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First published online 27 May 2008
doi: 10.1242/jcs.018432

Journal of Cell Science 121, 2027-2036 (2008)
Published by The Company of Biologists 2008
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Sorting nexin-1 defines an early phase of Salmonella-containing vacuole-remodeling during Salmonella infection

Miriam V. Bujny1,*, Phil A. Ewels1, Suzanne Humphrey2, Naomi Attar1, Mark A. Jepson2 and Peter J. Cullen1,{ddagger}

1 Henry Wellcome Integrated Signalling Laboratories, Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, BS8 1TD, UK
2 Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, BS8 1TD, UK


Figure 1
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  		Fig. 1. SNX1 is recruited to sites of Salmonella Typhimurium entry. HeLa cells were either fixed directly, or were infected with SL1344 for 15 min and fixed at indicated times. Cells were immunolabeled using anti-SNX1 (Alexa488, green) and stained with TRITC-phalloidin to visualize actin (red) and DAPI to label DNA (blue). (A) In uninfected cells, SNX1 displayed a cytoplasmic distribution with perinuclear enrichment. (B) SNX1 was recruited to invasion sites, readily identifiable by membrane ruffles, and (C) stayed associated with the bacteria. (D) After 180 minutes, SNX1 had resumed its original distribution. (E) For the kinetic analysis of SNX1 association with SCVs, between 50–150 SCVs were scored per assay per time point for the presence or absence of SNX1 (n>=6 for 15 minutes and 60 minutes ± standard deviation, s.d.; n=2 for 180 minutes and 360 minutes, error ± minimum or maximum). (F) SNX1 recruitment to SCVs is comparable in MDCK (n=4, ± s.d., 50–150 SCVs scored per assay) and HeLa cells (n>3, ± s.d.), analyzed after a 15-minute bacterial `pulse' or further 45 minutes incubation. Scale bar: 10 µm.

 

Figure 2
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  		Fig. 2. Further analysis of SNX1-recruitment to plasma membrane ruffles and SCVs. (A-C) MDCK cells were infected with the SL1344 strain for 15 minutes, then fixed and immunolabeled. Ten optical z-slices were deconvolved and 3D-image volume rendered (see also supplementary material Movie 1). (Ai-iii) SNX1 (green) clearly accumulates at membrane ruffles (arrows) and around bacteria (boxed area in Ai, magnification in Aii). (B) Magnification of the membrane ruffle shown in (Aiii), with the green (SNX1) channel omitted in (Biii). (C) A single optical z-section of the maximum projections shown in (Aiii) and (B), respectively, is displayed with insets providing the respective YZ- and XZ-view. Note that SNX1 is localized as a ring around the bacterium. (D) HeLa cells infected with SL1344 for 15 minutes were fixed, immunolabeled for endogenous SNX1 (Alexa594, red) and EEA1 (Alexa488, green), and labeled with DAPI (blue) and TRITC-phalloidin (magenta). Whereas SNX1 appears as globular or tubular at sites of infection, EEA1 displays a punctate distribution (arrowheads). (E) Quantification of SNX1 and EEA1 acquisition on SCVs, scoring between 40-100 SCVs in individual z-sections, expressed as percentage of all SNX1- and/or EEA1-positive bacteria (n=3 for 15 minutes, n=2 for 60 minutes, error ± minimum or maximum). Scale bar: 10 µm.

 

Figure 3
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  		Fig. 3. SNX1 recruitment induces long-range tubules appearing concomitant with vacuolar shrinking. MDCK cells expressing GFP-SNX1 were infected with the SL1344 strain and invasion was imaged live (see supplementary material Movie 2a-d). Selected frames of a region of interest are shown (time in seconds). (A) Phase contrast images. Note the size reduction of vacuoles (arrowhead) at vacuole containing bacteria (arrow). (B) GFP-SNX1 was readily recruited to bacteria and spacious SCVs and labeled long-rage tubules; these spacious vacuole-associated tubules (SVATs) were also occasionally observed to make contact with vesicular SNX1 pools (boxed area). (C-E) Analysis of vacuolar shrinking. (C) The changes of vesicle area (in pixels) per frame of one focal depth from movies of LCI infection assays were analyzed from randomly chosen large light-lucent vesicles (see D) in infected cells expressing GFP-SNX1 (vesicles 6-8, compare E) or non-transfected yet infected cells (1-5). Data are representative for over n=3 independent experiments for each cell type (HeLa or MDCK cells) with over ten cells imaged.

