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. 2010 Sep 1;12(9):1292-307.
doi: 10.1111/j.1462-5822.2010.01468.x. Epub 2010 Mar 25.

The Anaplasma phagocytophilum-occupied vacuole selectively recruits Rab-GTPases that are predominantly associated with recycling endosomes

Bernice Huang  1 , Andree Hubber, Justin A McDonough, Craig R Roy, Marci A Scidmore, Jason A Carlyon
Affiliations

The Anaplasma phagocytophilum-occupied vacuole selectively recruits Rab-GTPases that are predominantly associated with recycling endosomes

Bernice Huang et al. Cell Microbiol. .

Abstract

Anaplasma phagocytophilum is an obligate intracellular bacterium that infects neutrophils to reside within a host cell-derived vacuole. The A. phagocytophilum-occupied vacuole (ApV) fails to mature along the endocytic pathway and is non-fusogenic with lysosomes. Rab GTPases regulate membrane traffic. To better understand how the bacterium modulates the ApV's selective fusogencity, we examined the intracellular localization of 20 green fluorescent protein (GFP) or red fluorescent protein (RFP)-tagged Rab GTPases in A. phagocytophilum-infected HL-60 cells. GFP-Rab4A, GFP-Rab10, GFP-Rab11A, GFP-Rab14, RFP-Rab22A and GFP-Rab35, which regulate endocytic recycling, and GFP-Rab1, which mediates endoplasmic reticulum to Golgi apparatus trafficking, localize to the ApV. Fluorescently tagged Rabs are recruited to the ApV upon its formation and remain associated throughout infection. Endogenous Rab14 localizes to the ApV. Tetracycline treatment concomitantly promotes loss of recycling endosome-associated GFP-Rabs and acquisition of GFP-Rab5, GFP-Rab7, and the lysosomal marker, LAMP-1. Wild-type and GTPase- deficient versions, but not GDP-restricted versions of GFP-Rab1, GFP-Rab4A and GFP-Rab11A, localize to the ApV. Strikingly, GFP-Rab10 recruitment to the ApV is guanine nucleotide-independent. These data establish that A. phagocytophilum selectively recruits Rab GTPases that are primarily associated with recycling endosomes to facilitate its intracellular survival and implicate bacterial proteins in regulating Rab10 membrane cycling on the ApV.

