1. Volume 21, Issue 3, Pages 390-402 (March 2017)
Salmonella enterica Remodels the Host Cell Endosomal System for Efficient Intravacuolar Nutrition 
Viktoria Liss, A. Leoni Swart, Alexander Kehl, Natascha Hermanns, Yuying Zhang, Deepak Chikkaballi, Nathalie Böhles, Jörg Deiwick, Michael Hensel 
Cell Host & Microbe 
Volume 21, Issue 3, Pages (March 2017)
DOI: /j.chom
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2. Cell Host & Microbe 2017 21, 390-402DOI: (10.1016/j.chom.2017.02.005)
Copyright © 2017 Elsevier Inc. Terms and Conditions

3. Figure 1 The SCV Is Accessible to Endosomal Content
(A) Scheme for pulse-chase (P/C) experiments with fluid phase markers and nanoparticles (NPs) as endocytic probes (EPs).
(B) HeLa cells stably expressing LAMP1-GFP were infected with Salmonella wild-type (WT), ssaV, or sifA mutant strains expressing GFP with a multiplicity of infection (MOI) of 100. P/C with fluorescent EPs (BSA-rhodamine NPs) was performed overnight (O/N) prior infection, or for 1 hr at 6–7 hr post-infection (p.i.). Live-cell imaging (LCI) was performed at 4 and 8 hr p.i.
(C) RAW264.7 macrophages stably expressing LAMP1-GFP were pulse chased with Dextran-Alexa Fluor 647 (D647) and infected with WT or ssaV Salmonella, or mock infected. LCI was performed 12 hr p.i.
(D) Scheme for quantification of accumulation of fluorescent EPs in the lumen of SCVs. Two surfaces were created with surface 1 (S1) showing the bacterial body and surface 2 (S2) the SCV lumen. For S1 and S2, the fluorescence intensity of EP (FIEP) was measured and the sum was divided by volume S1: (FIEPS1 + FIEPS2)/VS1.
(E) Quantification of EP accumulation in the SCV lumen harboring Salmonella (n ≥ 48 cells per condition).
(F) Salmonella in the SCV is directly accessible to EPs. HeLa cells infected with Salmonella WT or ssaV strains, each expressing GFP, at MOI 75 and pulse chased with BSA-rhodamine for 4–7 hr p.i. After LCI at 8 hr p.i. (a, maximum intensity projections, MIPs; b, single Z plane), cells were immediately fixed. 3,3′-diaminobenzidine (DAB) photooxidation by endocytosed BSA-rhodamine was performed and cells were prepared for TEM. Details for LAMP1-positive and fluid tracer-labeled SCVs and SIFs are shown by live-cell CLEM (b and c). In higher magnification micrographs of Salmonella (d), arrowheads indicate DAB polymer deposition in direct contact to Salmonella within SCVs.
Scale bars, 10 (B overview, C, and Fa), 5 (B merge and D), 2 (Fc), and 1 μm (Fd).
Cell Host & Microbe  , DOI: ( /j.chom )
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4. Figure 2
Membranes and Luminal Content of SIFs Are Rapidly Interchanging
(A–C) HeLa cells were infected with Salmonella WT expressing mCherry at MOI 75 and pulse chased with Dextran Alexa Fluor 488 (D488) at 3–7 hr p.i. FLIP experiments were performed for SIF lumen at 7–9 hr p.i. The fluorescence intensity (FI) of D488 was monitored for a part of an SIF network bleached 20 times at 488 nm (ROI 1). As control, the FI of a part of an SIF network of a non-bleached cell (ROI 2) was measured simultaneously. Extracellular background FI (ROI 3) was subtracted from ROI 1 and ROI 2. See Movie S1.
(A) Representative field of view with selected bleaching area (red circle) and measurement ROIs (white rectangles). Scale bar, 20 μm.
(B) Time series showing the continuous bleaching of the SIF network.
(C) Relative fluorescence intensity (RFI) of SIF lumen during FLIP experiment. In total, 30 cells per condition were measured in four independent experiments.
