1. Volume 27, Issue 7, Pages 2147-2156.e5 (May 2019)
Salmonella Translocated Effectors Recruit OSBP1 to the Phagosome to Promote Vacuolar Membrane Integrity 
Anna M. Kolodziejek, Melissa A. Altura, Junping Fan, Erik M. Petersen, Matthew Cook, Peter S. Brzovic, Samuel I. Miller 
Cell Reports 
Volume 27, Issue 7, Pages e5 (May 2019)
DOI: /j.celrep
Copyright © 2019 The Authors Terms and Conditions

2. Cell Reports 2019 27, 2147-2156.e5DOI: (10.1016/j.celrep.2019.04.021)
Copyright © 2019 The Authors Terms and Conditions

3. Figure 1
SseJ Binds to the Coiled-Coil Region of OSBP1 in a RhoA-Independent Manner
(A) Domain structure of human OSBP1, including amino acid numbering of regions.
(B) SseJ can pulldown OSBP1 from a eukaryotic cell soluble extract with or without binding RhoA. Purified His-tagged SseJ, the RhoA-binding-deficient SseJ mutant (SseJF121D), or the RhoA/SseJ complex in the enzymatically activated form (GppNHp bound) was incubated with Ni beads, washed, and incubated with an equal amount of lysate of HeLa cells expressing HA-OSBP1191–432. Proteins were separated on SDS-PAGE and immunoblotted with anti-HA antibodies to detect OSBP1191–432 binding. Transferred proteins were western blotted with anti-HA antibodies to detect HA-OSBP1 while Coomassie blue-stained SDS-PAGE shows the presence of SseJ and the complexity of the pre-incubation lysate (lysate only).
(C) SseJ binds to both full-length OSBP1 and fragments corresponding to the OSBP1 coiled-coil domain. His-SseJ-bound Ni beads were incubated with equal amounts of HeLa cell lysates expressing HA-OSBP1 and its truncated forms and were immunoblotted with anti-HA antibodies. Coomassie blue-stained SDS-PAGE shows equal levels of lysate additions (His pulldown [PD]), as well as the presence of SseJ in pulldown samples released from Ni beads for western blotting.
(D) SseJ/OSBP1 form a complex that is stable in size exclusion chromatography. His-SseJ and His-OSBP1191–357 were mixed in a 1:1 molar ratio, incubated for 20 min., and separated on an S200 size exclusion column. The chromatography profile of the protein mixture (black) was plotted as elution volume versus UV absorbance. Collected fractions corresponding to the indicated region were run on SDS-PAGE and visualized with Coomassie blue. Blue and green plots represent separately run profiles of His-SseJ and His-OSBP1191–357, respectively.
Cell Reports  , e5DOI: ( /j.celrep )
Copyright © 2019 The Authors Terms and Conditions

4. Figure 2
SseJ/RhoA Complex Can Direct OSBP1 to the Endosomal Compartment
(A) HeLa cells were transfected with HA-OSBP1 (red) with or without myc-SseJ (green) and stained for LAMP1 (cyan), a late endosomal marker.
(B) HeLa cells were transfected with SseJ deficient in RhoA binding (SseJF121D) (red) and OSBP1191–432 (green) and stained for LAMP1 (cyan), a late endosomal marker.
(C) HeLa cells transfected with OSBP1 (green) with or without cotransfection with SseJ (red) and with or without treatment with 50 μM 25OH. Counterstaining was for either the endosome (LAMP1) or the Golgi (RCAS1) (cyan).
Scale bars, 5 μm.
Cell Reports  , e5DOI: ( /j.celrep )
Copyright © 2019 The Authors Terms and Conditions

5. Figure 3
OSBP1 Is Recruited to the SCV in a SPI2- and SseJ-SseL-Dependent Manner
HeLa cells were infected with Salmonella Typhimurium for 24 h to allow sufficient accumulation of host proteins. Cells were fixed; stained for native OSBP1 (green), LAMP1 (red), and salmonellae (cyan); and analyzed for the percentage of infected cells with OSBP1 present on SCV.
(A) Representative micrographs showing infection with a WT, ssaT::mTn5 (SPI2−), ΔsseJΔsseL, or ΔsseJΔsseL complemented strain. Scale bars, 5 μm.
(B) Quantification of the percentage of infected HeLa cells containing LAMP1-positive SCV with OSBP1 colocalization. Average of three separate experiments (n = 55 counted cells for each experiment). Statistical analysis was determined from the average of the three data points. Data are presented as mean ± SEM from the three experiments; ∗p < 0.05 (ANOVA).
Cell Reports  , e5DOI: ( /j.celrep )
Copyright © 2019 The Authors Terms and Conditions

