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Rab14 regulates maturation of macrophage phagosomes containing the fungal pathogen Candida albicans and outcome of the host-pathogen interaction.

Okai B, Lyall N, Gow NA, Bain JM, Erwig LP - Infect. Immun. (2015)

Bottom Line: Interference with endogenous Rab14 did not affect the migration of macrophages toward C. albicans cells, the rate of engulfment, the overall uptake of fungal cells, or early phagosome processing.This was associated with a significant increase in the level of macrophage killing by C. albicans.Therefore, Rab14 activity promotes phagosome maturation during C. albicans infection but is dysregulated on the phagosome in the presence of the invasive hyphal form, which favors fungal survival and escape.

View Article: PubMed Central - PubMed

Affiliation: Aberdeen Fungal Group, University of Aberdeen, Aberdeen, United Kingdom.

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Knockdown of Rab14 by siRNA does not affect the migration of macrophages toward C. albicans cells. (A to C) Tracking diagrams illustrate the distances traveled and the directionality and velocity of J774.1 macrophages that had been left untreated (A) or transfected with negative-control (B) or Rab14 (C) siRNA in response to live C. albicans cells. Tracks represent the movements of individual phagocytes relative to their starting positions; symbols mark the locations of phagocytes at 1-min intervals; and arrows indicate directionality. (D and E) Depletion of Rab14 either by siRNA-mediated knockdown (D) or by the expression of dominant negative Rab14 variants (E) does not affect the mean track velocity of macrophages in response to C. albicans cells. Data are means ± SEM for at least three independent experiments. ANOVA and Bonferroni post hoc tests were carried out. ns, no significant difference.
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Figure 3: Knockdown of Rab14 by siRNA does not affect the migration of macrophages toward C. albicans cells. (A to C) Tracking diagrams illustrate the distances traveled and the directionality and velocity of J774.1 macrophages that had been left untreated (A) or transfected with negative-control (B) or Rab14 (C) siRNA in response to live C. albicans cells. Tracks represent the movements of individual phagocytes relative to their starting positions; symbols mark the locations of phagocytes at 1-min intervals; and arrows indicate directionality. (D and E) Depletion of Rab14 either by siRNA-mediated knockdown (D) or by the expression of dominant negative Rab14 variants (E) does not affect the mean track velocity of macrophages in response to C. albicans cells. Data are means ± SEM for at least three independent experiments. ANOVA and Bonferroni post hoc tests were carried out. ns, no significant difference.

Mentions: Directional movement and velocity in response to live C. albicans cells did not differ between untreated macrophages, macrophages transfected with negative-control siRNA, and macrophages with siRNA-mediated Rab14 knockdown (Fig. 3A to C). Mean track velocities were not significantly different for untreated (1.01 ± 0.03 μm/min), negative-control siRNA-transfected (1.07 ± 0.03 μm/min), and Rab14 siRNA-transfected (1.07 ± 0.03 μm/min) macrophages (Fig. 3D). Similar results were also found in comparisons between macrophages transfected to express eGFP-tagged Rab14, eGFP-Rab14S25N, and eGFP-Rab14N124I (Fig. 3E). Next, we investigated whether Rab14 depletion affected the overall rate of engulfment of C. albicans cells by macrophages following cell-cell contact. The average times to engulfment (from the first cell-cell contact to complete internalization) of UV-killed C. albicans cells by untreated, negative-control siRNA-transfected, and Rab14 siRNA-transfected macrophages were not significantly different (3.7 ± 0.1 min, 3.9 ± 0.2 min, and 3.7 ± 0.1 min, respectively) (Fig. 4A and B). In keeping with previous observations, the times to engulfment of live C. albicans cells were longer than those for UV-killed cells (28) but, again, not significantly different for untreated, negative-control siRNA-transfected, and Rab14 siRNA-transfected macrophages. (5.1 ± 0.2 min, 5.3 ± 0.2 min, and 5.1 ± 0.2 min, respectively) (Fig. 4C and D). We also observed a similar trend in the engulfment kinetics of BMDMs and of macrophages transfected with eGFP-Rab14 and dominant negative variants (Fig. 4E and F). Therefore, Rab14 does not accelerate or delay macrophage migration toward fungal cells or the rate of their engulfment.


