Fig. 3

Death and Phagocytosis of Infected Macrophages

(A–C) DIC time lapse of death and phagocytosis of infected macrophage. (A) two focal planes at time zero showing bacteria in cell with healthy nucleus (large black arrow), with uninfected cell (small white arrow and brackets as in Figure 2D) passing by. Dashed arrow, direction of travel. (B) Same infected cell as in (A), before (top) and after (bottom) nuclear collapse. (C) Same cell after further nuclear collapse into apoptotic sphere and later time points showing complete phagocytosis by new macrophage (black and white brackets and white arrow). Small black arrowheads, vesicles in approaching macrophage.

(D) Hoechst (white) and bacterial GFP (green) overlay above, with matching DIC below, showing compact Hoechst-positive apoptotic nuclei (black arrows) associated with bacteria (white arrowheads), compared to diffuse Hoechst staining of live nuclei (white arrow).

(E) Infected Hoechst-positive apoptotic cells per granuloma in WT and ΔRD1 lesions over 24 hr. p from Mann-Whitney test.

(F–I) Apoptotic cells in WT granulomas detected by acridine orange (F and H) or annexin V (G and I). (F) Granuloma of red fluorescent Mm with acridine orange-positive cells (white arrows). Bracket indicates area shown in (H). (G) Granuloma of red fluorescent Mm with annexin V-positive cells (white arrows). Bracket indicates area shown in (I). (H) Acridine orange signal (left) corresponds with DIC appearance of apoptotic bodies (dotted circle, middle). Overlay, right. Scale bar, 10 μm. (I) Annexin V signal (left) corresponds with DIC appearance of apoptotic bodies (dotted circle, middle). Overlay, right. Scale bar, 10 μm.

(J) DIC time-lapse images of single macrophage (nucleus indicated by white arrow) pulling a small group of bacteria (white arrowheads) from a larger cluster (X). Below, sketches derived from these panels with additional details from other planes at same time point.

All scale bars, 5 μm.