular microbial killing (reviewed in reference8). A crucial role of neutrophils in controllingS. aureusinfection is evident through the study of patients with immune defects (reviewed in reference9); for example , cancer patients with neutropenia (10) or individuals with genetic DO-264 defects of neutrophil function (11, 12) demonstrate markedly increased rates ofS. aureusinfection. S. aureuscan enter the bloodstream through several routes (1), whereupon a sophisticated and synergistic group of neutrophil resistance mechanisms, including antioxidant systems, factors that bind or inactivate granule components, and mechanisms to avoid complement opsonization and phagocytosis, enhance thein vivofitness of the organism (6, 13, 14). Impairment of neutrophil intracellular killing allowsS. aureusto survive long enough within these cells in the bloodstream to travel to and infect distant sites (reviewed in references6and15). A higher prevalence of thesdrDgene amongS. aureusisolates from patients with bone infections has been reported (16, 17). In addition , the expression ofsdrDis upregulated upon incubation in fresh human bloodex vivo(18). These data raised the possibility that SdrD could play a role during systemic infection. To date the interaction of SdrD with host innate immune components has not been studied. The current work evaluates whether SdrD may aid the pathogen in immune evasion. Using an isogenic mutant with a deletion of thesdrDgene, we found a significant contribution of SdrD inS. aureusresistance to killing by innate immune components present in bloodin vitroand bacterial clearance from tissues and blood during systemic infections. == RESULTS == == Expression profile ofsdrD. == The expression profile ofsdrDwas assessed usingS. aureussubsp. aureusNCTC8325-4 expressing ansdrDpromoter-green fluorescent protein (GFP) fusion construct. In regular bacterial growth medium (tryptic soy broth [TSB]), the level ofsdrDpromoter-driven GFP expression was low and slightly decreased during early exponential growth. Thereafter, thesdrDpromoter activity increased when bacteria entered late exponential phase and continued to increase during the stationary phase (Fig. 1A). == FIG 1 . == sdrDpromoter activity and SdrD protein expression under different growth conditions. (A) Promoter activity ofsdrDduring growth in TSB using NCTC8325-4 harboring thesdrD-GFP reporter construct (S. aureussubsp. aureuspsdrD-GFP). Data represent the means SEMs from an individual experiment. The experiments were performed twice in triplicate. RFU, relative fluorescence units. (B) Immunoblotting of the bacterial lysates and the culture cell-free supernatant of NCTC8325-4 and its isogenic mutant, NCTC8325-4 sdrD, using anti-SdrD antibody on the bacterial lysates and the culture cell-free supernatant. A representative Western blot is shown. (C) Promoter activity ofsdrDevaluated by fluorescence microscopy using NCTC8325-4 harboring psdrD-GFP in the presence of freshly isolated human neutrophils. (Top) Live imaging was performed after 0. 2, 1 . 5, and 3. 5 h using fluorescence microscopy; (bottom) bright-field and GFP merged images. The white boxes in the first column labeled 3. 5 h are enlarged in the second column labeled 3. 5 h. The experiment was performed at an MOI of 20. Arrows, S. aureussubsp. aureuspsdrD-GFP. The sizes of the scale bars are indicated. We also tested SdrD protein expression by immunoblotting using SdrD-specific antibodies. SdrD was detected within the bacterial lysate of NCTC8325-4 and was detected most abundantly at early stationary phase but was not detected in the isogenic mutant, NCTC8325-4 sdrD(Fig. 1B). Similar to a previous report for SdrC (19), SdrD was detected in cell-free concentrated culture supernatants, particularly in late exponential phase (Fig. 1B), which may have been due to proteolytic release and/or cell death. A previous study showed thatsdrDexpression was upregulated in the presence of human whole blood (18). As neutrophils are.