Fig 3. PrfA inhibition facilitates survival of infected macrophages alongside rapid expansion but ultimate elimination of intracellular L. monocytogenes.

(A) Survival of BMM determined by LDH release assay upon infection with wild type (WT) L. monocytogenes EGD-e and 10403S, ΔprfA, PrfA^G145S, and Δhly in the presence of PrfA inhibitor IWP-2 (10 μM) or DMSO (solvent control) at the indicated time points post-infection. Data are means +/- sem of 3 independent experiments, each performed in technical triplicates. Two-way ANOVA, Sidak multiple comparison test; *p<0.05, **p<0.01. (B) Immune fluorescence images of BMM infected with wild type L. monocytogenes or left uninfected, in the presence of PrfA inhibitor IWP-2 (10 μM) or DMSO at 5 days post-infection. No imageable cells remained in cultures infected in the presence of DMSO. Data are representative of at least three independent experiments. (C) Intracellular bacterial burden upon infection with wild type (WT) L. monocytogenes EGD-e and 10403S, ΔprfA, PrfA^G145S, and Δhly in the presence of PrfA inhibitor IWP-2 (10 μM) or DMSO (solvent control) at the indicated time points post-infection. Data are means +/- sem of 3 independent experiments, each performed in technical triplicates. (D) For the data in (C), the time required for a 50% increase in CFU (WT L. monocytogenes EGD-e, 10403S 4–10 h post-infection; PrfA^G145S 4–6 h post-infection) and 50% decline in CFU (10–96 h post-infection) of L. monocytogenes in infected macrophage cultures in the presence of DMSO (D) and IWP-2 (I) were calculated. Data are means +/- sem of 3 independent experiments, each performed in technical triplicates. N/A not applicable; n.d. not determined. Unpaired two-tailed t-test, *p<0.05. (E) LLO expression in BMM infected with WT L. monocytogenes, ΔprfA, PrfA^G145S, and Δhly in the presence of DMSO or IWP-2 (10 μM) for the indicated times. Untreated starting cultures served as control (ctrl). Western blot representative of 2 independent experiments.