A genome-wide in vitro bacterial-infection screen reveals human variation in the host response associated with inflammatory disease

Abstract

Recent progress in cataloguing common genetic variation has made possible genome-wide studies that are beginning to elucidate the causes and consequences of our genetic differences. Approaches that provide a mechanistic understanding of how genetic variants function to alter disease susceptibility and why they were substrates of natural selection would complement other approaches to human-genome analysis. Here we use a novel cell-based screen of bacterial infection to identify human variation in Salmonella-induced cell death. A loss-of-function allele of CARD8, a reported inhibitor of the proinflammatory protease caspase-1, was associated with increased cell death in vitro (p = 0.013). The validity of this association was demonstrated through overexpression of alternative alleles and RNA interference in cells of varying genotype. Comparison of mammalian CARD8 orthologs and examination of variation among different human populations suggest that the increase in infectious-disease burden associated with larger animal groups (i.e., herds and colonies), and possibly human population expansion, may have naturally selected for loss of CARD8. We also find that the loss-of-function CARD8 allele shows a modest association with an increased risk of systemic inflammatory response syndrome in a small study (p = 0.05). Therefore, a by-product of the selected benefit of loss of CARD8 could be increased inflammatory diseases. These results demonstrate the utility of genome-wide cell-based association screens with microbes in the identification of naturally selected variants that can impact human health.

Figure 1

Hi-HOST Reveals that LCLs Undergo Salmonella-Induced Cell Death (A) A schematic diagram showing the steps involved in carrying out the Hi-HOST assay. LCLs are infected in 96-well plates with bacteria tagged with an inducible GFP plasmid. After waiting an hour to allow for invasion, one adds gentamicin for an additional hour to kill extracellular bacteria and then splits LCLs into plates for each time point. Adding IPTG 75 min prior to each time point induces GFP expression. Supernatants are collected so that cytokine release can be measured, and cells are stained with 7-AAD and analyzed by flow-cytometry so that viability and intracellular bacteria can be measured. (B) Phase image of LCLs overlaid with 7-AAD fluorescence (red) 2 hr after infection with S. typhimurium. The scale bar represents 10 μm. (C) Effects of bacterial mutations on Salmonella-induced cell death. Four different LCLs were infected at an MOI of 30 with wild-type, ΔprgH, ΔssaT, or fliC;fljB S. typhimurium. Cell death is greatly reduced in all LCLs with the prgH and fliC;fljB mutants, and the observed variation among the four LCLs is similar to that in uninfected cells. Data shown are the means and standard deviation from two sets of experiments. (D) Inhibition of cell death with the caspase-1 inhibitor, Ac-YVAD-cmk. Two LCLs were treated with 50 μM Ac-YVAD-cmk beginning 30 min prior to infection and assayed for cell death. Data shown are the means and standard deviation from two sets of experiments. (E) Fluorescence-labeled inhibitor of caspase (FLICA) measurement of caspase-1 2 hr after S. typhimurium infection. The fraction of cells labeled by FAM-YVAD-fmk increases with Salmonella infection. Data shown are for LCL 7357 but are representative of several LCLs tested. (F) Dose-response curves of inhibition of cell death in LCL 7357 via the caspase inhibitors Ac-YVAD-cmk and Ac-DEVD-cmk. Cells were treated with 0, 1, 5, or 50 μM of each inhibitor 30 min prior to infection and assayed for cell death. The percentage of cell death in uninfected cells was subtracted from the amount measured at 3 hr after the initial infection. The IC₅₀ for YVAD is 1.9 μM, and that for DEVD is 16.1 μM. Similar values were obtained when an additional LCL (18853) was assayed (not shown). Data points are the means and standard error of the mean (SEM) for three separate experiments. (G) Linear regression of Salmonella-induced cell death versus log₂ (CASP1 expression) in combined CEU-YRI parents shows that higher levels of CASP1 are correlated with increased levels of cell death (Pearson r = 0.28; p = 0.002).

Figure 2

Variation in Salmonella-Induced Cell Death among CEU and YRI LCLs (A) Scatter plots of cell death in CEU and YRI parental LCLs. Each dot represents the mean percentage of each LCL that stained positive for 7-AAD (uninfected cell death subtracted from background) assayed in three separate experiments. Mean and standard deviation of each population is displayed in red. (B) Parent-offspring regression for cell death in combined CEU-YRI populations. Heritability is estimated at 14.9%. (C) Parent-offspring regression for S. typhimurium viable invasion, measured as the percentage of LCLs containing GFP-Salmonella at 3.5 hr, in combined CEU-YRI populations. Heritability is estimated at 54%.

