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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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Rab14 associates transiently and differentially with phagosomes containing C. albicans yeast or hyphal cells. (A and B) Live RAW 264.7 macrophages were transfected with eGFP-Rab14, allowed to ingest C. albicans cells, and monitored by live-cell imaging using fluorescence (upper panels) and differential interfer ence contrast microscopy (lower panels). Bar, 5 μm. The corresponding movies can be viewed in Videos S1 and S2 in the supplemental material. (A) RAW 264.7 macrophages were transfected with eGFP-Rab14 for 16 h, treated with live C. albicans cells, and imaged using live-cell microscopy over a 6-h period. Selected frames depict events from the time of contact between a live C. albicans yeast cell and the macrophage to the time of Rab14 localization. The white arrows indicate colocalization between phagosomes containing C. albicans cells and Rab14. (B) Selected frames from a live-cell microscopy movie showing detailed events from the time of contact between a live C. albicans hyphal cell and the macrophage to the time of Rab14 localization. (C) Time from the engulfment of C. albicans yeast or hyphal cells to the start of Rab14 recruitment. A Mann-Whitney statistical test was performed because the data were skewed; ns, no significant difference. (D) Duration of Rab14 localization on phagosomes containing either yeast or hyphal cells. ANOVA and post hoc Bonferroni tests were performed to determine significant differences (***, P ≤ 0.001). (E) Positive correlation (Spearman's) between hyphal length and the duration of Rab14 retention on phagosomes containing C. albicans hyphal cells. Data are means ± SEM for at least three independent experiments.
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Figure 1: Rab14 associates transiently and differentially with phagosomes containing C. albicans yeast or hyphal cells. (A and B) Live RAW 264.7 macrophages were transfected with eGFP-Rab14, allowed to ingest C. albicans cells, and monitored by live-cell imaging using fluorescence (upper panels) and differential interfer ence contrast microscopy (lower panels). Bar, 5 μm. The corresponding movies can be viewed in Videos S1 and S2 in the supplemental material. (A) RAW 264.7 macrophages were transfected with eGFP-Rab14 for 16 h, treated with live C. albicans cells, and imaged using live-cell microscopy over a 6-h period. Selected frames depict events from the time of contact between a live C. albicans yeast cell and the macrophage to the time of Rab14 localization. The white arrows indicate colocalization between phagosomes containing C. albicans cells and Rab14. (B) Selected frames from a live-cell microscopy movie showing detailed events from the time of contact between a live C. albicans hyphal cell and the macrophage to the time of Rab14 localization. (C) Time from the engulfment of C. albicans yeast or hyphal cells to the start of Rab14 recruitment. A Mann-Whitney statistical test was performed because the data were skewed; ns, no significant difference. (D) Duration of Rab14 localization on phagosomes containing either yeast or hyphal cells. ANOVA and post hoc Bonferroni tests were performed to determine significant differences (***, P ≤ 0.001). (E) Positive correlation (Spearman's) between hyphal length and the duration of Rab14 retention on phagosomes containing C. albicans hyphal cells. Data are means ± SEM for at least three independent experiments.

Mentions: In order to visualize the temporal phagosomal localization of Rab14, RAW 264.7 macrophages were transfected to express enhanced green fluorescent protein (eGFP)-tagged Rab14 (Fig. 1). Live-cell video microscopy enabled detailed analysis of the temporal localization of Rab14 to phagosomes containing live C. albicans cells. Prior to the onset of phagocytosis, clusters of Rab14 vesicles were localized diffusely around the cell, concentrated around the perinuclear region, as shown previously for other cell types (27). A commercially available anti-Rab14 antibody was used to confirm the binding pattern of Rab14 identified by eGFP-Rab14-expressing macrophages (see Fig. S1 in the supplemental material). Following the internalization of C. albicans cells by macrophages, newly formed phagosomes transiently acquired Rab14 approximately 2 min after the completion of engulfment. Rab14 remained associated with phagosomes for an average of 6.0 ± 0.7, 5.6 ± 0.6, or 6.8 ± 1.0 min on phagosomes containing the WT strain (CAI4-CIp10) ingested in yeast form, a yeast-locked mutant (efg1Δ) strain, or 6-μm latex beads, respectively (Fig. 1). The presence of Rab14 on phagosomes containing C. albicans cells was confirmed by staining of primary cells (BMDM), which are not amenable to transfection, with an anti-Rab14 antibody (see Fig. S2 in the supplemental material). Next, we examined whether the phagosomal localization of Rab14 was influenced by the morphology of the ingested fungal particle. Live phagocytosis imaging permits the observation of Rab14 dynamics on individual phagosomes containing yeast-locked fungi, yeast cells that subsequently germinate into hyphae, and fungi that have already germinated prior to engulfment. As with phagosomes containing the yeast form of C. albicans, localization of Rab14 to phagosomes containing hyphae occurred within 2 min (Fig. 1). Interestingly, phagosomes containing hyphal fungal cells retained Rab14 for 16.8 ± 3.2 min—around 3-fold the duration seen on phagosomes containing yeast cells (Fig. 1D). Hyphal lengths were measured using a line tool (Volocity; PerkinElmer), and the correlation of the hyphal length at the time of engulfment with the duration of the retention of Rab14 on phagosomes was determined. A positive correlation was observed (Rs = 0.35) between hyphal length and the duration of Rab14 recruitment to phagosomes (Fig. 1E). Figure 1A and B show a time lapse sequence of images in macrophages transfected with eGFP-Rab14 and infected with live C. albicans cells in either the yeast or the hyphal form. (The corresponding movies can be viewed in Videos S1 and S2 in the supplemental material, respectively).


