1. Volume 21, Issue 6, Pages 769-776.e3 (June 2017)
Shigella sonnei Encodes a Functional T6SS Used for Interbacterial Competition and Niche Occupancy 
Mark C. Anderson, Pascale Vonaesch, Azadeh Saffarian, Benoit S. Marteyn, Philippe J. Sansonetti 
Cell Host & Microbe 
Volume 21, Issue 6, Pages e3 (June 2017)
DOI: /j.chom
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2. Cell Host & Microbe 2017 21, 769-776. e3DOI: (10. 1016/j. chom. 2017
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3. Figure 1 Schematic Organization of the S. sonnei T6SS Cluster
(A) Schematic showing the genetic organization of the region encompassing the T6SS of S. sonnei CIP and the remaining genes found in S. flexneri M90T. Letters denote the Tss nomenclature. Arrows indicate the predicted promotor locations.
(B and C) Western blot analysis of Hcp2-his secretion in S. sonnei (B) and S. flexneri (C). Where indicated with a +, isopropyl β-D-1-thiogalactopyranoside (IPTG) (1 mM final concentration) was added to induce Hcp2-his expression. T3SS chaperone IpgC was used as a cell lysis and loading control. IpaB was used as a secretion control. See also Figure S1.
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4. Figure 2
S. sonnei T6SS Targets E. coli and S. flexneri and Is Required for Colonization of the Mouse Gut
(A and B) Recovered CFUs and images of overnight colonies from competition assays using colicin-resistant E. coli (A) and S. flexneri (B) strains expressing dsRed from plasmid pMW211. Bars and error represent mean ± SEM of recovered CFUs. See also Figures S2 and S3.
(C and D) Cohorts of 6-week-old BALB/cJRj mice were orally gavaged with 1010 CFUs of the indicated strains. Animals were killed after 24 hr and CFUs recovered from tissue (C) and feces (D) were enumerated. Mice were pre-treated (+) (n = 7) or not (−) (n = 10) with streptomycin 24 hr prior to infection. Data are expressed as log CFUs/g of material. See also Figure S4.
(E) Colon samples from streptomycin-treated animals infected with the indicated strains were cut into 30 μM sections and stained; DAPI (cell nuclei), Phalloidin (Actin), and Mub40-Cy5 (neutrophils). Shigella express pMW211 plasmid encoded dsRed. Center and right images have DAPI and DAPI/Actin channels removed respectively to visualize intra-tissue bacteria.
Statistical analysis of all comparisons was carried out using the Mann-Whitney test. ∗∗p < 0.01; ∗∗∗p <
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5. Figure 3
E. coli Colonization Impairs T6SS-Deficient Shigella and Is an In Vivo T6SS Target
(A and B) Streptomycin-treated mice (n = 13) colonized with E. coli MG1655 for 24 hr and challenged with the indicated Shigella strains. CFUs associated with tissue and fecal compartments were enumerated for surviving Shigella (A) and E. coli (B) after 48 hr of competition. Bars represent the mean recovered CFUs.
(C) Box and whisker plots from qPCR analysis with primers targeting E. coli and Lactobacillus using DNA isolated from Shigella-infected mice. Results are displayed as the ratio of E. coli to Lactobacillus DNA amplification in mice infected with S. sonnei or its isogenic ΔtssF mutant and normalized to uninfected control mice using the ΔΔCt method (n = 7 mice per group). Bars denote the average ΔΔCt values obtained. Boxes indicate the 25th to 75th percentile ranges, and whiskers represent the minimum and maximum values obtained.
Statistical analysis of all comparisons was carried out using the Mann-Whitney test. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < NS, not significant.
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6. Figure 4 S. sonnei and S. flexneri In Vivo Competition
(A and B) Streptomycin-treated mice were gavaged with a 1:1 mixture of S. sonnei and S. flexneri and monitored for 48 hr. Mice were killed and colons and feces homogenized before plating on medium selective for either S. flexneri or S. sonnei. S. flexneri CFUs were enumerated using chloramphenicol plates selective for S. flexneri. S. sonnei CFUs were obtained by growth on ampicillin plates selective for S. sonnei. Bars represent the mean recovered CFUs. Statistical comparisons were obtained using the Mann-Whitney test (n = 13 mice per group). ∗p < 0.05; ∗∗∗p < 0.001; NS, not significant.
(C) Histopathology of infected colonic tissue showing increased S. flexneri in the presence of the ΔtssF strain. S. sonnei strains contained plasmid pMW211 expressing dsRed and S. flexneri contained plasmid pSU2_2rp expressing superfolding GFP. Three representative images for each condition are shown.
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