FIGURE 4.

Identification of S. aureus murB required for integrin βν-mediated phagocytosis. A, adult flies of the control (w¹¹¹⁸) line were injected with 5-carboxyfluorescein-labeled wild-type (parent) or murB mutant (murB^ts) S. aureus and analyzed for the level of phagocytosis in vivo. The mutant strain was also analyzed for susceptibility to phagocytosis with (murB) or without (vector) complementation of the wild-type gene. The data are aligned and presented as in Fig. 3A. Scale bars, 0.5 mm. B, an assay for phagocytosis in vitro was conducted with FITC-labeled S. aureus as targets and larval hemocytes prepared from control (w¹¹¹⁸) lines as phagocytes. The bacteria used are wild-type (parent) and murB mutant (murB^ts) strains with (murB) and without (vector) complementation of the wild-type gene. C, an assay for phagocytosis in vitro was conducted with FITC-labeled wild-type (parent) and murB mutant (murB^ts) S. aureus as targets and larval hemocytes prepared from control (w¹¹¹⁸) and integrin βν-deficient (betaInt-nu²) lines as phagocytes. The extent of phagocytosis is shown relative to that with wild-type bacteria taken as 100. D, larval hemocytes prepared from the indicated fly lines were incubated with FITC-labeled peptidoglycan prepared from wild-type S. aureus, washed, and examined by fluorescence microscopy. Left, fluorescence, bright field, and overlaid views of the same microscopic fields are shown as vertically aligned panels. Right, number of hemocytes bound by peptidoglycan was determined and is shown relative to the total number of hemocytes analyzed. Scale bar, 50 μm. E, binding of the extracellular region of integrin βν to S. aureus strains (left), insoluble peptidoglycan (PGN) (middle), and soluble peptidoglycan (right) was examined. A mixture of GST-fused integrin βν and GST at an equal molar ratio was incubated with bacteria (left) or insoluble peptidoglycan (middle) and centrifuged, and the resulting precipitates were subjected to Western blotting with anti-GST antibody. The open and closed arrowheads indicate the positions of GST-fused integrin βν and GST, respectively. In the right panel, GST-fused integrin βν (GST-βν) (open circles) and GST (closed circles) were incubated in culture dishes coated with soluble peptidoglycan prepared from parental S. aureus, and the amount of GST proteins attached to dishes was immunochemically determined. Data from one of three independent experiments that yielded similar results are presented.