GENETICS Correction for “Human genetic variation in VAC14 regulates Salmonella invasion and typhoid fever through modulation of cholesterol,” by Monica I. Alvarez, Luke C. Glover, Peter Luo, Liuyang Wang, Elizabeth Theusch, Stefan H. Oehlers, Eric M. Walton, Trinh Thi Bich Tram, Yu-Lin Kuang, Jerome I. Rotter, Colleen M. McClean, Nguyen Tran Chinh, Marisa W. Medina, David M. Tobin, Sarah J. Dunstan, and Dennis C. Ko, which was first published August 21, 2017; 10.1073/pnas.1706070114 (Proc Natl Acad Sci USA 114:E7746–E7755).
The authors note that the legend for Fig. 3 appeared incorrectly. The figure and its corrected legend appear below.
Fig. 3.
Loss-of-function studies and complementation indicate that VAC14 limits Salmonella invasion. (A) Reduction of VAC14 expression in LCLs by RNAi increases S. Typhi invasion. Percentages of S. Typhi invasion of LCL 18507 (YRI population) treated with either nontargeting (NT) or VAC14 siRNA demonstrated increased invasion with VAC14 depletion (P = 0.008). Data shown are the mean ± SEM of three experiments. Quantification of three Western blots of VAC14 knockdown showed 40% reduction in VAC14 protein levels (P = 0.01). (B) Reduction of VAC14 expression in HeLa cells by RNAi increased S. Typhi invasion. Shown are percentages of S. Typhi invasion in HeLa cells treated with either NT or VAC14 siRNA (P = 0.04). Data shown are the mean ± SEM from four experiments. (C) Representative Western blot of VAC14 protein demonstrated endogenous protein levels (WT), effective RNAi (siRNA VAC14), CRISPR knockout (vac14−/−), and plasmid overexpression (pVAC14) in HeLa cells. Protein extracted from each lane was collected from 300,000 cells, and α-tubulin was used as a loading control. Values below the blots show the mean ± SEM of three Western blots. (D) vac14−/− HeLa cells contain enlarged vacuoles, and transfection of pVAC14 rescued the vacuolated phenotype. Asterisks in the phase image denote cells that are transiently transfected with pVAC14-GFP. (E) Quantified (n = 100) vacuole-containing vac14−/− HeLa cells transfected with pVAC14-GFP demonstrated complementation (P = 0.001). (F) Complete loss of VAC14 protein expression in HeLa cells by CRISPR/Cas9 mutation increased S. Typhi invasion. S. Typhi invasion percentages demonstrated increased invasion in vac14−/− compared with WT cells (P = 0.005). Data shown are the mean ± SEM from four experiments. (G) Increase in Salmonella invasion is inversely correlated with VAC14 depletion (P = 0.05, r = −0.88). Increases in invasion percentage and the percentage of VAC14 protein depletion are calculated relative to Salmonella invasion with the GG allele in LCLs, NT siRNA controls, or WT HeLa cell controls. (H) Transient transfection of pVAC14 in vac14−/− cells complements invasion phenotype (P = 0.02). Data shown are the mean ± SEM from five experiments. All P values are calculated from paired t tests.
The authors also note that Fig. 5 appeared incorrectly. The corrected figure and its legend appear below.
Fig. 5.
Loss of VAC14 increases cholesterol at the plasma membrane. (A) vac14−/− cells have increased total cholesterol. WT and vac14−/− cells were fixed and stained with filipin, and fluorescence was measured by flow cytometry (P = 0.009). Fluorescent microscopy of WT and vac14−/− cells also shows increased filipin staining in the vac14−/− cells. Data shown are the mean ± SEM from three independent experiments. (B) Transient transfection of pVAC14 partially rescues cholesterol phenotype. Decreased filipin staining by flow cytometry was measured in vac14−/− cells transfected with pVAC14, while no difference was detected in transfected WT cells (P = 0.004). Data shown are the mean ± SEM from four independent experiments. ns, not significant. (C) vac14−/− cells have increased cholesterol at the plasma membrane. Imaging flow cytometry was used to image and measure WGA (cell membrane staining) and filipin staining in WT and vac14−/− cells. No difference is seen in WGA staining, while filipin staining at the plasma membrane is significantly increased in vac14−/− cells (P = 0.02). Data shown are the mean ± SEM from three independent experiments. (D) Expression of LDLR and HMG-Co-A Reductase mRNA are increased in vac14−/− cells. qPCR analysis of LDLR and HMGCR was done on WT and vac14−/− cells using 18S rRNA to normalize. Data shown are the mean ± SEM from three independent experiments for LDLR and four independent experiments for HMGCR. (E) rs8044133 is associated with free cholesterol levels in 48 CAP African American LCLs (P = 0.028). Cellular free cholesterol was measured using the Amplex Red Cholesterol Assay Kit, and rs8044133 genotypes were imputed. One heterozygous outlier was removed based on Grubbs’ test (P < 0.01). One-tailed P values are from linear regression. African American data without the outlier removed (P = 0.05) and European American data (P = 0.47) are shown in Fig. S5. (F) Cholesterol depletion with MβCD reduces S. Typhi invasion. The EC50 was significantly higher in vac14−/− cells than in WT cells (P = 0.02) indicating that greater amounts of MβCD are needed to overcome the higher cellular cholesterol in vac14−/− cells. Data shown are the mean ± SEM from nine independent experiments. (G) Repletion of cholesterol increases S. Typhi invasion. Exogenous cholesterol increases invasion in WT cells to levels similar to vac14−/− cells. Data shown are the mean ± SEM from four independent experiments. The P value is calculated from a paired t test.