 

Figure 4
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  		Fig. 4. Further characterization of SVATs. (A) HeLa cells expressing GFP-SNX1 were infected with SL1344, fixed after a 15-minute infection and treated with TRITC-phalloidin and DAPI. GFP-SNX1 (green) creates a meshwork around bacteria (blue). Images of a deconvolved stack are shown (see supplementary material Movie 3). (B,C) Tubulation is not an artifact of overexpressing GFP-SNX1. Cells infected for 15 minutes were fixed using 4% paraformaldehyde at 37°C for 15 minutes and immunolabeled for endogenous SNX1 (Alexa488, green). Scale bar: 10 µm.

 

Figure 5
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  		Fig. 5. No substantial recruitment of SNX1 was seen upon infection with a mutant strain that lacked SigD. (A) Cells expressing GFP-SNX1 were infected with a SL1344 strain lacking the inositol phosphatase SigD ({Delta}sigD) and infection was imaged for >20 minutes. Selected frames are shown (see also supplementary material Movie 4a,b). Although SNX1-positive tubules from endosomal structures were observed (arrows), there was no pronounced enrichment of GFP-SNX1 at the site of infection (arrowheads). (B) MDCK cells were infected a with wild-type SL1344 or with a SL1344-{Delta}sigD strain for 15 minutes, then fixed and labeled for endogenous SNX1 (Alexa568, red). Maximum projections of two z-sections (at 488 nm z-separation) are shown. Tubules up to 25 µm could be preserved in control cells (arrows). In SL1344-{Delta}sigD-infected cells, SNX1 is mainly vesicular with no pronounced recruitment to bacteria or even to very large vacuoles (arrowhead). Scale bar: 10 µm.

 

Figure 6
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  		Fig. 6. Characterization of infection of SNX1-suppressed cells. Control and SNX1-suppressed cells were infected with SL1344 for 15 minutes, fixed and immunolabeled with anti-SNX1 and treated with DAPI. (A) Additionally, cells were immunolabeled with anti-CI-MPR (Alexa488, green). In SNX1-suppressed cells, the CI-MPR is redistributed to the site of bacterial entry. (B) After removing bacteria, cells were either fixed directly or after further 45 minutes, and additionally labeled with TRITC-phalloidin (red). In SNX1-suppressed cells, S. Typhimurium displays reduced clustering. Scale bar: 10 µm.

 

Figure 7
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  		Fig. 7. In SNX1-suppressed cells, bacterial progress is slowed. (A,B) Analysis of intracellular progress of wild-type and {Delta}sigD bacteria in control and SNX1-suppressed cells. After 15 minutes, non-internalised bacteria were removed from siRNA-treated cells. Cells were subsequently fixed after a total of 180 minutes of incubation. After appropriate immunolabeling, cells were imaged and the distance covered by bacteria from the plasma membrane to the nucleus was measured for individual bacteria and expressed as a percentage of distance covered (at least 100-200 bacteria were measured). (C) Graphical representation of distance of bacteria from nucleus for wild-type and {Delta}sigD bacteria in control or SNX1 suppressed cells (± s.d). All P-values were calculated as comparisons with the wild-type value using a one-way ANOVA, followed by a post-hoc test (see Materials and Methods); **P<0.01.

 

Figure 8
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  		Fig. 8. In SNX1-suppressed cells, bacterial onset of replication is delayed. Analysis of bacterial replication kinetics in control and SNX1-suppressed cells for SL1344. The averaged number of bacteria per HeLa cell were determined at indicated times. Data are from a representative experiment with replicative efficiency being determined in triplicate (error bars shown ±s.d.). Similar data were observed in two additional independent assays.

 

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© The Company of Biologists Ltd 2008