Fig 1.
VAPA associates with L. amazonensis-harboring communal PVs.
BMM were infected with L. amazonensis or L. major metacyclic promastigotes for the indicated time points. Controls consisted of untreated BMM and BMM fed with zymosan. (A) Distribution of VAPA (green) and its association with PVs were assessed by confocal immunofluorescence microscopy. DNA is shown in blue. (B) Quantification of the association of VAPA to L. amazonensis- and L. major-harboring PVs and to zymosan-containing phagosomes. (C) Colocalization of LAMP1 with VAPA on communal PVs harboring L.amazonensis at 48 h post-infection. Colocalization (white pixels) of LAMP-1 (red) with VAPA (green) was assessed and quantified by confocal immunofluorescence microscopy. Insets display the PV area. DNA is shown in blue. (D) Western blot analysis of VAPA levels over time in L. amazonensis-infected BMM. Data are presented as the means ± standard errors of the means (SEM) of values from three independent experiments. White arrowheads denote internalized parasites. Representative images from three experiments are shown. **, P ≤ 0.01; ***, P ≤ 0.001.
Fig 2.
VAPA regulates the replication of L. amazonensis and PV expansion.
BMM (untreated, treated with siRNA to VAPA, or treated with scrambled siRNA) were infected with L. amazonensis or L. major metacyclic promastigotes and at various time points post-phagocytosis parasite replication and PV size were assessed. (A) Quantification of L. amazonensis parasite burden at 2, 24, 48, and 72 h post-infection. Data are presented as the means ± SEM of values from three independent experiments. **, P ≤ 0.01; ***, P ≤ 0.001. (B) Quantification of L. major parasite burden at 2, 24, 48, and 72 h post-infection. Data are presented as the means ± SEM of values from three independent experiments. *, P ≤ 0.05; **, P ≤ 0.01. (C) Labeling of L. amazonensis amastigotes with BrdU in BMM (untreated or treated with siRNA to VAPA) infected for 48 h and 72 h. (D) Percent of BrdU+ parasites in BMM (untreated, treated with siRNA to VAPA or with scrambled siRNA) at 48 h and 72 h post-phagocytosis. Data are presented as the means ± SEM of values from three independent experiments. ***, P ≤ 0.001. (E) Quantification of PV size in BMM infected with L. amazonensis at 48 and 72 h post-phagocytosis. Data are presented as a cloud with means ± standard deviations (SD) of values from three independent experiments for a total of 450 PVs. **, P ≤ 0.01; ***, P ≤ 0.001. Blots showing the efficacy the siRNA-mediated VAPA knockdowns are shown in S1 Fig.
Fig 3.
VAPA is required for the acquisition of sphingolipids by L. amazonensis.
BMM (untreated, treated with siRNA to VAPA, treated with scrambled siRNA, and mock transfected) were infected with L. amazonensis metacyclic promastigotes and at 48 h post-infection BMM were incubated with BODIPY FL-C5-Ceramide (green). (A) Images of live cells were acquired at 5 min and 120 min after the addition of BODIPY FL C5-Ceramide. (B) Quantification of BODIPYFL-C5 positive L. amazonensis amastigotes at 5 min and 120 min after the addition of BODIPY FL-C5-Ceramide. Data are presented with means ± standard deviations (SD) of values from three independent experiments. ***, P ≤ 0.001 (in comparison to controls, siScr and mock). Blots showing the efficacy the siRNA-mediated VAPA knockdowns are shown in S1 Fig.
Fig 4.
CERT and ORP1L associate with L. amazonensis-harboring communal PVs and regulate their expansion.
BMM were infected with L. amazonensis metacyclic promastigotes and at the indicated time points the localization of CERT (red) (A) and ORP1L (red) (B) was assessed by immunofluorescence confocal microscopy. Co-localization of VAPA (green) with CERT (red) (A) and ORP1L(red) (B) was assessed by immunofluorescence confocal microscopy at 72 h post-infection. DNA is shown in blue. White arrowheads denote internalized parasites. Representative images from three independent experiments are shown. BMM (normal, treated with siRNA to CERT or scramble siRNA) were infected with L. amazonensis metacyclic promastigotes and at the indicated time points post-infection, parasite burden (C) and PV size (D) were assessed. BMM (untreated, treated with siRNA to ORP1L or scramble siRNA) were infected with L. amazonensis metacyclic promastigotes and at the indicated time points post-infection parasite burden (E) and PV size (F) were assessed. Data are presented as the means ± SD of values from three independent experiments. **, P ≤ 0.01; ***, P ≤ 0.001. For the determination of PV surface area (D, F) data are presented as clouds with means ± SD of values from three independent experiments for a total of 450 PVs. **, P ≤ 0.01; ***, P ≤ 0.001. Blots showing the efficacy the siRNA-mediated CERT and ORP1L knockdowns are shown in S2 Fig.
Fig 5.
L. amazonensis hijacks VAPA. BMMs were infected or not with either L. amazonensis (A-D), Δlpg1 and Δlpg1 + LPG1 L. donovani (E) or Δgp63 and Δgp63+GP63 L. major (F) metacyclic promastigotes. At the indicated time points post-infection, in situ VAPA-CERT (A) and VAPA-ORP1L (C) complexes were detected by proximity ligation and visualized by confocal immunofluorescence microscopy (red dots). DNA is in blue. Representative images from 3 independent experiments are shown. Quantification of in situ complexes for VAPA-CERT (B) and VAPA-ORP1L (D) in uninfected BMM and in BMM infected with L. amazonensis. Quantification of in situ complexes for VAPA-ORP1L in uninfected BMM and in BMM infected with either (E) Δlpg1 and Δlpg1 + LPG1 L. donovani or (F) Δgp63 and Δgp63+GP63 L. major. Data are presented as clouds with means ± standard deviations (SD) of values from three independent experiments for a total of 75 cells in each group. **, P ≤ 0.01; ***, P ≤ 0.001. Controls for the proximity ligation assays are shown in S3 Fig.
Fig 6.
VAPA is required for the transfer of LPG to the host cell ER.
(A) BMM were infected with L. major metacyclic promastigotes and at 6 h post-phagocytosis, the localization of VAPA (green) and LPG (red) were assessed by confocal immunofluorescence microscopy. Colocalized pixels are in white and DNA is in blue. (B) BMM (untreated or treated with siRNA to VAPA, scrambled siRNA, or mock transfected) were infected with L. major metacyclic promastigotes and at 6 h post-phagocytosis, the localization of LPG (red) was assessed by confocal immunofluorescence microscopy. DNA is in blue. (C). Untreated BMM (left panel) or BMM treated with siRNA to VAPA (right panel) were infected with L. major metacyclic promastigotes and at 6 h post-phagocytosis, the co-localization of LPG (red) with ERSeeing (green) or LAMP1 (green) was assessed by confocal immunofluorescence microscopy. Colocalized pixels are in white, DNA is in blue. White arrowheads denote internalized parasites. Representative images from 3 independent experiments are shown. Blots showing the efficacy the siRNA-mediated VAPA knockdowns are shown in S1 Fig.
Fig 7.
Proposed model for the role of VAPA in macrophages infected with L. amazonensis.
VAPA is required for the acquisition of host sphingolipids by L. amazonensis amastigotes, as well as for the transfer of the Leishmania virulence glycolipid lipophosphoglycan (LPG) to the host cell endoplasmic reticulum. The identity of the lipid transfer protein(s) (LTP) involved in this bi-directional lipid transfer remains to be determined. Created with BioRender.