Figure 1.
Reverse transcription (RT)-PCR of bicP and bpss1522 in wild-type and bopA mutant B. pseudomallei.
RNA extracted from the wild-type K96243 or bopA deletion mutant strain was reverse-transcribed to obtain cDNA. Each cDNA preparation was used as a template for RT-PCR. (A) Schematic diagram showing the position of primers used for RT-PCR. (B) Electrophoretic separation of RT-PCR products amplified with primers for bicP (top panel) and bpss1522 (lower panel). These products were generated from reactions: (2) bopA mutant cDNA; (3) bopA mutant cDNA, no RT control; (4) wild-type cDNA, (5) wild-type cDNA, no RT control; (6) wild-type genomic DNA control; and (7) No DNA control. DNA size markers (bp) are shown in lane 1.
Figure 2.
Possible fates of B. pseudomallei in infected macrophages.
Following phagocytic uptake by macrophages, bacteria are first located within phagosomes. The majority of wild-type B. pseudomallei (Bp) can escape from the phagosome into the cytosol in a process which is largely uncharacterized but involves the TTSS3. Once free in the cytosol bacteria activate BimA-mediated actin-based motility, replicate and invade adjacent cells via membrane protrusions. Potentially some cytosolic bacteria may be sequestered in canonical autophagosomes (pathway A). Some bacteria which remain in phagosomes might be sequestered by double-membrane autophagosomes (pathway B). The autophagy marker protein LC3 can be recruited to bacteria-containing phagosomes, a process designated LC3-associated phagocytosis (LAP) which stimulates further phagosomal maturation via recruitment of other proteins including LAMP1, a late endosome/lysosome marker, and the subsequent fusion of phagosomes with lysosomes, leading to bacterial killing (pathway C). Finally phagosomes may mature to phagolysosomes without LC3 recruitment (pathway D).
Figure 3.
B. pseudomallei-containing vacuoles are bound by single membranes and the TTSS3 and the effector BopA are required for bacterial escape.
(A–F) Transmission electron micrographs show the intracellular location of B. pseudomallei in RAW 264.7 cells at 2–6 h post infection (p.i.). The scale bar is indicated. Boxed areas are shown as magnified images below each panel. Intracellular bacteria were observed either free in the cytosol and not membrane bound (panel A), or within single-membrane phagosomal compartments (panel B). Only one bacterium was found in a double-membrane compartment (panel C), which could be an autophagosome. Canonical autophagosomes having a double-membrane were observed in infected and uninfected RAW 264.7 cells (panels D–F). Arrows indicate detailed membrane ultrastructure. (G) The percentage of bacteria free in the cytosol of RAW 264.7 cells following infection with B. pseudomallei wild-type, bopA mutant and the bipD mutant at 2, 4, and 6 h p.i. Data represent the mean ± SEM of three separate experiments (n = 100 bacteria). Where shown * indicates p<0.05 relative to the wild-type strain at each time point.
Figure 4.
B. pseudomallei-associated actin-tails, host cell membrane protrusions and MNGC formation require TTSS3 translocator BipD, but not effector BopA.
(A) Representative confocal micrographs with DIC images of RAW 264.7 cells infected with the wild-type strain, the bopA mutant, or the bipD mutant at 6 h p.i. Bacteria were stained red, filamentous actin was stained green and nuclei were stained blue. Bacteria with associated actin-tails are marked with arrows. Scale bar = 5 µm. (B) Intracellular survival of B. pseudomallei wild-type and bipD mutant in RAW 264.7 cells at 2, 4, and 6 h p.i. Bacterial survival was normalized to CFU counts obtained at 2 h p.i. and presented as relative survival (%). Data represent the mean ± SEM of three separate experiments, each carried out in triplicate. Where shown * indicates p<0.05 relative to the wild-type strain at each time point.
Figure 5.
B. pseudomallei with defective TTSS3 show enhanced co-localization with autophagy marker protein LC3 and the lysosome marker LAMP1.
(A) Confocal images of RAW 264.7 cells expressing GFP-LC3 (green) and infected with B. pseudomallei (Bp). Cells were fixed at 2 h p.i., permeabilized, and stained for B. pseudomallei (blue) and LAMP1 (red). Arrows indicate bacteria associated with LC3 that are also within LAMP1-positive vacuoles. Bacterial co-localization with LC3 or LAMP1 was defined by the presence of labelled B. pseudomallei (blue) which were fully overlaid by intense green/red or fully contained within a green/red ring. Scale bar = 5 µm. (B–D) Quantitative analysis of bacterial co-localization with LC3 (B), LAMP1 (C) or both LC3 and LAMP1 (D) in RAW 264.7 cells infected with B. pseudomallei wild-type, bopA mutant or bipD mutant at 2, 4, and 6 h p.i. (E) The percentage of LC3-positive (LC3+ Bp) or LC3-negative bacteria (LC3− Bp) of the wild-type, bopA mutant or bipD mutant at 2 or 4 h p.i. that co-localized with LAMP1. Data represent the mean ± SEM of three separate experiments (n = 100 bacteria). Where shown * indicates p<0.05 relative to wild-type strain at each time point.
Figure 6.
The recruitment of LC3 to bacteria-containing phagosomes is diminished in RAW 264.7 cells infected with heat-killed B. pseudomallei.
Quantitative analysis of bacterial co-localization with LC3 in RAW 264.7 cells infected with live B. pseudomallei (wild-type strain, the bopA mutant or the bipD mutant) or heat-killed strains at 2 and 4 h p.i. The data represent the mean ± SEM of three separate experiments (n = 100 bacteria). Where shown * indicates p<0.05 relative to the wild-type strain at each time point.