The full-of-bacteria gene is required for phagosome maturation during immune defense in Drosophila

Abstract

Arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome is a fatal recessive disorder caused by mutations in the VPS33B or VPS16B genes. Both encode homologues of the Vps33p and Vps16p subunits of the HOPS complex necessary for fusions of vacuoles in yeast. Here, we describe a mutation in the full-of-bacteria (fob) gene, which encodes Drosophila Vps16B. Flies null for fob are homozygous viable and fertile. They exhibit, however, a defect in their immune defense that renders them hypersensitive to infections with nonpathogenic bacteria. fob hemocytes (fly macrophages) engulf bacteria but fail to digest them. Phagosomes undergo early steps of maturation and transition to a Rab7-positive stage, but do not mature to fully acidified phagolysosomes. This reflects a specific requirement of fob in the fusion of phagosomes with late endosomes/lysosomes. In contrast, cargo of autophagosomes as well as endosomes exhibit normal lysosomal delivery in fob cells. These findings suggest that defects in phagosome maturation may contribute to symptoms of ARC patients including recurring infections.

Figure 1.

A fob null allele is hypersensitive to infections with E. coli. (A) Schematic representation of the Drosophila fob gene and its neighbors. The targeting fragment generated in vivo (Gong and Golic, 2003) contains portions of neighboring genes around the mini-white gene (red box). (B) Southern hybridization with the entire fob gene yielded a signal with Ore-R (wt) but with any fob allele. Reprobing the same membrane with dVps33b confirmed the presence of DNA in all lanes. (C) qRT-PCR showed no expression of fob but similar levels of expression for neighboring genes in fob¹ compared with wild type. Gene expression levels were normalized with rp49 as an internal control and are shown relative to wild type. (D and E) Survival after infection was measured for wt, fob¹, and rescued fob¹ flies after injection with E. coli (D) or E. faecalis (E). (F) Induction of AMP genes 6 h after injection with E. coli (Drosocin, Diptericin, Cecropin, and Attacin) or 12 h after injection with E. faecalis (Defensin). (G) Bacterial load in injected flies at the indicated day after injection with E. coli. Error bars indicate standard deviation.

Figure 2.

fob mutants exhibit a defect in bacterial clearance. (A) 2 h after injection, pHrodo-labeled E. coli (arrows) were visible around the dorsal vessel in the thorax of wild-type and eater flies (Df(3R)D605/Df(3R)Tl-I). In contrast, diffuse weaker signals appeared in fob¹ flies. (B) Hemocytes were allowed to engulf FITC-labeled E. coli for 15 min, and, after quenching fluorescence of external bacteria with Trypan blue, the fluorescence of phagocytosed bacteria was visible in wild-type (B) and fob¹ (C) cells visualized by differential interference contrast microscopy (B′ and C′). (D and E) After 45 min of chase, bacteria were cleared from wild type (D) but accumulated inside fob¹ hemocytes (E). (F) Box and whisker plots display the number of bacteria detected in hemocytes of indicated genotypes after 45 min of chase. Bars: (A) 0.5 mm; (B and C) 25 µm; (D and E) 10 µm.

Figure 3.

fob phagosomes fail to mature. Double-labeled bacteria were allowed to internalize for 10 min (broken line) or 30 min (solid line), and images were captured for 15 min. The distribution of fluorescence ratios is shown for Ore-R (A) and fob¹ (B). The fluorescence ratio relates to pH as shown in Fig. S1. (C) Electron micrographs of phagosomes detected after a 30-min chase of phagocytosed E. coli. Phagosomal structures were broadly classified in three categories based on their ultrastructural appearance: phagosome (C), late phagosome (C′) and phagolysosomes (C′′). Bar, 1 µm. (D) Relative distribution of different categories of phagosomes in Ore-R and fob1. Data were collected from two independent sets of experiments with equivalent results. Quantification was performed in triplicate with a representative example shown in D.

Figure 4.

fob is required for the fusion of phagosomes with lysosomes. Micrographs show hemocytes isolated from wild-type (A–E) or fob¹ (A’–E’) larvae. (A–C) Hemocytes were allowed to phagocytose FITC-labeled E. coli, and were immunostained for Avl (red) and Rbsn-5 (blue; A), Rab7 (B), or Hook (C). (D) Hemocytes were allowed to internalize dextran–Alexa Fluor 488 (10 kD), which after a 90-min chase partially colocalized with LysoTracker in wild type (D) and fob¹ (D’), indicating functional labeling of lysosomes by dextran. (E) Hemocytes with lysosomes preloaded with internalized Texas red–dextran were allowed to phagocytose GFP-labeled E. coli. After 30–45 min of chase, the bacteria colocalized with dextran in wild-type (E) but not fob¹ hemocytes (E′). For display, images were imported into Photoshop (Adobe) and adjusted for gain, contrast, and gamma settings. Bar, 5 µm. (F) Bar graphs indicate percentages of bacteria in phagosomes positive for the indicated markers or the percentage of dextran in LysoTracker-positive structures (error bars indicate standard deviation). Hemocytes were harvested from larvae with indicated genotypes.