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Figures

Figure 1
Figure 1. Selective GFP- or RFP-Rab GTPase localization to the ApV
A. phagocytophilum infected (A to S) or uninfected control (T to V) HL-60 cells were transiently nucleofected with DNA constructs to express the indicated GFP- or RFP-tagged Rab-GTPases or GFP alone. At 5 h post-nucleofection, the cells were fixed and viewed by indirect immunofluorescence. A to S, Representative confocal micrograph images depicting localization of GFP-Rab10A (A to C), GFP-Rab11A (D to F), GFP-Rab14 (G to I), RFP-Rab22A (J to L), GFP-Rab35 (M to O), or GFP-Rab4A (P) or lack of recruitment of GFP-Rab18 (Q), GFP-Rab33A (R), or GFP alone (S) to the ApV are presented. Intravacuolar and HL-60 cell surface-associated A. phagocytophilum organisms were denoted by indirect immunofluorescence staining with mAb 20B4, which recognizes the A. phagocytophilum surface protein, Msp2 (P44), but not the AVM, followed by anti-mouse IgG conjugated to Alexa Fluor-594 (B, E, H, M, P, Q, R, S) or Alexa Fluor-488 (K). Panels A, B, D, E, G, H, J, K, M, and N present single channel GFP (A, D, G, M), RFP (J), or Msp2 (P44) (B, E, H, K, N) fluorescence. Panels C, F, I, L, O, P, Q, R, and S present merged fluorescent images. T to V, In the absence of infection, GFP-Rabs exhibit diffuse and/or organelle-like patterns of fluorescence. Asterisks demarcate HL-60 cell nuclei.
Figure 2
Figure 2. Percentages of ApVs exhibiting GFP- or RFP-Rab GTPase localization
A. phagocytophilum inclusions as demarcated by anti-Msp2 (P44) staining of intravacuolar bacteria were scored for GFP- or RFP-Rab GTPase localization as in Figure 1. The number of GFP- or RFP-positive ApVs was divided by the total number of ApVs, which was multiplied by 100 to determine the percentage of ApVs to which each GFP- or RFP-Rab GTPase localized. Results are presented as the mean percentages (±SD) of ApVs exhibiting GFP- or RFP-Rab GTPase and are derived from two to four separate experiments. Up to 972 bacterial inclusions were examined for localization of each GFP-or RFP-Rab GTPase.
Figure 3
Figure 3. Time course analyses of GFP- or RFP-Rab GTPase recruitment to the ApV
Percentages of ApVs to which GFP- or RFP-Rab GTPases associated are presented. Results are the mean (±SD) of two to five experiments. Up to 575 bacterial inclusions were examined for recruitment of each GFP- or RFP-Rab per time point. For assessment of GFP- or RFP-Rab GTPase association with the ApV at 4, 6, 8, and 12 h post-infection, HL-60 cells were transiently nucleofected with DNA constructs encoding GFP- or RFP-Rab GTPases. At 12 h post-nucleofection, the cells were synchronously infected with A. phagocytophilum for the time periods indicated and then fixed and examined by indirect immunofluorescence microscopy. To assess GFP- or RFP-Rab GTPase recruitment to the ApV at 12, 18, and 24 h, HL-60 cells were synchronously infected with A. phagocytophilum. At 7 h post-infection, the cells were nucleofected to express GFP- or RFP-Rab GTPases and fixed and examined at 12, 18, and 24 h post-infection.
Figure 4
Figure 4. Endogenous Rab14 localizes to the ApV
A. phagocytophilum infected HL-60 cells were fixed and viewed by indirect immunofluorescence. Rab14 was detected using rabbit polyclonal antisera against Rab14 followed by anti-rabbit IgG conjugated to Alexa Fluor-488. Intravacuolar and HL-60 cell surface-associated A. phagocytophilum organisms were denoted by indirect immunofluorescence staining with mAb 20B4, which recognizes the A. phagocytophilum surface protein, Msp2 (P44), but not the AVM, followed by anti-mouse IgG conjugated to Alexa Fluor-594. Panels A, B, and C present single channel Rab14 detection, single channel Msp2 (P44) detection, and merged fluorescent images, respectively. Arrows denote ApVs that exhibit pronounced Rab14 staining. Results are representative of a total of 1031 inclusions examined for two separate experiments.
Figure 5
Figure 5. GFP-Rab10 and GFP-Rab14 localization to the ApV is insensitive to BFA
A. phagocytophilum infected HL-60 cells expressing GFP-Rab10 or GFP-Rab14 were treated with BFA or vehicle control (methanol) for 3 h, after which the cells were fixed, processed for immunofluorescence microscopy, and examined by LSCM. Panels A–L, confocal micrograph images depicting GFP-Rab10 and immunofluorescent staining for the Golgi marker, GM130 or A. phagocytophilum Msp2 (P44). Panels A to C and G to I, BFA-treated cells. Panels D to F and J to L, vehicle control-treated cells. Panels A, D, G, and J, GFP-Rab10. Panels B and E, GM130. Panels H and K, Msp2 (P44). Panels C, F, I, and L, merged images. Panel M, Percentages of ApVs to which GFP-Rab10 or GFP-Rab14 associates in the absence or presence of BFA. Results are the mean (± SD) of two separate experiments. At least 361 ApVs inclusions were examined for GFP-Rab GTPase localization.
Figure 6
Figure 6. Localization of GFP-Rab1, GFP-Rab4A, & GFP-Rab11A to the ApV is guanine nucleotide-dependent, while GFP-Rab10 association with the ApV is guanine nucleotide-independent
(A) A. phagocytophilum infected HL-60 cells expressing GFP-fusions of wild-type (WT), constitutively active GTPase-restricted (CA), and dominant negative GDP-restricted (DN) forms of Rab1, Rab4A, Rab10, and Rab11A were assessed for GFP-Rab recruitment to the ApV, which was demarcated by anti-Msp2 (P44) staining of intravacuolar bacteria. The number of GFP-positive ApVs was divided by the total number of ApVs, which was multiplied by 100 to determine the percentage of ApVs to which each GFP-Rab GTPase localized. Results are presented as the mean percentages (±SD) of ApVs exhibiting GFP-Rab GTPase recruitment and are derived from at least three experiments. A total of at least 642 bacterial inclusions were examined for recruitment of each GFP-Rab. (B) Comparison of the colocalization of GFP-Rab10 WT and GFP-Rab10N122I, which is a nucleotide-free mutant that is incapable of binding GTP or GDP (no-nucleotide, NN), to the ApV. Results are presented as the mean percentages (±SD) of ApVs exhibiting GFP-Rab GTPase recruitment and are derived from two experiments. Totals of 363 and 332 inclusions were examined for HL-60 cells expressing GFP-Rab10 and GFP-Rab10N122I, respectively.
Figure 7
Figure 7. Selective recruitment of GFP- or RFP-Rab GTPases to the ApV is dependent on bacterial protein synthesis
A. phagocytophilum infected HL-60 cells were transiently nucleofected with DNA constructs encoding the indicated GFP- or RFP-Rab GTPase. At 5 h post-nucleofection, tetracycline or vehicle control (70% ethanol) was added and incubation was continued. At 30 min, the cells were fixed and examined for GFP- or RFP-Rab GTPase association with the ApV. Intravacuolar A. phagocytophilum organisms were denoted by indirect immunofluorescence staining of Msp2 (P44). (A) GFP- or RFP-Rab localization to the ApV in the absence (−Tet) or 30 min post-addition of tetracycline (±Tet). Up to 817 bacterial inclusions were examined for GFP-Rab GTPase association per condition. Results are the mean (±SD) of at least three experiments. (B) GFP-Rab GTPase-positive A. phagocytophilum inclusions were examined for lysosomal fusion 30 min following the addition of tetracycline or vehicle control using a mAb directed against the lysosomal marker, LAMP-1. Up to 406 bacterial inclusions were examined for the localization of each GFP-Rab GTPase. Results are the mean (±SD) of at least three experiments. Statistically significant (* P<0.05; **P<0.01; ***P<0.001) values are indicated.
Figure 8
Figure 8. Model indicating the Rab GTPases and host cell membrane traffic pathways that are hijacked by the A. phagocytophilum-occupied vacuole (ApV)
ER, endoplasmic reticulum; cG, cis-Golgi; ERC, endocytic recycling center; IC, pre-Golgi intermediate compartment; LE, late endosome; LYS, lysosome; mG, medial-Golgi; NUC, nucleus; RE, recycling endosome; SG, secretory granule; SV, synaptic vesicle; tG, trans-Golgi. This figure is partially adapted from Figure 1 presented by Grant and Donaldson.
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