Journal: Cell Genomics
Article Title: Human variation impacting MCOLN2 restricts Salmonella Typhi replication by magnesium deprivation
doi: 10.1016/j.xgen.2023.100290
Figure Lengend Snippet: Increased mucolipin-2 expression restricts S . Typhi replication in human immune cell lines (A) The rs10873679 C-allele associates with less MCOLN2 mRNA expression in 1000 Genomes Project LCLs. Linear regression of 448 LCLs (238 females and 210 males) is significant (β = 5.8 ± 0.6; p < 2 × 10 −16 ; adjusted r 2 = 0.166). (B) The rs10873679 genotype does not associate with MCOLN3 mRNA expression in the same dataset (β = −0.05 ± 0.03; p = 0.07; adjusted r 2 = 0.005). LCLs in (A) and (B) are from four European populations (91 CEU, 92 FIN, 86 GBR, and 91 TSI) and one African population (88 YRI) and consist of 124 CC, 217 TC, and 107 TT individuals. (C) The rs10873679 C-allele associates with less MCOLN2 protein expression in 33 LCLs (19 female and 14 male) measured with quantitative mass spectrometry. LCLs are from four populations: 18 CEU, 10 YRI, 4 CHB, and 1 JPT. Linear regression is significant for MCOLN2 (β = 0.15 ± 0.05, p = 0.01; adjusted r 2 = 0.017). In (A)–(C), bars are mean ± SD. (D) Both interferon treatment and S . Typhi (STy) infection (MOI 10 for 24 h) stimulate MCOLN2 expression in THP-1s. Expression measured by qRT-PCR and quantified by ΔΔC T (ΔC T stimulated – ΔC T untreated). Seven replicates from three experiments. p values are from Dunnett’s T3 comparison after Welch’s ANOVA (p < 0.0001). (E) In the LCL GM18540 (derived from a female CHB), MCOLN2 knockdown increases S . Typhi replication, while MCOLN3 knockdown modestly decreases S . Typhi replication in comparison with non-targeting (NT) siRNA. Seven replicates from three experiments. Knockdown qPCR: 0.33-fold (±0.14) of NT MCOLN2 expression and 0.53-fold (±0.04) of NT MCOLN3 expression. p values are from Dunnett post-hoc comparison with NT following a one-way ANOVA (main effect p < 0.0001). (F) MCOLN2 knockdown (0.09-fold [±0.02] of NT) increases S . Typhi replication in THP-1s. Ten replicates from two experiments. (G) CRISPR-Cas9 knockout of MCOLN2 increases S . Typhi replication in THP-1s. Ten replicates from two experiments. (H) S . Typhimurium has a minor growth advantage in MCOLN2 knockout THP-1s. Six replicates from two experiments. In (E)–(H), ratios are mean in siRNA-treated/NT or knockout/wild-type cells. In (D)–(H), bars are mean ± SEM and all statistics are calculated with log 2 -transformed data. (I) Mcoln2 knockout does not significantly increase burden in C57BL/6J mice spleens 4 days post i.p. infection with 1,000 CFUs of late-log S. Typhimurium (14028s) tagged with p67GFP3.1. Eighteen wild types (10 female and 8 male) and 22 knockouts (13 female and 9 male) from six experiments. Lines are geometric means and log 10 (geo. mean) is shown above each genotype. p value calculated with log 10 -transformed data. Without the low outlier (log 10 [CFU] = 3.6; identified at Grubbs’ α = 0.01), the Mcoln2 +/+ log 10 (geo. mean) is 7.72 and p = 0.2. In (E)–(I), p values are from Welch’s t test.
Article Snippet: THP-1 knock out pools were confirmed to maintain ≥85% frameshift indels by Sanger sequencing that was analyzed with the inference of CRISPR editing (ICE) webtool v2.0 ( https://ice.synthego.com ) from Synthego.
Techniques: Expressing, Mass Spectrometry, Infection, Quantitative RT-PCR, Comparison, Derivative Assay, Knockdown, CRISPR, Knock-Out, Transformation Assay
![Increased mucolipin-2 expression restricts S . Typhi replication in human immune cell lines (A) The rs10873679 C-allele associates with less MCOLN2 mRNA expression in 1000 Genomes Project LCLs. Linear regression of 448 LCLs (238 females and 210 males) is significant (β = 5.8 ± 0.6; p < 2 × 10 −16 ; adjusted r 2 = 0.166). (B) The rs10873679 genotype does not associate with MCOLN3 mRNA expression in the same dataset (β = −0.05 ± 0.03; p = 0.07; adjusted r 2 = 0.005). LCLs in (A) and (B) are from four European populations (91 CEU, 92 FIN, 86 GBR, and 91 TSI) and one African population (88 YRI) and consist of 124 CC, 217 TC, and 107 TT individuals. (C) The rs10873679 C-allele associates with less MCOLN2 protein expression in 33 LCLs (19 female and 14 male) measured with quantitative mass spectrometry. LCLs are from four populations: 18 CEU, 10 YRI, 4 CHB, and 1 JPT. Linear regression is significant for MCOLN2 (β = 0.15 ± 0.05, p = 0.01; adjusted r 2 = 0.017). In (A)–(C), bars are mean ± SD. (D) Both interferon treatment and S . Typhi (STy) infection (MOI 10 for 24 h) stimulate MCOLN2 expression in THP-1s. Expression measured by qRT-PCR and quantified by ΔΔC T (ΔC T stimulated – ΔC T untreated). Seven replicates from three experiments. p values are from Dunnett’s T3 comparison after Welch’s ANOVA (p < 0.0001). (E) In the LCL GM18540 (derived from a female CHB), MCOLN2 knockdown increases S . Typhi replication, while MCOLN3 knockdown modestly decreases S . Typhi replication in comparison with non-targeting (NT) siRNA. Seven replicates from three experiments. Knockdown qPCR: 0.33-fold (±0.14) of NT MCOLN2 expression and 0.53-fold (±0.04) of NT MCOLN3 expression. p values are from Dunnett post-hoc comparison with NT following a one-way ANOVA (main effect p < 0.0001). (F) MCOLN2 knockdown (0.09-fold [±0.02] of NT) increases S . Typhi replication in THP-1s. Ten replicates from two experiments. (G) <t>CRISPR-Cas9</t> knockout of MCOLN2 increases S . Typhi replication in THP-1s. Ten replicates from two experiments. (H) S . Typhimurium has a minor growth advantage in MCOLN2 knockout THP-1s. Six replicates from two experiments. In (E)–(H), ratios are mean in siRNA-treated/NT or knockout/wild-type cells. In (D)–(H), bars are mean ± SEM and all statistics are calculated with log 2 -transformed data. (I) Mcoln2 knockout does not significantly increase burden in C57BL/6J mice spleens 4 days post i.p. infection with 1,000 CFUs of late-log S. Typhimurium (14028s) tagged with p67GFP3.1. Eighteen wild types (10 female and 8 male) and 22 knockouts (13 female and 9 male) from six experiments. Lines are geometric means and log 10 (geo. mean) is shown above each genotype. p value calculated with log 10 -transformed data. Without the low outlier (log 10 [CFU] = 3.6; identified at Grubbs’ α = 0.01), the Mcoln2 +/+ log 10 (geo. mean) is 7.72 and p = 0.2. In (E)–(I), p values are from Welch’s t test.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_3047/pmc10203047/pmc10203047__gr2.jpg)