Rab14 regulates maturation of macrophage phagosomes containing the fungal pathogen Candida albicans and outcome of the host-pathogen interaction.

Okai B, Lyall N, Gow NA, Bain JM, Erwig LP - Infect. Immun. (2015)

Knockdown of Rab14 by siRNA does not affect the migration of macrophages toward C. albicans cells. (A to C) Tracking diagrams illustrate the distances traveled and the directionality and velocity of J774.1 macrophages that had been left untreated (A) or transfected with negative-control (B) or Rab14 (C) siRNA in response to live C. albicans cells. Tracks represent the movements of individual phagocytes relative to their starting positions; symbols mark the locations of phagocytes at 1-min intervals; and arrows indicate directionality. (D and E) Depletion of Rab14 either by siRNA-mediated knockdown (D) or by the expression of dominant negative Rab14 variants (E) does not affect the mean track velocity of macrophages in response to C. albicans cells. Data are means ± SEM for at least three independent experiments. ANOVA and Bonferroni post hoc tests were carried out. ns, no significant difference.
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Figure 3: Knockdown of Rab14 by siRNA does not affect the migration of macrophages toward C. albicans cells. (A to C) Tracking diagrams illustrate the distances traveled and the directionality and velocity of J774.1 macrophages that had been left untreated (A) or transfected with negative-control (B) or Rab14 (C) siRNA in response to live C. albicans cells. Tracks represent the movements of individual phagocytes relative to their starting positions; symbols mark the locations of phagocytes at 1-min intervals; and arrows indicate directionality. (D and E) Depletion of Rab14 either by siRNA-mediated knockdown (D) or by the expression of dominant negative Rab14 variants (E) does not affect the mean track velocity of macrophages in response to C. albicans cells. Data are means ± SEM for at least three independent experiments. ANOVA and Bonferroni post hoc tests were carried out. ns, no significant difference.
Mentions: Directional movement and velocity in response to live C. albicans cells did not differ between untreated macrophages, macrophages transfected with negative-control siRNA, and macrophages with siRNA-mediated Rab14 knockdown (Fig. 3A to C). Mean track velocities were not significantly different for untreated (1.01 ± 0.03 μm/min), negative-control siRNA-transfected (1.07 ± 0.03 μm/min), and Rab14 siRNA-transfected (1.07 ± 0.03 μm/min) macrophages (Fig. 3D). Similar results were also found in comparisons between macrophages transfected to express eGFP-tagged Rab14, eGFP-Rab14S25N, and eGFP-Rab14N124I (Fig. 3E). Next, we investigated whether Rab14 depletion affected the overall rate of engulfment of C. albicans cells by macrophages following cell-cell contact. The average times to engulfment (from the first cell-cell contact to complete internalization) of UV-killed C. albicans cells by untreated, negative-control siRNA-transfected, and Rab14 siRNA-transfected macrophages were not significantly different (3.7 ± 0.1 min, 3.9 ± 0.2 min, and 3.7 ± 0.1 min, respectively) (Fig. 4A and B). In keeping with previous observations, the times to engulfment of live C. albicans cells were longer than those for UV-killed cells (28) but, again, not significantly different for untreated, negative-control siRNA-transfected, and Rab14 siRNA-transfected macrophages. (5.1 ± 0.2 min, 5.3 ± 0.2 min, and 5.1 ± 0.2 min, respectively) (Fig. 4C and D). We also observed a similar trend in the engulfment kinetics of BMDMs and of macrophages transfected with eGFP-Rab14 and dominant negative variants (Fig. 4E and F). Therefore, Rab14 does not accelerate or delay macrophage migration toward fungal cells or the rate of their engulfment.

Bottom Line: Interference with endogenous Rab14 did not affect the migration of macrophages toward C. albicans cells, the rate of engulfment, the overall uptake of fungal cells, or early phagosome processing.This was associated with a significant increase in the level of macrophage killing by C. albicans.Therefore, Rab14 activity promotes phagosome maturation during C. albicans infection but is dysregulated on the phagosome in the presence of the invasive hyphal form, which favors fungal survival and escape.

View Article: PubMed Central - PubMed

Affiliation: Aberdeen Fungal Group, University of Aberdeen, Aberdeen, United Kingdom.

Show MeSH
Related in: MedlinePlus