6. Figure 4
Loss of Both SseJ and SseL Destabilizes the SCV, Resulting in Increased Levels of Bacteria Escaping to the Cytoplasm
(A) Representative micrographs of HeLa cells infected with teal fluorescent protein (TFP)-expressing Salmonella Typhimurium (green) for 7 h, selectively permeabilized with digitonin, incubated with primary anti-Salmonella antibodies (red), and fixed. Green bacteria indicate vacuolar cells, yellow bacteria indicate cytoplasmic cells, and actin (cyan) was visualized with phalloidin. Scale bars, 10 μM.
(B) Cells were infected with WT Salmonella, ΔsseJ, ΔsseL, or ΔsseJΔsseL mutants and analyzed for the percentage of infected cells with cytoplasmic bacteria at 7 h post-infection. ΔsifA was used as a positive control for bacteria released into the cytoplasm. The ΔsseJΔsseL strain was complemented using the pAK222 plasmid expressing both SseJ and SseL. Average of three separate experiments, with three coverslips (n = 50 counted cells per coverslip) quantified in each experiment. A single average data point was derived from each experiment, and statistical analysis was conducted on the averages from the three separate experiments.
(C) Cells were infected with WT Salmonella, ΔsseJΔsseL mutant, or ΔsifA-positive control and analyzed for the percentage of infected cells with cytoplasmic bacteria at the indicated time points. Shown is quantification from three separate biological replicate coverslips conducted on the same day. Data are presented as mean ± SEM (n = 50 counted cells per coverslip); ∗p < 0.05, ∗∗∗∗p < (ANOVA) between indicated strain.
Cell Reports  , e5DOI: ( /j.celrep )
Copyright © 2019 The Authors Terms and Conditions

7. Figure 5
OSBP1 and the OSBP1-Binding Proteins VAPA/B Are Required for Maintenance of the SCV
(A) Representative western blot of HeLa cells left untransfected or transfected with anti-OSBP1 siRNA or nonsense siRNA with actin loading control.
(B) Cells left untransfected or transfected with anti-OSBP1 siRNA or nonsense siRNA for 96 h and then infected with WT Salmonella Typhimurium and analyzed for the percentage of infected cells with cytoplasmic bacteria at the indicated time points. Shown is a representative experiment conducted three times, with each experiment consisting of three biologically separate coverslips. Data are presented as mean ± SEM (n = 50 counted cells per coverslip); ∗p < 0.05 (ANOVA) among the three biologically separate coverslips within a single experimental day.
(C) Cotransfection of myc-SseJ (red) and HA-OSBP1 (green), followed by staining for native VAPA/B (purple).
(D) VAPA/B double knockout (VAPDKO) or its parental HeLa line were infected with WT Salmonella or ΔsseJΔsseL mutants and analyzed for the percentage of infected cells with cytoplasmic bacteria. Average of three separate experiments, with three coverslips (n = 50 counted cells per coverslip) quantified in each experiment. A single average data point was derived from each experiment, and statistical analysis was conducted on the averages from the three separate experiments. Data are presented as mean ± SEM; ∗p < 0.05 (Student’s t test) between WT and VAPDKO conditions.
Cell Reports  , e5DOI: ( /j.celrep )
Copyright © 2019 The Authors Terms and Conditions

8. Figure 6
Hypothesized Interactions among SseJ, SseL, OSBP1, and VAPA/B that Lead to SCV Stability
SseJ binding to GTP-bound RhoA directs the RhoA/SseJ complex to the SCV, where SseJ GCAT activity esterifies cholesterol, causing it to form lipid droplets. OSBP1 binding to SseJ also directs OSBP1 to the SCV, where it can recruit the VAPA/B proteins, facilitating lipid transfer to the SCV from a donor membrane. Meanwhile, the action of membrane remodeling, SseJ GCAT activity, or some other SPI2-related function causes the host to respond by ubiquitinating various factors on the SCV, potentially including OSBP1, the VAPA/B proteins, or other OSBP1-bound kinases and phosphatases. This ubiquitination and direction to the autophagy pathway is countered by the deubiquitinase activity of SseL, which is simultaneously recruiting OSBP1 to the SCV to further lipid transfer. Combined, these actions stabilize the SCV membrane.
Cell Reports  , e5DOI: ( /j.celrep )
Copyright © 2019 The Authors Terms and Conditions