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)

Rab14 associates transiently and differentially with phagosomes containing C. albicans yeast or hyphal cells. (A and B) Live RAW 264.7 macrophages were transfected with eGFP-Rab14, allowed to ingest C. albicans cells, and monitored by live-cell imaging using fluorescence (upper panels) and differential interfer ence contrast microscopy (lower panels). Bar, 5 μm. The corresponding movies can be viewed in Videos S1 and S2 in the supplemental material. (A) RAW 264.7 macrophages were transfected with eGFP-Rab14 for 16 h, treated with live C. albicans cells, and imaged using live-cell microscopy over a 6-h period. Selected frames depict events from the time of contact between a live C. albicans yeast cell and the macrophage to the time of Rab14 localization. The white arrows indicate colocalization between phagosomes containing C. albicans cells and Rab14. (B) Selected frames from a live-cell microscopy movie showing detailed events from the time of contact between a live C. albicans hyphal cell and the macrophage to the time of Rab14 localization. (C) Time from the engulfment of C. albicans yeast or hyphal cells to the start of Rab14 recruitment. A Mann-Whitney statistical test was performed because the data were skewed; ns, no significant difference. (D) Duration of Rab14 localization on phagosomes containing either yeast or hyphal cells. ANOVA and post hoc Bonferroni tests were performed to determine significant differences (***, P ≤ 0.001). (E) Positive correlation (Spearman's) between hyphal length and the duration of Rab14 retention on phagosomes containing C. albicans hyphal cells. Data are means ± SEM for at least three independent experiments.
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Figure 1: Rab14 associates transiently and differentially with phagosomes containing C. albicans yeast or hyphal cells. (A and B) Live RAW 264.7 macrophages were transfected with eGFP-Rab14, allowed to ingest C. albicans cells, and monitored by live-cell imaging using fluorescence (upper panels) and differential interfer ence contrast microscopy (lower panels). Bar, 5 μm. The corresponding movies can be viewed in Videos S1 and S2 in the supplemental material. (A) RAW 264.7 macrophages were transfected with eGFP-Rab14 for 16 h, treated with live C. albicans cells, and imaged using live-cell microscopy over a 6-h period. Selected frames depict events from the time of contact between a live C. albicans yeast cell and the macrophage to the time of Rab14 localization. The white arrows indicate colocalization between phagosomes containing C. albicans cells and Rab14. (B) Selected frames from a live-cell microscopy movie showing detailed events from the time of contact between a live C. albicans hyphal cell and the macrophage to the time of Rab14 localization. (C) Time from the engulfment of C. albicans yeast or hyphal cells to the start of Rab14 recruitment. A Mann-Whitney statistical test was performed because the data were skewed; ns, no significant difference. (D) Duration of Rab14 localization on phagosomes containing either yeast or hyphal cells. ANOVA and post hoc Bonferroni tests were performed to determine significant differences (***, P ≤ 0.001). (E) Positive correlation (Spearman's) between hyphal length and the duration of Rab14 retention on phagosomes containing C. albicans hyphal cells. Data are means ± SEM for at least three independent experiments.
Mentions: In order to visualize the temporal phagosomal localization of Rab14, RAW 264.7 macrophages were transfected to express enhanced green fluorescent protein (eGFP)-tagged Rab14 (Fig. 1). Live-cell video microscopy enabled detailed analysis of the temporal localization of Rab14 to phagosomes containing live C. albicans cells. Prior to the onset of phagocytosis, clusters of Rab14 vesicles were localized diffusely around the cell, concentrated around the perinuclear region, as shown previously for other cell types (27). A commercially available anti-Rab14 antibody was used to confirm the binding pattern of Rab14 identified by eGFP-Rab14-expressing macrophages (see Fig. S1 in the supplemental material). Following the internalization of C. albicans cells by macrophages, newly formed phagosomes transiently acquired Rab14 approximately 2 min after the completion of engulfment. Rab14 remained associated with phagosomes for an average of 6.0 ± 0.7, 5.6 ± 0.6, or 6.8 ± 1.0 min on phagosomes containing the WT strain (CAI4-CIp10) ingested in yeast form, a yeast-locked mutant (efg1Δ) strain, or 6-μm latex beads, respectively (Fig. 1). The presence of Rab14 on phagosomes containing C. albicans cells was confirmed by staining of primary cells (BMDM), which are not amenable to transfection, with an anti-Rab14 antibody (see Fig. S2 in the supplemental material). Next, we examined whether the phagosomal localization of Rab14 was influenced by the morphology of the ingested fungal particle. Live phagocytosis imaging permits the observation of Rab14 dynamics on individual phagosomes containing yeast-locked fungi, yeast cells that subsequently germinate into hyphae, and fungi that have already germinated prior to engulfment. As with phagosomes containing the yeast form of C. albicans, localization of Rab14 to phagosomes containing hyphae occurred within 2 min (Fig. 1). Interestingly, phagosomes containing hyphal fungal cells retained Rab14 for 16.8 ± 3.2 min—around 3-fold the duration seen on phagosomes containing yeast cells (Fig. 1D). Hyphal lengths were measured using a line tool (Volocity; PerkinElmer), and the correlation of the hyphal length at the time of engulfment with the duration of the retention of Rab14 on phagosomes was determined. A positive correlation was observed (Rs = 0.35) between hyphal length and the duration of Rab14 recruitment to phagosomes (Fig. 1E). Figure 1A and B show a time lapse sequence of images in macrophages transfected with eGFP-Rab14 and infected with live C. albicans cells in either the yeast or the hyphal form. (The corresponding movies can be viewed in Videos S1 and S2 in the supplemental material, respectively).

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