Figure 3

Linkage Disequilibrium around rs2043211 in CEU and YRI Populations (A) CEU and (B) YRI populations. Boxes are colored on the basis of D′/LOD; red represents high LD. Numbers within boxes are r² values. The two SNPs identified by the screen (rs6503965 and rs6503966) are highlighted in yellow, rs2043211 is highlighted in red, and other SNPs in strong LD with rs6503966 (r² > 0.8) are highlighted in orange. Only 20 kb of the CARD8 gene (41.4 kb) is shown. Images were exported from HaploView.

Figure 4

A Polymorphism in CARD8 Causes Increased Cell Death in Response to Salmonella (A) The derived allele of rs2043211 correlates with decreased CARD8 expression levels. A is the ancestral allele, and T is the derived nonsense allele (designated based on the + strand of chromosome 19, as is the convention for the HapMap Project; CARD8 coding sequence is on the – strand). Expression levels for LCLs are from Stranger et al., 2007. All LCLs are displayed, and when family structure is controlled for in PLINK (QFAM-parents), a p value of 3.72 × 10⁻⁵ is obtained for the correlation between rs2043211 genotype and CARD8 expression. Median and interquartile ranges are displayed in red. (B) The derived allele of rs2043211 correlates with increased cell death. All LCLs were assayed for S. typhimurium cell death in three separate experiments, and mean values for each are shown. Background-subtracted values reflect the subtraction of uninfected cell death. All LCLs are displayed, and when family structure is controlled for in PLINK (QFAM-parents), a p value of 0.013 is obtained for the correlation between rs2043211 genotype and Salmonella-induced cell death. Median and interquartile ranges are displayed in red. (C) Overexpression of the ancestral CARD8 allele inhibits cell death. LCL 7357 was transfected with 2 μg empty vector or ancestral or derived alleles of CARD8. 6 hr after transfection, cells were infected with S. typhimurium at an MOI of 30, and cell death was measured by 7-AAD staining 2 hr after the initial infection. Only the ancestral allele inhibits the amount of detected cell death. The p value from repeated-measures ANOVA is 0.002. Similar results were obtained for LCL 18507 (not shown). Expression of HA-CARD8 was verified with a rabbit CARD8 antibody. Error bars show the SEM for three separate experiments. (D) Overexpression of the ancestral CARD8 allele reduces caspase-1 activity in Salmonella-infected cells. LCL 7357 was transfected with 2 μg empty vector or ancestral or derived alleles of CARD8. For CARD8 ancestral dose-response measurements, 0.5, 1, and 2μg were used. Western blot inset demonstrates increasing HA-CARD8 protein detected with CARD8 antibody. 6 hr after transfection, cells were infected with S. typhimurium at an MOI of 30, and caspase-1 activation was measured by FLICA. No difference was noted in caspase-1 activation for uninfected cells, but infected cells show that the ancestral allele inhibits caspase-1 activation. The p values on the graph are from two-tailed t tests. Errors bars are SEM for assays conducted four separate times (except HA-CARD8 [A] 0.5 μg and 1 μg, which were assayed twice). (E) RNAi against CARD8 causes increased cell death in LCLs with the ancestral allele. Each LCL was stably transduced with lentivirus expressing nonsilencing shRNA or two different CARD8 shRNAs. Knockdown was quantified in stably transduced lines via real-time RT-PCR, and expression levels normalized to LCL 7357 stably transduced with nonsilencing shRNA are shown. Each LCL was assayed in three separate experiments, and means with SEM are displayed. The p values on the graph are from paired two-tailed t tests. All other comparisons of nonsilencing versus CARD8 RNAi constructs give p > 0.05.

Figure 5

Evidence of Natural Selection Involving CARD8 and Cell Death in Mammals and Humans (A) Absence of CARD8 in a 15-mammal phylogenetic tree correlates with large group size. A mirror tree of 15 mammalian species displays color-coded group size on the left and the presence of CARD8 on the right. Details regarding putative orthologs can be found in Table S1. From Pagel's test of independent evolution, a p value of 0.002 is obtained from 1000 simulations. This tree contains only species where the presence or absence of CARD8 could be determined with high confidence (>5× coverage to call absence; presence requiring at least half of protein sequence found). An additional mirror tree of 24 mammalian species is presented as Figure S1. Image was exported from Mesquite. (B) Reduced Salmonella-induced cell death in humans from hunter-gatherer versus agricultural populations. Cells from Native American individuals were grouped into populations that had traditionally lived as hunter-gatherers (Pima, Karitiana, and Surui) and those that had lived in denser agricultural communities (Maya and Quechuan) at the time of contact with Europeans. The aggregated hunter-gatherer populations exhibit on average 4.2% less cell death (a 21% reduction) than the CEU, YRI, ASN, and agricultural Native American groups. Error bars show means and standard deviations for each population. One-way ANOVA gives a p value of 0.017 for the means' being different among the populations. The data suggest adaptation of inflammatory caspase activation in response to changes in human societies.