(D–G) HeLa cells were infected with Salmonella WT strain expressing mCherry at MOI 25 and FRAP of SIF membrane protein LAMP1-GFP (D and F) or SIF luminal content labeled by D488 (E and G) was performed at various time points p.i. Parts of single SIFs at various positions within the SIF network were imaged before and after a 488 nm laser pulse for photobleaching of GFP or D488. Recovery of green fluorescence was measured over time, and RFIs are displayed as a fraction of the intensity before bleaching. Representative image series are shown in Figures S1A and S2A.
(D) FRAP of SIF membranes.
(E) FRAP of SIF lumen.
(F) The final mobile fraction of LAMP1-GFP within SIF membrane as a percent of initial signal intensity.
(G) The final mobile fraction of D488 within SIF lumen as a percent of initial signal intensity.
For statistical analysis, Student’s t test was performed. Significance is indicated as n.s., not significant; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < At least 30 events per condition were measured.
Cell Host & Microbe  , DOI: ( /j.chom )
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5. Figure 3
Interchange of SCV Membranes and Lumen Depends on Function of the SPI2-T3SS
HeLa cells were infected with Salmonella WT, ssaV, or sifA sseJ strains at MOI 25, each expressing mCherry. FRAP experiments at 7–9 hr p.i. for SCV lumen (A and B) and SCV membranes (C and D) were basically performed as described for Figure 2. At least 30 events per condition were measured. Representative image series are shown in Figures S1B and S2B.
(A) Infected HeLa cells were pulse chased with D488 at 3–7 hr p.i. For WT-infected cells, SCVs with or without connection to SIFs were located.
(B) The final mobile fraction of D488 within SCV lumen as a percent of initial signal intensity.
(C) HeLa cells stably transfected with LAMP1-GFP were used for FRAP of SCV membranes.
(D) The final mobile fraction of LAMP1-GFP within SCV membrane as a percent of initial signal intensity.
Statistical analysis was performed as for Figure 2.
Cell Host & Microbe  , DOI: ( /j.chom )
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6. Figure 4 Dimensions of the SCV/SIF Continuum
HeLa cells expressing LAMP1-GFP were infected with Salmonella expressing GFP (A) or mCherry (B) and pulse chased with rhodamine-labeled NPs (A) or BSA (B).
(A) Quantification of endosomal compartments by LCI at various time points p.i. with Salmonella WT as indicated. 3D volume reconstruction was performed, and number of rhodamine-positive objects and relative increase in surface area of Salmonella-modified membranes (SMMs, yellow) were quantified for representative cells.
(B) CLEM analyses of SIF and SCV continuity in a Salmonella-infected HeLa cell. LCI was performed and cell fixed and processed for TEM. Several ultra-thin sections were stitched to generate a cell section with complete SIF network. SIFs were pseudo-colored orange, and magnified details (partially pseudo-colored) show the double-membrane organization of SIFs. Inner and outer SIF membranes were indicated with open and filled arrowheads, respectively. Scale bars, 10 μm (a) and 250 nm (d–f).
(C) Determination of the luminal volume and overall endosomal membrane area of the SCV/SIF continuum. The dimensions of bacteria and of SCV and SIF network were measured by means of TEM micrographs and 3D fluorescence images. Volumes and membrane areas were calculated by simplification of the bacterial body, the SCVs, and SIFs to spheres and cylinders as described in Figure S3. For defining the volume of SCVs, the volume of Salmonella within SCVs was subtracted. For the outer volume of WT double-membrane SIFs, the volume of the inner tubule was subtracted. For comparison of SIF networks, cells with similar SIF length of 350 μm were used, either harboring WT or sseF strains.
(D) Overall membrane areas and volumes of the SCV/SIF continuum were determined for cells harboring WT Salmonella with or without connection to SIFs, or cells infected with the sseF strain. Values for SCVs without SIFs were set to 1 and x-fold increase of SCV/SIF continuum was calculated.
Statistical analysis was performed as for Figure 2 comparing SCVs without SIFs to SCVs with SIFs. SDs were calculated from error propagation of the measured length and width.
