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. 2018 May 22;86(6):e00048-18. doi: 10.1128/IAI.00048-18

FIG 4.

FIG 4

Intracellular zinc toxicity within human neutrophils. (A) Relative expression levels of GAS metal transporter genes in 5448 WT after contact with human neutrophils. Genes tested included genes coding for zinc export (czcD), zinc import (adcA, adcB, and adcAII), copper export (copA), manganese import (mtsA), manganese export (mntE), and iron export (pmtA). Error bars show standard deviations of the results of 3 independent experiments; relative expression is expressed as fold change of gene expression in GAS in contact with neutrophils divided by gene expression in control GAS. Relative gene expression was calculated using the 2−ΔΔCT method, with gyrA as the reference gene. (B) Human neutrophil killing assay showing the percent survival of 5448 WT and zinc export (ΔczcD) and zinc import (ΔadcA ΔadcAII) mutants following coculture with human neutrophils in vitro. Error bars show standard deviations of the results of 3 independent experiments. One-way ANOVA of the results was performed for the WT versus the mutants. ***, P < 0.001; ns, not significant. (C) Competitive human neutrophil killing assay was performed using 50:50 mixtures of WT and ΔczcD mutant or WT and ΔadcA ΔadcAII mutant. Colonies were enumerated on selective THY agar plates after 30 min of incubation with human neutrophils. Competitive indices were calculated as total numbers of mutant colonies divided by total numbers of 5448 WT colonies. One-way ANOVA was performed on the competitive indices for both mutants compared to the WT. **, P < 0.005; ns, not significant. (D) Human neutrophil killing assay in the absence and presence of 10 μg/ml cytochalasin D. Cytochalasin D inhibits actin polymerization, hence preventing phagocytosis and intracellular uptake of bacteria. Results show the percent survival of 5448 WT and zinc export (ΔczcD), zinc import (ΔadcA ΔadcAII), and DNase1 (Δsda1) deletion mutants following coculture with human neutrophils in vitro. Error bars show standard deviations of the results of 3 independent experiments. One-way ANOVA of the results was performed for the WT versus the mutants. ****, P < 0.0001; ns, not significant.