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7. Figure 5
Access of Intracellular Salmonella to Extracellular Carbon Sources Is Dependent on SPI2-T3SS Function
(A) Carbohydrate-starved RAW264.7 cells were infected with Salmonella WT, ssaV, or sifA mutant strains with an MOI of 10 each harboring a plasmid encoding the lac operon. After 1 hr in medium containing 100 μg × mL−1 gentamicin and lacking carbohydrates, infected cells were incubated in medium containing 10 μg × mL−1 gentamicin and either 25 mM lactose or glucose. At 8 hr p.i., cells were lysed to determine β-Gal activity and colony-forming unit (CFU) counts, and activity was normalized by CFU.
(B) The x-fold induction is the ratio of β-Gal activity of bacteria cultured with lactose to bacteria cultured with glucose, either recovered from RAW cells or cultured in medium.
(C) Intracellular replication of Salmonella as function of availability of extracellular glucose was determined. RAW264.7 macrophages were set on medium without FCS and glucose 1 hr prior to infection, and FCS was out for the remainder of the experiment. Cells were infected with Salmonella WT, ssaV, sifA sseJ, or sseF strains at an MOI of 1 and afterward medium with 100 μg × mL−1 gentamicin without glucose was added for 1 hr. Next, this medium was replaced by medium containing gentamicin at 10 μg × mL−1 without (−Glc) or with 4.5 g × L−1 glucose (+Glc).
(D) Intracellular replication is the ratio of CFU recovered 8 hr p.i. to 1 hr p.i.
Depicted are means and SD of representative results of three independent experiments (B), or of three biological replicates each performed in triplicate (D). Statistical analysis was performed as for Figure 2.
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8. Figure 6
SPI2-T3SS-Dependent Endosomal Remodeling Increases Exposure of Intracellular Salmonella to Endocytosed Antibiotics
Intracellular replication of Salmonella was determined by the gentamicin protection assay. HeLa cells (A) or RAW264.7 macrophages (B) were infected with Salmonella WT, ssaV, or sifA sseJ strains at an MOI of 1. After infection, medium with 100 μg × mL−1 gentamicin was used to kill extracellular bacteria for 1 hr, followed by incubation with medium containing gentamicin at 10 μg × mL−1 (standard condition), or at 100 or 200 μg × mL−1. Intracellular CFU counts were determined 2 and 16 hr p.i. and intracellular replication is the CFU ratio of 16 to 2 hr. Depicted are means and SD of four biological replicates, each performed in triplicate. Statistical analysis was performed as for Figure 2.
Cell Host & Microbe  , DOI: ( /j.chom )
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9. Figure 7
Metabolic Activity of Intracellular Salmonella Depends on SPI2-T3SS-Mediated Endosomal Remodeling
HeLa or IFNγ-activated RAW264.7 cells were pulsed chased O/N with Dextran Alexa Fluor 647 (D647) and subsequently infected with Salmonella WT, ssaV, or sifA sseJ strains at MOI of 75 and 25 for HeLa and RAW264.7 cells, respectively. All strains constitutively expressed mKikumeG. Infected cells were imaged directly before and after a 405 nm laser pulse for photoconversion from KikumeG to KikumeR, and green and red FI were measured over the indicated time frames. As control, chloramphenicol (Cm) was added to block protein biosynthesis. At least 50 events per condition were analyzed, except 30 events for Cm controls. The D647 FI was recorded to select bacteria within intact SCVs and connection to SIFs for WT Salmonella. The ratio of green and red fluorescence of vacuolar bacteria was calculated and normalized values are displayed referring to the value before conversion.
(A) KikumeGR for probing metabolic activity. In proliferating, metabolically active bacteria, dilution of KikumeR, and new synthesis of KikumeG are anticipated. Non-proliferating, metabolically inactive bacteria are expected to maintain KikumeR.
(B) Infection of HeLa cells. Representative stills of the time series of WT-infected cells are shown in Figure S7.
(C) The slope of the green/red ratio was calculated as percent (%) × hr−1 for infected HeLa cells.
(D) Infection of IFNγ-activated RAW264.7 cells.
(E) The slope of the green/red ratio was calculated as percent (%) × hr−1 for infected RAW264.7 cells.
Statistical analysis was performed as for Figure 2.
Cell Host & Microbe  , DOI: ( /j.chom )
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